COMPOSITION AND METHODS FOR DETECTING ADENINE MODIFICATIONS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/417,245 filed October 18, 2022, which is hereby incorporated by reference in its entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on October 18, 2023, is named ARCD_P0764WO_Sequence_Listing.xml and is 176,639 bytes in size. TECHNICAL FIELD [0003] The present invention relates generally to the field of molecular biology. More particularly, it concerns at least methods and compositions for detecting, evaluating, sequencing, and/or mapping modified adenosines. BACKGROUND [0004] A central question of biology is how the flow of genetic information from DNA to RNA to protein is regulated. While transcriptional regulation—the production of messenger RNA (mRNA)—plays major roles, and has been extensively studied, protein expression ultimately determines biological phenotypes. Protein production is augmented by various post- transcriptional regulations such as mRNA structure, microRNA, and mRNA translation; each of these processes fundamentally affects the protein levels and localizations that eventually impact every biological process. [0005] Reversible and dynamic mRNA and long non-coding RNA (lncRNA) modifications were recently discovered as being a fundamental mechanism that broadly controls protein expression at the post-transcriptional level (see e.g., Jia G, et al., Nat Chem Biol. 2011;7(12):885-7; Liu J, et al., Nat Chem Biol. 2014;10(2):93-5; Wang X, et al., Nature. 2014;505(7481):117-20; Zheng G, et al., Mol Cell.2013;49(1):18-29; and Fu Y, et al., Nat Rev Genet. 2014;15(5):293-306). [0006] Since then, there has been extensive research interest in profiling various mRNA/lncRNA modifications such as N ⁶ -methyladenosine (m ⁶ A) based on antibodies or pseudouridine (Ψ) based on a chemical reaction (see e.g., Carlile TM, et al, Nature. 2014;  515(7525):143-6; Schwartz S, et al., Cell. 2014;159(1):148-62; Dominissini D, et al., Nature. 2012;485(7397):201-6; and Meyer K, Cell.2012;149(7):1635-46). These studies have identified the presence of a very large number of modification sites, leading to the current high interests in the epitranscriptome field. Functional explorations of RNA modifications in various biological processes have so far uncovered several new gene expression regulatory mechanisms (see e.g., Liu J, et al., Nat Chem Biol. 2014;10(2):93-5; Wang X, et al., Nature. 2014;505(7481):117-20; Zheng G, et al., Mol Cell.2013;49(1):18-29; Batista PJ, et al., Cell Stem Cell. 2014;15(6):707-19; Chen T, et al., Cell Stem Cell. 2015;16(3):289-301; Geula S, et al., Science. 2015;347(6225):1002-6; Ping X-L, et al., Cell Res. 2014;24(2):177-89; Schwartz S, et al., Cell. 2013;155(6):1409-21; and Wang Y, et al., Nat Cell Biol. 2014;16(2):191-8). RNA modification is a highly fertile ground where additional regulatory mechanisms will be discovered. In particular, mRNA/lncRNA modifications are expected to be increasingly associated with human health and diseases as the field progresses. Additionally, mapping 6mA in mammalian gDNA can be considered exceedingly challenging due do the low levels of 6mA reported, and associated high rates of false positives/negatives. [0007] Despite the functional significances and potential associations with human diseases, mRNA/lncRNA modifications have generally been studied with methods significantly limited in resolution and sensitivity. Therefore, there is a need in the art for new methods of detecting RNA modifications. Additionally, there is a need in the art for new methods of detecting DNA modifications with ultrahigh efficiency. SUMMARY [0008] The current disclosure addresses the aforementioned need in the art and describes a new generation of base modification technologies, such as sequencing technology, that can be applied generally in order to obtain highly sensitive and single-base-resolution mapping of nucleic acid modifications. [0009] In some embodiments, technologies described herein include but are not limited to nucleic acids, polynucleotides, peptides, proteins, enzymes, protein-oligonucleotide complexes (including enzyme-oligonucleotide complexes), methods of use, methods of manufacturing, and/or kits. [0010] In some embodiments, provided herein are methods for detecting a methylated adenines in a target nucleic acid, such as ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) comprising an adenine and a methylated adenine, the method comprising: contacting the target nucleic acid with a tRNA-specific adenosine deaminase (TadA) enzyme to generate

137741474.1 - 2 - a deaminated target nucleic acid (e.g., DNA and/or RNA) comprising an inosine and the methylated adenine, and detecting the deaminated target nucleic acid. In certain embodiments, detecting the deaminated target nucleic acid comprises sequencing the nucleic acid. In certain embodiments, the TadA enzyme can deaminate adenines in single stranded RNA or in RNA duplexed with an at least partially complementary strand of RNA or DNA, or in single stranded DNA or in DNA duplexed with an at least partially complementary strand of RNA or DNA. In certain embodiments, the TadA enzyme is an evolved and/or mutant hypermorph relative to a wild type TadA enzyme, and/or has at least 80% sequence identity to any one of SEQ ID NOs: 1 to 33, or 105-113. In certain embodiments, the TadA enzyme is TadA8.20 (SEQ ID NO: 3). In certain embodiments, the TadA enzyme is TadA8r (SEQ ID NO: 25). In some embodiments, the TadA enzyme is TadA8e (SEQ ID NO:4). In certain embodiments, the TadA enzyme is TadA088c (SEQ ID NO: 30), TadA088d (SEQ ID NO: 31), TadA088e (SEQ ID NO: 32), or TadA088f (SEQ ID NO: 33). In some embodiments, the target nucleic acid comprises or consists of RNA. In some embodiments, the target nucleic acid comprises or consists of DNA. In some embodiments, the target nucleic acid comprises or consists of single stranded DNA (ssDNA). In some embodiments, the target nucleic acid is produced through DNA denaturation with NaOH, DMSO, high pH, salt, and/or heat. [0011] In certain embodiments, a method for detecting a methylated adenine in a target nucleic acid comprising an adenine and a methylated adenine, further comprises contacting an additional nucleic acid comprising an adenine and a methylated adenine with a demethylating enzyme to generate a demethylated nucleic acid; contacting the demethylated nucleic acid with the TadA enzyme to produce control nucleic acid comprising an inosine; and sequencing the control nucleic acid. In some embodiments, the additional nucleic acid is in vitro transcription (IVT) created RNA or in vitro created DNA. In some embodiments, the method further comprises comparing the sequence of the target nucleic acid to the sequence of the control nucleic acid. In some embodiments, the demethylating enzyme is an N6-methyladenosine (m6A)-specific demethylating enzyme and/or N ⁶ -deoxyadenosine methylation (6mA)-specific demethylating enzyme. In some embodiments, the demethylating enzyme is a fat mass and obesity-associated (FTO) demethylase or a AlkB Homolog 5 (ALKBH5) demethylase. In some embodiments, the target nucleic acid is in a duplex with a complementary strand of RNA or DNA. In some embodiments, the target nucleic acid is single stranded and is not in duplex with a complementary strand of RNA or DNA. In some embodiments, the TadA enzyme is derived from an Escherichia coli enzyme. In some embodiments, the target nucleic acid is an RNA and comprises mRNA, lncRNA, pri-microRNA, pre-piRNA, rRNA, tRNA, snoRNA, or snRNA.

137741474.1 - 3 - In some embodiments, the target RNA is mRNA. In some embodiments, the target RNA is pre- miRNA. In some embodiments, the target RNA is pri-miRNA. In some embodiments, the target RNA is miRNA. In some embodiments, the target RNA is not tRNA. [0012] In some embodiments, the deaminated target nucleic acid comprises in situ hybridization of the nucleic acid. In some embodiments, the method further comprises isolating nucleic acids. In some embodiments, prior to contacting the target nucleic acid with the TadA enzyme, the method does not comprise generating a nucleic acid strand that is complementary with the target nucleic acid and hybridizing the complementary nucleic acid strand with the target nucleic acid. In some embodiments, prior to contacting the target nucleic acid with the TadA enzyme, the method comprises generating a nucleic acid strand that is complementary with the target nucleic acid and hybridizing the complementary nucleic acid strand with the target nucleic acid. In some embodiments, prior to contacting the target nucleic acid with the TadA enzyme, the method comprises denaturing the target nucleic acid to a single stranded molecule. In some embodiments, the modified adenine is modified at the N6 position. In some embodiments, the modified adenine is N6-methyladenosine (m ⁶ A) and/or N ⁶ -deoxyadenosine methylation (6mA). In some embodiments, modified adenine is N6-dimethyladenosine (m ⁶ ₂ A). In some embodiments, the target nucleic acid is immobilized on a solid support. In some embodiments, the target nucleic acid is labeled. In some embodiments, the label is biotin. In some embodiments, the label is a phosphorothioate group. In some embodiments, the target nucleic acid is immobilized by reaction of the phosphorothioate group with a thiol-reactive group. In some embodiments, the thiol-reactive group is iodoacetamide, maleimide, or methanethiosulfonate. In some embodiments, the target nucleic is fragmented prior to immobilization on the solid support. In some embodiments, the target nucleic acid is fragmented into molecules about 50-300 nucleotides in length. In some embodiments, the average nucleic acid molecule fragment is about 100 nucleotides. In some embodiments, sequencing the target nucleic acid comprises construction of a library of nucleic acid molecules comprising the target nucleic acid sequence. In some embodiments, the method further comprises contacting the target nucleic acid with reverse transcriptase and/or polymerase to synthesize a complementary DNA strand. In some embodiments, the target nucleic acid is about 10-1000 nucleic acids in length. In some embodiments, the sequence of the target nucleic acid is known. In some embodiments, the method further comprises comparing the known sequence of the target nucleic acid with the sequence of the deaminated nucleic acid. In some embodiments, contacting the target nucleic acid with the TadA enzyme is done in the presence

137741474.1 - 4 - of Zn2+, H+, and H2O. In some embodiments, the method further comprises purification of the deaminated nucleic acid prior to sequencing. [0013] In certain embodiments, a method for detecting a methylated adenine in a target nucleic acid comprising an adenine and a methylated adenine, further comprises providing a quantification nucleic acid control comprising a known percentage of modified adenine; contacting the quantification control nucleic acid with the TadA enzyme to generated deaminated quantification control nucleic acid; and sequencing the deaminated quantification control nucleic acid. In some embodiments, greater than about 10% of the adenine in the quantification nucleic acid control is modified. In some embodiments, greater than about 50% of the adenine in the quantification nucleic acid control is modified. In some embodiments, greater than about 80% of the adenine in the quantification nucleic acid control is modified. In some embodiments, the target nucleic acid is in a composition comprising equal to or less than about 2.5 ng total RNA and/or DNA. In some embodiments, the target nucleic acid is in a composition comprising equal to or less than about 250 pg total RNA and/or DNA. In some embodiments, the target nucleic acid is in a composition comprising equal to or less than about 25 pg total RNA and/or DNA. In some embodiments, the target nucleic acid is in a composition comprising equal to or less than about 5 pg total RNA and/or DNA. In some embodiments, the target nucleic acid is obtained from less than or equal to about 50 cells. In some embodiments, the target nucleic acid is obtained from less than or equal to about 30 cells. In some embodiments, the target nucleic acid is obtained from less than or equal to about 10 cells. In some embodiments, the target nucleic acid is obtained from less than or equal to about 5 cells. [0014] In some embodiments, the target cell type is an embryonic stem cell, cell undergoing induced reprogramming, neuron, and/or neuronal precursor cell. In some embodiments, the TadA enzyme recognizes adenines within all DRACH sequences. In some embodiments, the TadA enzyme deaminates greater than or equal to about 85% of non- modified adenines in a target nucleic acid. In some embodiments, the TadA enzyme deaminates greater than or equal to about 90% of non-modified adenines in a target nucleic acid. In some embodiments, the TadA enzyme deaminates greater than or equal to about 96% of non- modified adenines in a target nucleic acid. In some embodiments, the TadA enzyme deaminates greater than or equal to about 98% of non-modified adenines in a target nucleic acid. In some embodiments, the TadA enzyme deaminates greater than or equal to about 99% of non- modified adenines in a target nucleic acid. In some embodiments, the TadA enzyme does not deaminate greater than or equal to about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%

137741474.1 - 5 - of cytosines. In some embodiments, the TadA enzyme deaminates substantively no cytosines, or little-to-no cytosines. In some embodiments, the TadA enzyme is insensitive to sequence context. In some embodiments, the TadA enzyme is contacted with the target nucleic acid for less than or equal to about 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, or 0.1 hours. In some embodiments, the TadA enzyme is contacted with the target nucleic acid for less than or equal to about 2 hours. In some embodiments, the TadA enzyme is contacted with the target nucleic acid for less than or equal to about 0.5 hours. In some embodiments, the TadA enzyme is contacted with the target nucleic acid at about or exactly 44 °C. In some embodiments, the TadA enzyme is contacted with the target nucleic acid at about or exactly 53 °C. In some embodiments, the TadA enzyme is contacted with the target nucleic acid at about or exactly 53 °C, and then the TadA enzyme is contacted with the target nucleic acid at about or exactly 44 °C. In some embodiments, the TadA enzyme is contacted with the target nucleic acid at a pH of about 5.5 to about 8.5. In some embodiments, the TadA enzyme is contacted with the target nucleic acid at a pH of about 7.5. In some embodiments, the TadA enzyme is contacted with the target nucleic acid more than one time. In some embodiments, the TadA enzyme is contacted with the target nucleic acid at least 3 times. [0015] In some embodiments, methods described herein further comprise identification and/or quantification of a biomarker of a disease state and/or a biomarker of likelihood of development of a disease state in a sample from a subject. In some embodiments, methods described herein further comprise modification of a regimen and/or a treatment of the subject as a function of the biomarker of a disease state and/or the biomarker of likelihood of development of a disease state. [0016] Also disclosed herein are kits comprising: a TadA enzyme and instructions for detecting modified adenines in target nucleic acids. In some embodiments, the TadA enzyme is an evolved and/or mutant hypermorph relative to a wild type TadA enzyme. In some embodiments, the TadA enzyme is fused and/or conjugated to a tag and/or label. In some embodiments, the tag and/or label comprises a maltose-binding protein (MBP). In some embodiments, the TadA enzyme has at least about 80% sequence identity to any one of SEQ ID NOs: 1 to 33, or 105-113. In some embodiments, the TadA enzyme is TadA8.20. In some embodiments, the TadA enzyme is TadA8r. In some embodiments, the TadA enzyme is TadA8e. In some embodiments, the TadA enzyme is TadA088c, TadA088d, TadA088e, or TadA088f. In some embodiments, a kit further comprises a control nucleic acid. In some embodiments, the control nucleic acid is a non-naturally occurring nucleic acid or non-widely

137741474.1 - 6 - present nucleic acid. In some embodiments, the control nucleic acid comprises a non-naturally occurring sequence. In some embodiments, the control nucleic acid comprises RNA and/or DNA comprising modified adenines. In some embodiments, the percentage of adenines that are modified in the control nucleic acid is known. In some embodiments, a kit further comprises an adenosine and/or deoxyadenosine demethylase. In some embodiments, the adenosine and/or deoxyadenosine demethylase is specific for N6-methyladenosine and/or 6mA. In some embodiments, the kit further comprises a reverse transcriptase and/or polymerase. In some embodiments, the kit further comprises NaOH and/or DMSO. In some embodiments, the kit comprises instructions for detecting modified adenosines in target RNA. In some embodiments, the kit comprises instructions for detecting modified deoxyadenosines in target DNA. [0017] Also disclosed herein are methods for detecting modified adenines in a target nucleic acid (e.g., DNA and/or RNA) comprising: a) contacting a target nucleic acid with a TadA enzyme to generate deaminated nucleic acid; and c) sequencing the deaminated nucleic acid. In some embodiments, the method does not comprise generating a DNA and/or RNA strand that is complementary with the target nucleic acid before contacting the target nucleic acid with the TadA enzyme. In some embodiments, a method comprises generating a single stranded target nucleic acid before contacting the target nucleic acid with the TadA enzyme. In some embodiments, the TadA enzyme has at least about 80% sequence identity to any one of SEQ ID NOs: 1 to 33, or 105-113. In some embodiments, the TadA enzyme is TadA8.20. In some embodiments, the TadA enzyme is TadA8r. In some embodiments, the TadA enzyme is TadA8e. In some embodiments, the TadA enzyme is TadA088c, TadA088d, TadA088e, or TadA088f. In some embodiments, the target nucleic acid is in a composition comprising at most about 2.5 ng total RNA and/or DNA. [0018] Also disclosed herein are methods for diagnosing a disease, comprising: measuring a methylated adenine in a target nucleic acid (e.g., RNA and/or DNA) comprising an adenine and a methylated adenine, the method comprising: contacting the target nucleic acid with a tRNA-specific adenosine deaminase (TadA) enzyme to generate a deaminated target nucleic acid comprising an inosine and the methylated adenine, and detecting the deaminated target nucleic acid. In some embodiments, detecting the deaminated target nucleic acid comprises sequencing the nucleic acid. In some embodiments, the TadA enzyme can deaminate adenosines in single stranded RNA or in RNA duplexed with an at least partially complementary strand of RNA or DNA, or deoxyadenosines in single stranded DNA or in DNA duplexed with an at least partially complementary strand of RNA or DNA. In some embodiments, the TadA enzyme is an evolved and/or mutant hypermorph relative to a wild

137741474.1 - 7 - type TadA enzyme, and/or has at least 80% sequence identity to any one of SEQ ID NOs: 1 to 33, or 105-113. In some embodiments, the TadA enzyme is TadA8.20. In some embodiments, the TadA enzyme is TadA8r. In some embodiments, the TadA enzyme is TadA8e. In some embodiments, the disease is cancer. In some embodiments, the target RNA comprises a sequence complementary to or identical to SEQ ID NO: 114, SEQ ID NO: 115, and/or SEQ ID NO: 116. In some embodiments, the target nucleic acid is from a eukaryotic organism. In some embodiments, the target nucleic acid is from a prokaryotic organism. In some embodiments, the target nucleic acid is from a pathogen. In some embodiments, the target nucleic acid is from a mammal. [0019] Also disclosed herein are methods for treating a disease, comprising: measuring a methylated adenine in a target nucleic acid comprising an adenine and a methylated adenine, the method comprising: contacting the target nucleic acid with a tRNA-specific adenosine deaminase (TadA) enzyme to generate a deaminated target nucleic acid comprising an inosine and the methylated adenine, and detecting the deaminated target nucleic acid, and treating the disease. In some embodiments, detecting the deaminated target nucleic acid comprises sequencing the nucleic acid. In some embodiments, the TadA enzyme can deaminate adenosines in single stranded RNA or in RNA duplexed with an at least partially complementary strand of RNA or DNA, or deoxyadenosines in single stranded DNA or in DNA duplexed with an at least partially complementary strand of RNA or DNA. In some embodiments, the TadA enzyme is an evolved and/or mutant hypermorph relative to a wild type TadA enzyme, and/or has at least 80% sequence identity to any one of SEQ ID NOs: 1 to 33, or 105-113. In some embodiments, the TadA enzyme is TadA8.20. In some embodiments, the TadA enzyme is TadA8r. In some embodiments, the TadA enzyme is TadA8e. In some embodiments, the disease is cancer. In some embodiments, the target nucleic acid comprises a sequence complementary to or identical to SEQ ID NO: 114, SEQ ID NO: 115, and/or SEQ ID NO: 116. In some embodiments, the treatment comprises inhibition of one or more target miRNAs. In some embodiments, the treatment comprises reversal of overexpression of one or more target mature miRNAs. In some embodiments, the treatment comprises inhibition of METTL3 function. [0020] In some embodiments, methods described herein further comprise contacting target RNA with a demethylating enzyme; contacting the demethylated target RNA with the adenosine deaminase enzyme to produce control RNA; and sequencing the control RNA. In further embodiments, the demethylated target RNA is made by methods and steps described herein for generating controls. In some embodiments, the method further comprises comparing

137741474.1 - 8 - the sequence of the target RNA with deaminated adenosines to the sequence of the demethylated target RNA. In some embodiments, methods described herein further comprise contacting target DNA with a demethylating enzyme; contacting the demethylated target DNA with the adenosine and/or deoxyadenosine deaminase enzyme to produce control DNA; and sequencing the control DNA. In further embodiments, the demethylated target DNA is made by methods and steps described herein for generating controls. In some embodiments, the method further comprises comparing the sequence of the target DNA with deaminated deoxyadenosines to the sequence of the demethylated target DNA. In some embodiments, the demethylating enzyme is an N6-methyladenosine-specific demethylating enzyme and/or N6- methyldeoxyadenosine-specific demethylating enzyme. In some embodiments, the demethylating enzyme is ALKBH5 or FTO. FTO (fat mass and obesity associated) and ALKBH5 (AlkB homolog 5, RNA demethylase or AlkB, alkylation repair homolog 5) are demethylating enzymes specific for N ⁶ -methyladenosine and/or N ⁶ -methyldeoxyadenosine. FTO and ALKBH5 are known in the art. The enzyme may be recombinantly made or synthetic, and may be from any species. In some embodiments, the enzyme is the mammalian enzyme. The human ALKBH5 is represented by GenBank Accession Nos: NM_017758.3 (mRNA) and NP_060228.3 (protein). The mouse ALKBH5 is represented by GenBank Accession Nos.: NM_172943.4 (mRNA) and NP_766531.2 (protein). The human FTO is represented by GenBank Accession Nos.: XM_011523313.1 (mRNA), XP_011521615.1 (protein), XM_011523316.1 (mRNA), XP_011521618.1 (protein), XM_011523314.1 (mRNA) XP_011521616.1 (protein), XM_011523315.1 (mRNA), and XP_011521617.1 (protein). In some embodiments, the demethylating enzyme is ALKBH5. The sequences associated with each of these GenBank accession numbers is herein incorporated by reference for all purposes. In some embodiments, the demethylating enzyme is from insects. In some embodiments, the demethylating enzyme is from Drosophila melanogaster. [0021] In some embodiments, the target nucleic acid comprises RNA, and the RNA may be any type of RNA in a cell. In some embodiments, the target RNA is pre-mRNA, mRNA, lncRNA, pri-microRNA, pre-miRNA, miRNA, pre-piRNA, rRNA, tRNA, snoRNA, or snRNA. In some embodiments, the target RNA is mRNA or lncRNA. In some embodiments, the method further comprises isolating RNA. In some embodiments, the method comprises isolating a specific RNA. The term isolating, in this context, refers to the separation of one type of RNA from other types of RNA. Therefore, the isolated RNA fraction may contain at least, at most, or exactly about 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% (or any derivable range

137741474.1 - 9 - therein) of a specific RNA type. In some embodiments, the term isolated also refers to something that is separated from cellular components and/or is free of cellular materials. [0022] When the term “modified adenosines” is used herein, the modified adenosine may be any modified adenosine known in the art and/or described herein. In some embodiments of the methods, compositions, and kits of the disclosure, the modified adenosine is N ⁶ - methyladenosine. In some embodiments, the nucleic acid may comprise more than one type of adenosine modification. In some embodiments, the method further comprises determining the type of modification in a type of RNA. [0023] When the term “modified adenine” is used herein, the modified adenine may be any modified adenine known in the art and/or described herein. In some embodiments of the methods, compositions, and kits of the disclosure, the modified adenine is N ⁶ -methyladenosine and/or N ⁶ -methyldeoxyadenosine. In some embodiments, the nucleic acid may comprise more than one type of adenine modification. In some embodiments, the method further comprises determining the type of modification in a type of nucleic acid. [0024] In some embodiments, the target RNA is immobilized on a solid support. Solid supports are known in the art and include, for example, glass, plastics, polymers, metals, metalloids, ceramics, organics, beads, agarose, cellulose, dextran (commercially available as, i.e., Sephadex, Sepharose) carboxymethyl cellulose, polystyrene, polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchange resins, plastic films, polyaminemethylvinylether maleic acid copolymer, glass beads, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. [0025] In some embodiments, the target nucleic acid (e.g., RNA and/or DNA) strand is labeled. As used herein, the term “label” intends a directly or indirectly detectable compound or reactable functional group useful for attachment of nucleic acids to solid supports. The label may also be conjugated directly or indirectly to the composition to be detected, e.g., polynucleotide or protein such as an antibody so as to generate a "labeled" composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A response that is simply

137741474.1 - 10 - detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. [0026] In some embodiments, the label is a compound or reactable functional group useful for attachment of a nucleic acid to a solid support. In some embodiments, the labelling may comprise biotinylation. In some embodiments, the label comprises, consists essentially of, or consists of biotin. In some embodiments, the label is a phosphorothioate group. In some embodiments, the target nucleic acid (e.g., DNA and/or RNA) is immobilized by reaction of the phosphorothioate group with a thiol-reactive group. In some embodiments, the thiol- reactive group is iodoacetamide, maleimide, or methanethiosulfonate. [0027] In some embodiments, the target nucleic acid (e.g., RNA and/or DNA) is fragmented prior to immobilization on the solid support. In some embodiments, the nucleic acid is fragmented into molecules 50-300 nucleotides in length. In some embodiments, the nucleic acid is fragmented RNA molecules 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 350, 400, 450, or 500 to 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 350, 400, 450, 500, 1000, 1500, or 2000 (or any range derivable therein) nucleotides in length. In some embodiments, the average nucleic acid molecule fragment is about 25, 50, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 325, 350, 375, 400, 450, 500, or 600 nucleotides in length. In some embodiments, the target nucleic acid is fragmented prior to immobilization. [0028] The target and/or demethylated target nucleic acid (e.g., RNA and/or DNA) may be of any length. In some embodiments, the target nucleic acid is 10-1000 nucleic acids in length. In some embodiments the target nucleic acid is at least, at most, or exactly about 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 7500, or 10000 nucleic acids in length, or any derivable range therein. In some embodiments, nucleic acid molecules may be DNA, RNA, or a combination of both. Nucleic acids may be recombinant, genomic, or synthesized. In additional embodiments, methods involve nucleic acid molecules that are isolated and/or purified. The nucleic acid may be isolated from a cell or biological sample in some embodiments. Certain embodiments involve isolating nucleic acids from a eukaryotic, mammalian, or human cell. In some cases,

137741474.1 - 11 - they are isolated from non-nucleic acids. In some embodiments, the nucleic acid molecule is eukaryotic; in some cases, the nucleic acid is mammalian, which may be human. This means the nucleic acid molecule is isolated from a human cell and/or has a sequence that identifies it as human. In particular embodiments, it is contemplated that the nucleic acid molecule is not a prokaryotic nucleic acid, such as a bacterial nucleic acid molecule. In particular embodiments, it is contemplated that the nucleic acid molecule is a prokaryotic nucleic acid, such as a bacterial nucleic acid molecule. In additional embodiments, isolated nucleic acid molecules are on an array. In particular cases, the array is a microarray. In some cases, a nucleic acid is isolated by any technique known to those of skill in the art, including, but not limited to, using a gel, column, matrix or filter to isolate the nucleic acids. In some embodiments, the gel is a polyacrylamide or agarose gel. [0029] In some embodiments, disclosed herein are methods directed to the detection of adenine modifications (e.g., quantification of levels and/or binary determination of presence or absence of adenosine modification) associated with disease states, e.g., biomarkers, e.g., biomarkers of a diseased state and/or likelihood of developing a diseased state. In certain embodiments, methods of treatment of a subject are modified according to biomarkers identified and/or quantified using methods described herein. [0030] In some embodiments, the sequence of the target and/or demethylated target nucleic acid (e.g., RNA and/or DNA) is known. The term sequence as used herein refers to the nucleotide sequence such as “A” for adenine, “G” for guanine, “C” for cytosine, “T” for thymine, and “U” for uracil. Even though the sequence is known, it may not be known whether the nucleic acid bases are modified or unmodified. [0031] In some embodiments, the method further comprises comparing the known sequence of the target RNA with the sequence of the target RNA with deaminated adenosines. [0032] In some embodiments, the method further comprises providing a quantification nucleic acid control comprising a known percentage of modified adenines; contacting the quantification control nucleic acid with the adenosine deaminase enzyme; and sequencing the deaminated quantification control nucleic acid. In some embodiments, 0, 25, 50, 75, or 100% of the adenine in the quantification nucleic acid control is modified. In some embodiments, the method comprises at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 quantification controls (or any derivable range therein). In each of the quantification controls, at least, at most, or exactly 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% (or any derivable range therein) of the adenines may be modified.

137741474.1 - 12 - [0033] Further aspects of the disclosure relate to a kit comprising: an adenosine deaminase enzyme and instructions for detecting modified adenines in target nucleic acids. In some embodiments, the kit further comprises a control nucleic acid. The control nucleic acid may be any embodiment described herein. In some embodiments, the control nucleic acid comprises a non-naturally occurring nucleic acid or non-widely present nucleic acid. In some embodiments, the control nucleic acids are a non-naturally occurring nucleic acid sequence. In some embodiments, the control nucleic acid comprises RNA or DNA comprising modified adenines. In some embodiments, the percentage of adenines that are modified in the control nucleic acid is known. In some embodiments, at least, at most, or exactly 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% (or any derivable range therein) of the adenines in the control nucleic acid are modified. In some embodiments, the kit further comprises an adenine demethylase. The adenine demethylase may be one known in the art and/or described herein. In some embodiments, the adenine demethylase is specific for N ⁶ - methyladenosine and/or N ⁶ -methyldeoxyadenosine. In some embodiments, the kit further comprises a DNase. In some embodiments, the kit further comprises an RNase inhibitor. In some embodiments, the kit further comprises a reverse transcriptase. In some embodiments, the kit further comprises a polymerase. In some embodiments, the kit further comprises NaOH and/or DMSO. In some embodiments, the kit further comprises a molecule or embodiment described herein in the methods and compositions. In some embodiments, the kit further comprises reagents to perform the method steps described herein, such as immobilization reagents, enzymes, and buffers described throughout the disclosure. [0034] In certain embodiments, the enzymes and/or nucleic acids used in the methods, kits, and compositions described herein may comprise one or more detectable moieties and/or modification. A detectable moiety refers to a chemical compound or element that is capable of being detected. In certain embodiments, a detectable moiety is fluorescent, radioactive, enzymatic, electrochemical, or colorimetric. In some embodiments, the detectable moiety is a fluorophore or quantum dot. In some embodiments, a modification moiety may be a linker that allows one or more functional or detectable moieties or isolation tags to be attached to the molecules. In some embodiments the linker is an azide linker or a thiol linker. In further embodiments, the modification moiety may be an isolation tag, which means the tag can be used to isolate a molecule that is attached to the tag. In certain embodiments, the isolation tag is biotin, Flag, or a histidine tag. In some cases, the tag is modified, such as with a detectable moiety. It is contemplated that the linker allows for other chemical compounds or substances to be attached to the molecule.

137741474.1 - 13 - [0035] Methods and compositions may also involve one or more enzymes. In some embodiments, the enzyme is a restriction enzyme or a polymerase. In certain cases, embodiments involve a restriction enzyme. The restriction enzyme may be methylation- insensitive. In other embodiments, the enzyme is polymerase. [0036] Methods may involve identifying adenine modifications in the nucleic acids by comparing modified nucleic acids with unmodified nucleic acids or to nucleic acids whose modification state is already known. Detection of the modification can involve a wide variety of recombinant nucleic acid techniques. In some embodiments, a modified nucleic acid molecule is incubated with polymerase, at least one primer, and one or more nucleotides under conditions to allow polymerization of the modified nucleic acid. In additional embodiments, methods may involve sequencing a modified nucleic acid molecule. In other embodiments, a modified nucleic acid is used in a primer extension assay. [0037] Methods and compositions may involve a control nucleic acid. In addition to the controls described herein, control may also be used to evaluate whether modification or other enzymatic or chemical reactions are occurring. Alternatively, the control may be used to compare modification states. The control may be a negative control or it may be a positive control. It may be a control that was not incubated with one or more reagents in the modification reaction. Alternatively, a control nucleic acid may be a reference nucleic acid, which means its modification state (based on qualitative and/or quantitative information related to modification at adenines, or the absence thereof) is used for comparing to a nucleic acid being evaluated. In some embodiments, multiple nucleic acids from different sources provide the basis for a control nucleic acid. In some embodiments, the control is a pool of target RNA that has undergone demethylation. Moreover, in some cases, the control nucleic acid is from a normal sample with respect to a particular attribute, such as a disease or condition, or other phenotype. In some embodiments, the control sample is from a different patient population, a different cell type or organ type, a different disease state, a different phase or severity of a disease state, a different prognosis, a different developmental stage, etc. [0038] Embodiments also concern kits, which may be in a suitable container, that can be used to achieve the described methods. In further embodiments, a kit may include one or more buffers, such as buffers for nucleic acids or for reactions involving nucleic acids. Other enzymes may be included in kits in addition to the adenosine deaminase enzyme. In some embodiments, an enzyme is a polymerase. Kits may also include nucleotides for use with the polymerase. In some cases, a restriction enzyme, (e.g. DNase) is included in addition to or instead of a polymerase.

137741474.1 - 14 - [0039] Other embodiments also concern an array or microarray containing nucleic acid molecules that have been modified at adenines. [0040] In certain embodiments, any one or more of the embodiments disclosed herein may be specifically excluded from any one or more of another embodiment disclosed herein. [0041] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” [0042] It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention. [0043] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method. [0044] As used herein, the terms “or” and “and/or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment. [0045] As used herein, the term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The phrase “consisting of” excludes any element, step, or ingredient not specified. The phrase “consisting essentially of” limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of the term “comprising” may also be implemented in the context of the term “consisting of” or “consisting essentially of.” [0046] As used herein, the terms “individual," “subject,” and “patient” are used interchangeably and can refer to a human or non-human. [0047] As used herein, a “protein” “peptide” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild- type versions of a protein or polypeptide are employed, however, in many aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure,

137741474.1 - 15 - particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity. [0048] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antibody or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule. [0049] The following is a discussion of changing the amino acid subunits of a protein to create an equivalent, or even improved, second-generation variant polypeptide or peptide. For example, certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity. [0050] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids. [0051] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,

137741474.1 - 16 - 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type (or any range derivable therein). A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids. [0052] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Description of the Drawings. [0053] A variety of embodiments are discussed throughout this application. Any embodiment discussed with respect to one aspect applies to other aspects as well and vice versa. Each embodiment described herein is understood to be embodiments that are applicable to all aspects. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition, and vice versa. Furthermore, compositions and kits can be used to achieve methods disclosed herein. [0054] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of” any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect. [0055] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DESCRIPTION OF THE DRAWINGS [0056] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better

137741474.1 - 17 - understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0057] FIGs.1A-1J. Global A deamination by TadA8.20. (A) Proposed m ⁶ A detection scheme. An adenosine deaminase (e.g., TadA-8.20) selectively converts A into I, without acting on m ⁶ A. I is recognized as G by reverse transcriptases. Persistent A post-TadA8.20 treatment corresponds to m ⁶ A. (1B and 1C). In vitro deamination of RNA probes hosting A or m ⁶ A in “CGAUC” (1B) and “GGACU” (1C) motifs by TadA8.20. Unmethylated and methylated RNA sequences were prepared through in vitro transcription using ATP and N ⁶ - methyl-ATP as starting materials, respectively. Treated RNA was reverse transcribed, amplified, and subjected to Sanger sequencing. (1D and 1E) TadA8.20-catalyzed A-to-I conversion rates in “CGAUC” (1D) and “GGACU” (1E) probes quantified by next-generation sequencing. (1F) Deamination of synthetic A/m ⁶ A RNA probes by TadA8.20. 53-nt RNA probes hosting NNANN (RNA #1 spike-in probe, SEQ ID NO: 97) and NNm ⁶ ANN (RNA #8 spike-in probe, SEQ ID NO: 104) motifs were treated by TadA-8.20. Deaminated RNA underwent RT and next-generation sequencing. (1G) Correlation of persistent A signals captured by eTAM-seq and m ⁶ A contents in RNA probes. (1H) Capillary gel electrophoresis analysis of fragmented HeLa mRNA treated with or without TadA8.20 at different temperatures for 3 h. RNA size distribution is plotted on the right. For exemplary eTAM-seq, RNA was incubated with TadA8.20 at 53 °C for 1 h followed by 2 h treatment at 44 °C. Experiments were repeated independently with similar results. (1I) Transcriptome-wide A-to- I conversion rates in two independent replicates. 10% of A sites with ^ 100 counts were randomly sampled to make the scatter plot. Pearson’s r was calculated for all A sites with ^ 100 counts. (1J) Two m ⁶ A sites in human rRNA. Positions 1829-1835 of 18S rRNA and positions 4217-4223 of 28S rRNA are plotted. [0058] FIGs. 2A-2I. Transcriptome-wide m ⁶ A profiling in HeLa mRNA by eTAM- seq. (2A) Overlap analysis of m ⁶ A sites identified in three biological replicates of eTAM-seq (HeLa/IVT). (2B) Methylation levels of common sites detected in eTAM-seq (HeLa/IVT-1) and eTAM-seq (HeLa/IVT-2). Correlative analyses on methylation levels reported by replicate 1 vs 3 and 2 vs 3 are provided in FIG.12. (2C) Hit distributions in DRACH and non-DRACH sequences at different methylation levels. (2D) Cumulative m ⁶ A signals from highly methylated sites to lowly methylated sites (right to left). m ⁶ A constitutes 0.41% of all A subject to evaluation in the HeLa transcriptome. (2E) Overlap analysis of m ⁶ A sites identified by eTAM-seq and peak clusters generated via MeRIP-seq. The overlap between eTAM-seq and

137741474.1 - 18 - MeRIP-seq increased with higher read depth and methylation levels. (2F) Metagene plot of transcriptome-wide distribution of m ⁶ A. m ⁶ A distributions across different RNA regions are provided in the inserted pie chart (IGR = intergenic region; ncRNA = non-coding RNA). (2G) Major sequence motifs hosting m ⁶ A. DRACH motifs are in black and non-DRACH motifs are colored in red. The consensus sequence hosting m ⁶ A was inserted. (2H) m ⁶ A sites co- discovered by eTAM-seq and m ⁶ A-SAC-seq. Hits in DGACU captured by both methods are subject to overlap analysis. M6A-SAC-seq dataset: GSE198246 (see e.g., Ge, R. et al. m(6)A- SAC-seq for quantitative whole transcriptome m(6)A profiling. Nat. Protoc., 2022). (2I) m ⁶ A positions and fractions in MALAT1, TPT1, MYC, and ZBED5. eTAM-seq signals were plotted as methylation levels (%)alongside meRIP-seq peaks in normalized read coverage. Note that eTAM-seq (HeLa/IVT) had slightly higher coverage than eTAM-seq (HeLa/FTO) and may therefore capture more m ⁶ A sites. MALAT1_2515, 2577, 2611 and TPT1_687, 703 are indicated by arrows. The coding sequence for TPT1 is on the minus strand of the genome. [0059] FIGs.3A-3E. m ⁶ A profiling in mouse embryonic stem cells (mESCs) by eTAM- seq. (3A) Hit distributions in DRACH and non-DRACH sequences at different methylation levels. m ⁶ A sites identified by eTAM-seq (mESC/IVT) were plotted. (3B) Metagene plot of transcriptome-wide distribution of m ⁶ A. m ⁶ A distributions across different RNA regions were inserted. (IGR = intergenic region; ncRNA = non-coding RNA). (3C) Overlap analysis of m ⁶ A sites identified by eTAM-seq and peak clusters generated via MeRIP-seq. Hits detected by eTAM-seq (mESC/IVT) were overlapped with a published MeRIP-seq dataset (top) (see e.g., Zhang et al., 2021). One MeRIP-seq peak covers multiple m ⁶ A sites (bottom). Similar analyses using the eTAM-seq (mESC/FTO) dataset are provided in FIG. 23. (3D) Methylation levels reported by eTAM-seq (mESC/IVT) and eTAM-seq (mESC/FTO). (E) m ⁶ A positions and fractions in selected regions of Nanog, Sox2, and Klf4. eTAM-seq hits were plotted in methylation levels (%) and were juxtaposed with MeRIP-seq peaks in normalized read coverage. For a zoomed-out view of m ⁶ A distribution in full-length Nanog, Sox2, and Klf4, see FIG.24. [0060] FIGs. 4A-4F. m ⁶ A is strongly depleted in Mettl3 KO mESCs, and impacts transcript stability. (4A) Venn diagram showing the overlap of eTAM-seq detected m ⁶ A sites in ctrl and Mettl3 KO mESCs. (4B) Hit distributions in DRACH and non-DRACH sequences. (4C) Methylation levels of eTAM-seq captured m ⁶ A sites in ctrl and Mettl3 KO mESCs. Lower and upper hinges in the box plot represented first and third quartiles with the center line and red dot representing the median and mean, respectively. Whiskers cover ±1.5x of the interquartile range. (4D) Scatter plot of methylation levels for m ⁶ A sites jointly identified in

137741474.1 - 19 - ctrl and Mettl3 KO mESCs. (4E) Changes of methylation levels in ctrl and Mettl3 KO mESCs. Methylation difference for a given A site equals (=) methylation level in ctrl mESCs minus (-) methylation level in Mettl3 KO mESCs. (4F) Cumulative distributions for transcripts of different half-lives in HeLa cells treated with control and METTL3-targeting siRNA. Transcripts methylated to different levels were analyzed in separate bins (high m ⁶ A: n = 1,593; medium m ⁶ A: n = 1,594; low m ⁶ A: n = 1,593; no m ⁶ A: n = 1,758) . Box violin plots of transcript half-lives were inserted. Lower and upper hinges represented the first and third quartiles. The center line and the red dot denoted the median and the mean, with whiskers covering ±1.5x of the interquartile range. P-values were determined by one-tailed Wilcoxon rank-sum test using the unmethylated group as a reference. HeLa mRNA half-life dataset: GSE49339 (see e.g., Wang, X et al., 2014). [0061] FIGs. 5A-5E. Site-specific, deep sequencing-free m ⁶ A detection and quantification. (5A) Workflow for eTAM-seq-enabled site-specific quantification of m ⁶ A. mRNA is fragmented, ligated to a DNA adapter, treated by TadA8.20, and reverse transcribed into cDNA. Site-specific primers are designed to recognize post-deamination RNA sequences and amplify the loci of interest. m ⁶ A quantification can be achieved by both Sanger sequencing and amplicon deep sequencing. (5B) Methylation quantification for 4 m ⁶ A sites in HeLa mRNA (ACTB_1427, EIF2A_994, HDAC2_1815, and ZBED5_1575) by Sanger sequencing, amplicon deep sequencing, and RNA-seq. TadA8.20-treated IVT samples are provided for reference only. (5C) Methylation quantification for 4 m ⁶ A sites in HeLa mRNA (Malat1_2577, MYC_1841, ILF3_3108, and CLCN3_3332) by Sanger sequencing, amplicon deep sequencing, and RNA-seq. TadA8.20-treated IVT samples are provided for reference only. (5D) Methylation quantification for ACTB_1427 and EIF2A_994 with 5 ng, 500 pg, 50 pg, and 5 pg mRNA respectively. (5E) Methylation quantification for ACTB_1427 and EIF2A_994 with 25 ng, 2.5 ng, and 250 pg total RNA respectively. [0062] FIGs. 6A-6C. In vitro activity of TadA8.20. (6A) Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE, left) and size exclusion chromatography (schematic right) analyses of TadA8.20. Three independent experiments were conducted and produced similar results. (6B) Unmethylated and methylated E. coli tRNA ^Arg (ACG) (tRNA structure and sequence shown top left; see SEQ ID NO: 37) treated with wildtype TadA and TadA8.20. tRNA ^Arg (ACG) is the natural substrate of TadA, e.g., likely the RNA sequence best accepted by TadA. The fact that a TadA enzyme accepts this substrate does not necessarily translate into function on other RNA, particularly RNA of user-defined sequences. However, tRNA ^Arg (m ⁶ ACG) may be an appropriate substrate to probe whether TadA and TadA

137741474.1 - 20 - derivatives accept m ⁶ A. Unmethylated and methylated tRNA were prepared through in vitro transcription using ATP and N ⁶ -methyl-ATP as starting materials, respectively. Treated RNA was reverse transcribed, amplified by PCR, and subjected to Sanger sequencing (top right) and next-generation sequencing (bottom right). (6C) RT-qPCR analysis of RNA sequences pre- and post-TadA8.20 treatment. [0063] FIGs. 7A-7E. Identification of assay conditions for TadA8.20-mediated global A deamination. (7A) Predicted secondary structure of RNA #3 (SEQ ID NO: 40). RNA #3 is an in vitro transcribed RNA probe with multiple A sites shielded by secondary structure (see Table 6). This probe was utilized as a surrogate to evaluate efficiency of TadA8.20-mediated global A deamination. (7B-7D) TadA8.20-mediated A-to-I conversion under different (7B) enzyme concentrations, (7C) temperatures, and (7D) pH. (7E) Remaining activity of TadA post-1 h incubation at different temperatures on RNA #2 aka Y1D (SEQ ID NO: 39). [0064] FIG. 8. Adenosine deamination activity of TadA8.20 prepared in different batches. All reactions were carried out using the same RNA probe (RNA #2; SEQ ID NO: 39) under identical assay conditions (e.g., pH 7.5, 37 °C, 1 h). [0065] FIG. 9. Deamination of synthetic RNA probes. Deamination of synthetic RNA probes carrying 0%, 25%, 50%, 75%, and 100% m ⁶ A in NNA/m ⁶ ANN (0% = RNA #1 spike- in probe (SEQ ID NO: 97); 25% = mix of RNA #2 spike-in probe (SEQ ID NO: 98) and #5 (SEQ ID NO: 101); 50% = mix of RNA #3 spike-in probe (SEQ ID NO: 99) and #6 (SEQ ID NO: 102); 75% = mix of RNA spike-in probe #4 (SEQ ID NO: 100) and #7 (SEQ ID NO: 103); and 100% = RNA #8 spike-in probe (SEQ ID NO: 104)) by TadA8.20. RNA was treated by TadA8.20, reverse transcribed, and analyzed by next-generation sequencing. [0066] FIGs. 10A-10C. HeLa mRNA treated by TadA8.20. (10A) RNA abundances reported by canonical RNA-seq (see e.g., Liu, J. et al., 2014) and eTAM-seq. In the box plots, lower and upper hinges represented first and third quartiles; the center line represented the median; the red dot represented the mean; and whiskers represented ± 1.5x of the interquartile range. n = 38,600. (10B) Transcriptome-wide A-to-I conversion rates in three replicates. (10C) Correlation of A-to-I conversion rates in three biological replicates. Scatter plots cover 10% of randomly sampled A sites with ≥ 100 counts. Pearson’s r was calculated for all A sites with ≥ 100 counts. [0067] FIGs.11A-11E. Preparation of an in vitro transcribed transcriptome (IVT) and HeLa rRNA treated by TadA8.20. (11A) Preparation of a methylation-free transcriptome by in vitro transcription (see e.g., Zhang, Z. et al., 2021; and Hagemann-Jensen, M. et al., 2020) and predicted behaviors of fully, partially, and non-accessible A and m ⁶ A sites in eTAM-seq.

137741474.1 - 21 - (11B) Selected unmodified A sites resistant to TadA8.20 in human rRNA due to secondary structures. HeLa and in vitro transcribed (IVT) RNA samples (SEQ ID NOs: 129-137) were both treated with TadA8.20, reverse transcribed, and sequenced. (11C) Two N ⁶ , N ⁶ - dimethyladenosine (m ⁶ 2A) sites in human rRNA (SEQ ID NOs: 138-139). (11D) Selected 2ʹ- O-methyladenosine (Am) sites in human rRNA. eTAM-seq data of HeLa rRNA and in vitro transcribed RNA are plotted side by side for (11B-11D) (SEQ ID NOs: 140-147). (11E) Quantification of m ⁶ A and m ⁶ Am in poly(A)-free, fragmented, and ligated mRNA by triple quadrupole LC/MS. Results shown were averaged from two independent injections. Error bars represented the standard deviations. [0068] FIGs. 12A-12D. Reproducibility of eTAM-seq (HeLa/IVT). (12A) Hit distributions in DRACH and non-DRACH sequences. Hits are called out using independent IVT controls. (12B) Contour plots of methylation levels reported by three biological replicates. (12C) Overlap analysis of m ⁶ A sites identified in three biological replicates of eTAM-seq (HeLa/IVT) with merged IVT controls. (12D) Overlap analysis of m ⁶ A sites identified in deep sequenced eTAM-seq (HeLa/IVT-1) and independently processed biological replicates (for more information, see methods section “Comparison of three biological replicates of eTAM- seq (HeLa/IVT) using merged IVT controls”). [0069] FIGs.13A-13G. m ⁶ A sites captured by deep sequenced eTAM-seq (HeLa/IVT). (13A) Hit distributions among different methylation levels. (13B) Overlap analysis of m ⁶ A sites identified by eTAM-seq and peak clusters generated via MeRIP-seq (see e.g., Liu, J. et al., 2014). (13C) Numbers of m ⁶ A sites identified per MeRIP-seq peak and per gene. (13D) Major sequence contexts of m ⁶ A detected by eTAM-seq and miCLIP (see e.g., Linder, B. et al., 2015). (13E) Accessibility of all A sites in the HeLa transcriptome.92% of all evaluated A sites in the HeLa transcriptome showed accessibility ≥ 0.9. (13F) Hit distribution across different accessibility bins. (13G) Comparison of methylation levels for DGACU sites co- discovered by eTAM-seq and m ⁶ A-SAC-seq. In the box plots, lower and upper hinges represented first and third quartiles; the center line represented the median; and whiskers represent ± 1.5× interquartile range. [0070] FIGs.14A-14B. Persistent A signals detected in mRNA and IVT samples. (14A) Box plot displaying persistent A signals detected in mRNA and IVT samples. (14B) Scatter plot showing persistent A signals in mRNA and IVT samples. Hits are binned by methylation levels reported by eTAM-seq (HeLa/IVT) (10-20: n = 10,250; 20-30: n = 8,884; 30-40: n = 7,623; 40-50: n = 6.655; 50-60: n = 5,953; 60-70: n = 5,799; 70-80: n = 5,813; 80-90: n = 6,403; 90-100: n = 12,454). In the box plots, lower and upper hinges represented first and third

137741474.1 - 22 - quartiles; the center line represented the median; and whiskers represented ± 1.5x interquartile range. [0071] FIGs.15A-15C. Endogenous RNA editing poses minimal impact on eTAM-seq. (15A) Exemplary workflow to call RNA-editing sites. (15B) RNA-editing types and distribution. (15C) Overlap of RNA-editing sites with eTAM-seq hits. Note that RNA editing frequently occurs in repeats. eTAM-seq has lower sensitivity in repeats as only reads uniquely mapped to the genome were considered. [0072] FIG. 16. Schematic of transcriptome-wide m ⁶ A profiling. Schematic of transcriptome-wide m ⁶ A profiling by eTAM-seq assisted by an N ⁶ -demethylated control transcriptome. m ⁶ A sites are identified and quantified by comparing deamination patterns of RNA treated with or without FTO. [0073] FIG.17. Sequential demethylation and deamination of synthetic RNA probes. Sequential demethylation and deamination of synthetic RNA probes by FTO and TadA8.20. 53-nt RNA probes that contain 0%, 25%, 50%, 75%, and 100% m ⁶ A in NNA/m ⁶ ANN were treated by FTO, TadA8.20, reverse transcribed, and analyzed by next-generation sequencing (0% = RNA #1 spike-in probe (SEQ ID NO: 97); 25% = mix of RNA #2 spike-in probe (SEQ ID NO: 98) and #5 (SEQ ID NO: 101); 50% = mix of RNA #3 spike-in probe (SEQ ID NO: 99) and #6 (SEQ ID NO: 102); 75% = mix of RNA spike-in probe #4 (SEQ ID NO: 100) and #7 (SEQ ID NO: 103); and 100% = RNA #8 spike-in probe (SEQ ID NO: 104)). [0074] FIGs. 18A-18D. Reproducibility of eTAM-seq with an FTO- treated control transcriptome. (18A) Overlap analysis of m ⁶ A sites identified in three biological replicates of eTAM-seq (HeLa/FTO). (18B) Hit distributions in DRACH and non-DRACH sequences. (18C) Comparison of methylation levels reported by three biological replicates of eTAM-seq (HeLa/FTO). (18D) Correlation of methylation levels reported by three biological replicates of eTAM-seq (HeLa/IVT) and eTAM-seq (HeLa/FTO) in Pearson’s r. [0075] FIGs. 19A-19F. m ⁶ A profiling in HeLa cells referenced to an FTO-treated transcriptome. (19A) Hit distributions across different methylation levels. (19B) Hit distributions in DRACH and non-DRACH sequences at different methylation levels. (19C) Overlap analysis of eTAM-seq (HeLa/IVT) and eTAM-seq (HeLa/FTO). The two reference transcriptomes (IVT and FTO) were prepared separately with orthogonal protocols, thereby serving as independent controls. Only A sites sampled in both datasets were considered. (19D) Methylation levels reported by eTAM-seq (HeLa/IVT) and eTAM-seq (HeLa/FTO). (19E) Overlap analysis of m ⁶ A sites identified by eTAM-seq (HeLa/FTO) and peak clusters

137741474.1 - 23 - generated by MeRIP-seq (see e.g., Peer, E. et al., 2017). (19F) Numbers of m ⁶ A sites identified per MeRIP-seq peak and per gene. [0076] FIG.20. Methylation level comparisons. Methylation levels at MALAT1_2515, 2577, 2611 and TPT1_687, 703 reported by eTAM-seq (HeLa/IVT), eTAM-seq (HeLa/FTO), and m ⁶ A-SAC-seq (see e.g., Ge, R. et al., 2022). Error bars represented the standard deviations when results from three biological replicates were considered. [0077] FIGs.21A-21F. m ⁶ A positions and fractions in 16 HeLa transcripts. Transcripts (21A) SLC7A5, ACTB, and CAND1; (21B) CIAO1, CLCN3, and CXCR4; (21C) EIF2A, GRWD1, and H2AFX; (21D) HDAC2, HOXB7, and ILF3; (21E) JUNB, OGT, and PPIB; and (21F) TPX2. eTAM-seq results are plotted below MeRIP-seq peaks. Normalized read coverage is plotted in MeRIP-seq tracks. Note that eTAM-seq (HeLa/IVT) has slightly higher coverage than eTAM-seq (HeLa/FTO) and may therefore capture more m ⁶ A sites. [0078] FIGs. 22A-22J. Biological replicates for mESCs. (22A) Correlation of A-to-I conversion rates in two replicates.10% of A sites with ≥ 100 counts were randomly sampled to make the scatter plot. Pearson’s r was calculated using all A sites with ≥ 100 counts. (22B) Transcriptome-wide A-to-I conversion rates. (22C) Overlap analysis of m6A sites identified in eTAM-seq (mESC/IVT-1) and eTAM-seq (mESC/IVT-2). (22D) Hit distributions in DRACH and non-DRACH motifs for eTAM-seq (mESC/IVT). (22E) Correlation of methylation levels reported by eTAM-seq (mESC/IVT-1) and eTAM-seq (mESC/IVT-2). (22F) Overlap analysis of m6A sites identified by eTAM-seq (mESC/FTO-1) and eTAM-seq (mESC/FTO-2). (22H) Hit distributions in DRACH and non-DRACH motifs for eTAM-seq (mESC/FTO). (22I) Correlation of methylation levels reported by eTAM-seq (mESC/FTO-1) and eTAM-seq (mESC/FTO-2). (22J) Correlation of methylation levels reported by two biological replicates of eTAM-seq (mESC/IVT) and eTAM-seq (mESC/FTO) in Pearson’s r. [0079] FIGs.23A-23E. m ⁶ A profiling in mESCs. (23A) Hit distributions among different methylation levels. Hits identified by eTAM-seq (mESC/IVT) and eTAM-seq (mESC/FTO) are presented side by side. (23B) Distributions of m ⁶ A sites identified by eTAM-seq (mESC/FTO) in DRACH and non-DRACH motifs at different methylation levels. (23C) Overlap analysis of m ⁶ A sites identified by eTAM-seq (mESC/IVT) and eTAM-seq (mESC/FTO). A sites sampled in both datasets are considered. (23D) Overlap analysis of m ⁶ A sites identified by eTAM-seq (mESC/IVT) and peak clusters generated via MeRIP-seq (see e.g., Zhang, Z. et al., 2021). (23E) Overlap analysis of m ⁶ A sites identified by eTAM-seq (mESC/IVT) and peak clusters generated via MeRIP-seq (see e.g., Zhang, Z. et al., 2021). Single MeRIP-seq peaks and individual genes cover multiple m ⁶ A sites.

137741474.1 - 24 - [0080] FIG. 24. m ⁶ A positions and fractions in Oct4 and Rex1 (top) and full-length Nanog, Sox2, and Klf4 (bottom) in mESCs. eTAM-seq results are plotted below MeRIP-seq peaks. Normalized read coverage was plotted in MeRIP-seq tracks. [0081] FIGs.25A-25C. m ⁶ A profiling in ctrl and Mettl3 knock-out (KO) mESCs. (25A) Western blot showing successful knock out of Mettl3. Independent experiments were repeated at least three times with similar results. (25B) Overlap analysis of m ⁶ A sites detected in ctrl mESCs and wildtype mESCs, both by eTAM-seq (mESC/FTO). (25C) Metagene plot of transcriptome-wide distribution of m ⁶ A in ctrl and Mettl3 KO mESCs. [0082] FIGs.26A-26B. Simulated and observed distances of one m ⁶ A site to its nearest neighbor. (26A) Two simulations were carried out with no context constraint. (26B) Two simulations were carried out by forcing m ⁶ A-carrying 5-nt motifs to match frequencies observed in eTAM-seq. [0083] FIGs.27A-27B. YTHDF2 is a core regulator for the stability of m ⁶ A-modified mRNA in HeLa cells. (27A) Cumulative half-life distributions for transcripts in HeLa cells treated with control and YTHDF2-targeting siRNA. (27B) Cumulative distribution for changes of transcript half-lives in HeLa cells treated with control and YTHDF2-targeting siRNA. Transcripts methylated to different levels (high m ⁶ A: n = 2,385; medium m ⁶ A: n = 2,386; low m ⁶ A: n = 2,385; and no m ⁶ A: n = 3,108) were plotted separately with box violin plots inserted. Lower and upper hinges represented first and third quartiles; the center line represented the median; the red dot represented the mean; and whiskers represented ± 1.5x interquartile range. P-values were determined by one-tailed Wilcoxon rank-sum test using the unmethylated group as a reference. HeLa mRNA half-life dataset: GSE49339 (see e.g., Wang, X. et al., 2014). [0084] FIGs.28A-28F. Detection and quantification of m ⁶ A by eTAM-seq. (28A) RT- qPCR products covering m ⁶ A-bearing regions (MALAT1, ACTB, EIF2A, ILF3, HDAC2, ZBED5, MYC, HOXB7, CAND1, OGT, TPX2, and CXCR4) analyzed by agarose gel electrophoresis. (28B-28D) Quantification of m ⁶ A sites in HeLa mRNA (28B) HOXB7 and SLC7A5 (SEQ ID NOs: 148 and 149), (28C) CAND1 and TPX2 (SEQ ID NOs: 150 and 151), and (28D) CIAO1 and OGT (SEQ ID NOs: 152 and 153), by Sanger sequencing, amplicon deep sequencing, and RNA-seq. (28E-28F) m ⁶ A quantification of additional m ⁶ A sites in HeLa mRNA (28E) JUNB and PPIB (SEQ ID NOs: 154 and 155), and (28F) GRWD1 and H2AFX (SEQ ID NOs: 156 and 157), by sanger sequencing in the absence of IVT controls. Independent experiments were carried out twice with similar results. [0085] FIGs. 29A-29D. Amplification of m ⁶ A-bearing transcripts from TadA8.20- treated mRNA and total RNA. (29A) RT-qPCR traces showing the amplification of

137741474.1 - 25 - fragments covering ACTB-1427 and EIF2A-994 from diluted cDNA. DNA standards were prepared by diluting ssDNA encoding the EIF2A sequence with all A replaced by G. std: standard. (29B) RT-qPCR amplification of fragments covering ACTB-1427 and EIF2A-994 from limited amounts of total RNA. (29C-29D) Analysis of RT-qPCR products by agarose gel electrophoresis. Independent experiments were carried out twice with similar results. [0086] FIGs. 30A-30F. Site-specific quantification of m ⁶ A using TadA8r. (30A) TadA8r was used for site-specific quantification of m ⁶ A from 50 ng total RNA, sites ACTB_1426, SLC7A5, JUNB and ACTB_1216 were tested (SEQ ID NOs: 158, 149, 154, and 159). Left: the Sanger traces of sites ACTB_1426, SLC7A5, JUNB and ACTB_1216 after treatment with TadA8r. Right: Sanger bar is the m ⁶ A fraction calculated by EditR based on Sanger sequencing data from treatment with TadA8r; Amplicon bar is the m ⁶ A fraction calculated from site specific amplicon-deep sequencing data by treatment with TadA8.20; RNA-seq bar is the m ⁶ A fraction calculated from RNA-seq data by treatment with TadA8.20. TadA8r gave the similar m ⁶ A fractions of sites ACTB_1426, SLC7A5, JUNB and ACTB_1216 compared with the m ⁶ A quantification using TadA8.20 from both Amplicon-deep sequencing and RNA-seq data. (30B) Shows TadA8r deaminated non-modified A, but not m6A at nucleotide 52 of exemplary in vitro deamination RNA2, aka Y1D, SEQ ID NO: 39 and 160. (30C) TadA8r effectively deaminated adenosines in RNA with secondary structure, e.g., exemplary in vitro deamination RNA3, aka Y1G, SEQ ID NO: 40. In some conditions, TadA8r resulted in lower levels of cytosine deamination relative to other TadA enzymes. (30D) TadA8r effectively deaminated adenosines in ACTB, CXCR4, SLC7A5, and JUNB target RNA molecules. (30E) Analysis of global deamination rate (y axis) on rRNA after 1X, 2X, and 3X TadA treatments (without FTO treatment) or FTO treatment followed by 3X TadA treatment on rRNA from sequencing library (e.g., Takara SMARTer v3 pico library construction kit). An optional filter (removing reads that had less than 50% deamination rates overall) was applied which showed that differences between the rate of filtered vs. non-filtered libraries was minimal, except for the samples with FTO treatment, potentially due to sequencing bias that may be introduced when reads were longer. (30F) The global deamination rate on mRNA after 1X, 2X, and 3X TadA treatments (without FTO treatment) or FTO treatment followed by 3X TadA treatment on mRNA for sequencing library creation (e.g., Takara SMARTer v3 pico library construction kit). [0087] FIGs.31A-31D. TadA enzymes were compatible with commercial sequencing library construction kits. Exemplified are quality control traces when TadA8r was coupled

137741474.1 - 26 - with (31A) NEB Small RNA Library, (31B) NEB Directional Library, (31C) Ligation Library, or the (31D) Takara SMARTer Library protocols/kits. [0088] FIGs. 32A-32B TadA8r quantitatively deaminated A in ssDNA but 6mA remained intact. TadA8r (SEQ ID NO: 25) treatment deaminated deoxyadenosine but not 6mA in ssDNA oligos. (FIG. 32A) TadA8r treatment deaminated adenine to inosine quantitatively as revealed by MALDI-Time of flight (TOF) Mass Spec (MS), analyzed before (top) and after (bottom) TadA8r treatment. (FIG.32B) 6mA remained intact before (top) and after (bottom) TadA8r treatment as revealed by MALDI-TOF MS. [0089] FIGs. 33A-33B. TadA8r deaminated dA in DNA with greater efficiency than rA in RNA. Deamination of 6mA in DNA by TadA8r was significantly more efficient than that of m ⁶ A in RNA. (33A) Adenines in ssDNA were converted to inosine within 10 minutes of TadA8r treatment. (33B) Adenines in ssRNA were deaminated to inosine by TadA8r at a slower rate than ssDNA, taking approximately 16 hours in the tested conditions. [0090] FIG.34. Schematic showing differentiation of 6mA from unmodified A sites in DNA. Upon treatment with a TadA enzyme, adenine (A) in DNA was converted into inosine (I), while the modified A (6mA) remained unaffected. During the PCR amplification step, inosine preferentially paired with cytosine as the complementary base, leading to an A-to-G mutation in the template strand. [0091] FIG. 35. Sanger Sequencing results confirmed TadA8r deamination activity when tested using a 100 mer ssDNA oligo. Shown are Sanger sequencing traces displaying TadA8r deamination activity in a 100 mer ssDNA oligo. Sanger sequencing results revealed that all adenines on a 100-nt single-stranded DNA oligo (SEQ ID NO: 163) underwent deamination and were detected as G, whereas the 6mA site remained unaltered and was read as A. [0092] FIG.36. TadA8r treatment deaminated unmodified adenines but not 6mA in ssDNA. Shown is the unconverted rate of A and 6mA in ssDNA spike-ins. ssDNA oligo treated with TadA8r displayed an A unconverted ratio at each site of less than 1% (e.g., less than 1% not deaminated to inosine), while the 6mA site exhibited a ratio of approximately 100% (e.g., no adenines deaminated to inosine). The single-stranded DNA (ssDNA) oligo treated by TadA8r and was sequenced on the Illumina platform. The y-axis represents the pile-up unconverted ratio of 6mA and A sites on the oligo (SEQ ID NO: 163), while the underlying sequence of the oligo was labeled. [0093] FIG.37. TadA8.20 or TadA8e treatment deaminated unmodified adenine but not 6mA in ssDNA. Shown is the average unconverted rate of A and 6mA in ssDNA spike-

137741474.1 - 27 - ins. The single-stranded DNA (ssDNA) oligo (SEQ ID NO: 163) was subjected to treatment with either (top) TadA8.20 (SEQ ID NO: 3) or (bottom) TadA8e (SEQ ID NO: 4) and subsequently sequenced using the Illumina platform. The y-axis depicts the pile-up unconverted ratio for both 6mA and A sites on the oligo. The results showed that additional variants of the TadA enzyme also demonstrated efficacy in DNA eTAM-seq methods. [0094] FIG. 38. TadA8r treatment potently deaminated adenine in lambda phage DNA. Shown is the average unconverted ratio of unmodified A sites on lambda DNA. Double- stranded lambda DNA was fragmented and denatured prior to TadA enzyme treatment. In two replicates, the average unconverted ratio of unmodified A sites was exceptionally low, at approximately ~0.15%, signifying an impressive conversion efficiency of nearly 99.9%. [0095] FIG. 39. TadA8r treatment potently deaminated adenine in lambda phage DNA across different motifs. Shown is the ratio of unconverted A sites across various motifs in lambda DNA. The unconverted ratio for all 16 motifs (N-A-N) ranged from 0.08% to 0.34%, demonstrating that DNA-eTAM-seq reliably detected all A motifs in the genome with high accuracy. [0096] FIG. 40. Provides a statistical model for high confidence 6mA site detection. To identify high-confidence 6mA sites, two technical replicates of one sample were conducted, along with the preparation of two replicates of FTO-treated control libraries. A high-confidence 6mA site was categorized as meeting three criteria: 1) the detected ratio was consistent between the two treated replicates; 2) the detected ratio was consistent between the two FTO control replicates; and 3) both replicates exhibited a significant decrease in the unconverted A level. [0097] FIG.41.6mA sites associated with GATC motif in E. coli genome were called with high confidence. Shown is an exemplary unconverted A ratio on the positive (+) (SEQ ID NO: 168) or negative (-) (SEQ ID NO: 169) DNA strand, where the central 6mA motif was unconverted while all adjacent A sites on both strands showed high levels of conversion following treatment with TadA8r. [0098] FIG. 42. DNA eTAM-seq uncovered nearly all 6mA sites in the DAM+ bacterial genome with minimal false positives. Two replicates of DAM+ E.coli samples were sequenced using the eTAM-seq method. All adenine (A) sites located on GATC motifs were identified as 6mA sites and were represented in blue. Every other A site, with the exception of those on EcoK motifs (AACNNNNNNGTGC/GCACNNNNNNGTT) (SEQ ID NOs: 166- 167), was considered as an unmodified A site. The count of A and 6mA sites with a coverage surpassing 10 was annotated in the title of every panel. A threshold of 50% was set; 6mA sites exhibiting an unconverted ratio below 50% were labeled as false negatives (FN), while

137741474.1 - 28 - unmodified A sites displaying an unconverted ratio above 50% were categorized as false positives (FP). The results showed FN% of less than 0.04% in each replicate. The results showed FP% of less than 0.002% in each replicate. [0099] FIG. 43. 6mA sites identified were effectively erased by control FTO protein treatment. Two replicates of DAM+ E.coli samples were treated with FTO protein and sequenced using the eTAM-seq method. The count of A and 6mA sites with a coverage surpassing 10 was annotated in the title of every panel. A threshold of 50% was set; Unmodified A sites displaying an unconverted ratio above 50% were regarded as false positives. The results showed FP% of less than 0.004% in each replicate. [0100] FIG.44. 6mA identification with eTAM-seq had a significantly reduced false positive rate. eTAM-seq was compared head-to-head with SMRT-seq in E. coli K12 samples. The results indicated that SMRT-seq had an~25-fold increase in false positives when identifying 6mA sites relative to eTAM-seq. [0101] FIGs. 45A-45B. eTAM-seq detected m6A and non-6mA sites overlapped almost completely between replicates. Each treatment condition had three replicates. Only sites with a sequencing depth >10 in both replicates were used for comparison. An unconverted ratio greater than 50% was used to detect 6mA sites, while a ratio less than 50% was used to detect non-m6A sites. There was a high overlap between replicates in both detected 6mA (45A) and non-6mA (45B) sites. [0102] FIG. 46. Exemplary detection limit and enrichment strategies. In contrast to highly modified 6mA sites, those with lower modification levels tended to have a greater overlap with the distribution of background noise (A sites). This overlapping pattern could potentially lead to false positives, and raising the detection cutoff could potentially compromise sensitivity. Utilizing an anti-6mA antibody to enrich fragments containing the 6mA modification could elevate the 6mA signal, thereby reducing the probability of false positives. [0103] FIG.47. Unconverted ratio of 6mA and A sites in 20% modified E. coli strain. In the distribution of the detected 6mA signal for an E. coli sample with approximately 20% modification, GATC motifs were depicted in blue and displayed in the upper panel. Non-6mA motifs were colored in red presented in the lower panel. [0104] FIG. 48. Unconverted ratio of 6mA and A sites E. coli strains with diverse modification levels. The unconverted ratio of 6mA (top) and A (bottom) sites in E. coli strains with diverse modification levels, ranging from 100%, 20%, 10%, 5%, and 2.5%, was analyzed. Two replicates were conducted for each sample, and an additional FTO-treated control was also included in the analysis.

137741474.1 - 29 - [0105] FIGs. 49A-49B. Detection sensitivity showed a dramatic drop at the level of 5% 6mA modification. (49A) Is an ROC curve illustrating the high performance of eTAM- seq across samples with diverse 6mA modification levels. Specifically, the ROC scores for samples with 100%, 20%, and 10% modification levels were all close to 1, signifying excellent detection accuracy. However, the ROC scores for samples with 5% and 2.5% modification levels exhibited a slight decrease. (49B) The Precision-Recall Curves revealed a sharp drop in performance at the 5% 6mA modification level, indicating that the detection limit of the eTAM- seq method was approximately 5%. This suggested that the method's reliability potentially decreased when dealing with samples below this threshold. [0106] FIG. 50. Sequencing coverage showing ~10-fold elevation at 6mA sites after anti-6mA pulldown. Sequencing coverage showed an ~10-fold elevation at 6mA sites after anti-6mA pulldown/antibody mediated enrichment. [0107] FIG.51. Detected signal of 6mA sites reached 100% after anti-6mA pulldown. The detected signal of 6mA sites from 2.5% input samples reached 100% following anti-6mA pulldown. DETAILED DESCRIPTION [0108] N6-methyladenosine (m ⁶ A) is the most abundant internal mRNA modification in higher eukaryotes, and serves a myriad of roles in regulating cellular processes. Functional dissection of m ⁶ A, however, has historically been at least in part obstructed by the lack of high- resolution and quantitative detection methods. Provided herein, in some embodiments, are improved methods and compositions for m ⁶ A RNA modification detection and/or N ⁶ - deoxyadenosine methylation (6mA) DNA modification detection. In some embodiments, provided herein is evolved TadA-assisted N6-methyladenine sequencing (eTAM-seq), an enzyme-assisted sequencing technology for simultaneous deconvolution of the genome, transcriptome and/or epitranscriptome. In some embodiments, eTAM-seq attains a global adenine deamination rate up to 99% and detects m ⁶ A and/or 6mA as persistent adenine (A). In some embodiments, the enzymatic deamination route employed by eTAM-seq preserves nucleic acid (e.g., RNA and/or DNA) integrity, facilitating m ⁶ A and/or 6mA detection from limited input samples. In some embodiments, provided herein are technologies for transcriptome-wide and/or genome-wide m ⁶ A and/or 6mA profiling. [0109] In some embodiments, provided herein are technologies for site-specific, deep sequencing-free m ⁶ A and/or 6mA quantification. In some embodiments, provided herein are technologies for site-specific, deep sequencing-free m ⁶ A and/or 6mA quantification with as

137741474.1 - 30 - few as about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cells, or any range derivable therein. In some embodiments, provided herein are technologies for site-specific, deep sequencing-free m ⁶ A and/or 6mA quantification with an input requirement of at least about 1, 2, 3, 4, or 5 orders of magnitude lower than existing quantitative profiling methods. In some embodiments, eTAM- seq represents an important technology advance in the genomics and/or epitranscriptomics field, enabling researchers to not only survey the m ⁶ A and/or 6mA landscape at unprecedented resolution, but also detect m ⁶ A and/or 6mA at user-specified loci with a simple workflow. [0110] In certain embodiments, technologies provided herein maintain nucleic acid (e.g., RNA and/or DNA) integrity with no measurable degradation. In certain embodiments, technologies provided herein maintain nucleic acid (e.g., RNA and/or DNA) integrity, with less than or equal to about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, or any range derivable therein, measurable nucleic acid (e.g., RNA and/or DNA) degradation. In certain embodiments, technologies provided herein maintain nucleic acid (e.g., RNA and/or DNA) integrity, with greater than or equal to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, reduction in nucleic acid (e.g., RNA and/or DNA) degradation relative to chemical deamination conditions. [0111] Certain embodiments are directed to methods and compositions for detecting modified adenine (A) in a target nucleic acid (e.g., RNA and/or DNA) by contacting the target nucleic acid with a tRNA-specific adenosine deaminase enzyme. In some embodiments, the adenosine deaminase enzyme is TadA. In some embodiments, the adenosine deaminase enzyme is an evolved enzyme. In some embodiments, the adenosine deaminase enzyme is TadA8.20 (SEQ ID NO: 3). In some embodiments, the adenosine deaminase enzyme is TadA8r (SEQ ID NO: 25). In some embodiments, the adenosine deaminase enzyme is TadA8e (SEQ ID NO: 4). In some embodiments, the adenosine deaminase enzyme deaminates unmodified adenines, converting them to inosines. [0112] In some embodiments, described herein is a novel m ⁶ A and/or 6mA sequencing method, (e.g., eTAM-seq), that functions through enzyme-assisted A deamination. In some embodiments, m ⁶ A and/or 6mA maps generated by eTAM-seq enable inspection of the distribution and function of m ⁶ A and/or 6mA at unprecedented resolution. In some embodiments, use of technologies provided herein reveals a close-to-even distribution for m ⁶ A

137741474.1 - 31 - sites across different methylation levels in both HeLa and mES cells. In some embodiments, technologies provided herein capture the majority of m ⁶ A signals (e.g., 0.35% of all A in HeLa cells). In some embodiments, eTAM-seq may be particularly sensitive and attuned to detection of sites with moderate to high methylation levels. In some embodiments, technologies provided herein provide means for detection and/or quantification of moderately to highly methylated A sites that are likely major contributors to the cellular m ⁶ A pool. In some embodiments, technologies provided herein provide means for detection and/or quantification of, clustered m ⁶ A sites. In some embodiments, technologies provided herein provide means for detection and/or quantification of clustered m ⁶ A sites that may have more prominent functions than isolated m ⁶ A sites, for example, these sites may interact more strongly with m ⁶ A reader proteins. [0113] One critical challenge faced by existing m ⁶ A profiling methods is their reliance on bulk input materials, for example, most protocols start with millions of cells. In contrast, provided herein are technologies that facilitate m ⁶ A profiling with significantly lower input levels when compared to traditional methodologies. For example in some embodiments, eTAM-seq employs an enzymatic deamination mechanism, which, unlike chemical deamination conditions that tend to degrade the input RNA (see e.g., Schaefer M. et al., 2009). maintains RNA integrity with no and/or minimal measurable RNA degradation levels. In some embodiments, technologies provided herein are suitable for m ⁶ A detection and/or quantification with as few as 10 cells. In some embodiments, technologies provided herein are suitable for m ⁶ A detection and/or quantification with 1 cell. In some embodiments, technologies provided herein are suitable for m ⁶ A detection and/or quantification for cell free RNA species. In some embodiments, technologies provided herein (e.g., eTAM-seq) sustains gene expression information captured by canonical RNA-seq. In some embodiments, technologies provided herein (e.g., single-cell eTAM-seq) may report transcript abundances and m ⁶ A modifications simultaneously in the same cell. [0114] As an mRNA modification, m ⁶ A is subject to cellular regulation via active deposition and removal. There is high demand in the epitranscriptomics field for a robust method that informs on the presence and stoichiometry of methylation at sites of interest. In some embodiments, provided herein are technologies suitable for work site-specific, deep sequencing-free m ⁶ A detection, e.g., with methods comprising eTAM-Sanger. In some embodiments, technologies described herein may comprise a workflow that employs routine molecular biology assays, such as reverse transcription, PCR, and Sanger sequencing, without relying on specialized laboratory techniques. In some embodiments, technologies provided

137741474.1 - 32 - herein can significantly lower the financial and/or technical barriers associated with m ⁶ A detection and/or quantification. In some embodiments, by bypassing deep sequencing, technologies provided herein reduce costs-per-assay. In some embodiments, technologies provided herein comprise m ⁶ A quantification with less than or equal to 250 pg total RNA. In some embodiments, technologies provided herein comprise m ⁶ A quantification with RNA inputs greater than or equal to four orders of magnitudes lower than existing quantitative profiling methodologies. In some embodiments, technologies provided herein facilitate routine survey of m ⁶ A dynamics in any gene or biological processes of interest. [0115] In some embodiments, deaminase accessibility to a target nucleic acid (e.g., RNA and/or DNA) site is a prerequisite for m6A detection. In some embodiments, technologies provided herein (e.g., eTAM-seq) work particularly well for less highly structured DNA and/or RNA. In some embodiments, control transcriptomes and/or control genomes are utilized as a component of technologies described herein. In some embodiments, a control transcriptome is an in vitro transcribed modification-free transcriptome. In some embodiments, a control genome is a genome from an organism with a mutated m6A deposition and/or removal pathway. In some embodiments, a control transcriptome is an FTO-treated N6-demethylated transcriptome and/or genome. In some embodiments, more than one control transcriptome and/or genome is utilized. In some embodiments, a control sample is prepared and sequenced only once for each assay condition (e.g., cell type, environmental input, cell age, etc.). In some embodiments, provided herein are control accessibility estimates for m6A in HeLa cells. In some embodiments, provided herein are control accessibility estimates for m6A in mESC cells. I. Nucleic acid modification [0116] Dynamic chemical modifications of DNA and histone proteins represent fundamental mechanisms of biological regulation. Post-transcriptional modifications are also ubiquitous in RNA. To date, over 100 different RNA modifications have been identified with a wide variety of chemical diversities. Examples of such modifications can be found on the world wide web at rna-mdb.cas.albany.edu/RNAmods/, and the contents of this website publication are herein incorporated by reference. Unlike genomic DNA that tends to have limited variation of chemical modifications, the wide variety of RNA modifications appears to be a strategy used by nature to entail and facilitate a much greater diversity of structures and cellular functions for different RNA species. The m ⁶ A modification in mRNA/lncRNA alone is known to modulate the affinity for RNA-binding proteins, control subcellular localization,

137741474.1 - 33 - lifetime, storage, transport, and translation of mRNAs, switch secondary structures of RNAs, as well as to affect innate immune response. [0117] The explosive discoveries of functional RNAs in the last decade have changed the current views on the functions of RNA and biological mechanisms that control RNA. However, the exact roles of most RNA modifications remain unknown. Certain RNA modifications are essential for life, with defects of RNA-modifying enzymes known to be associated with diverse human diseases. Most studies before 2011 on RNA modifications were limited to the abundant RNAs such as rRNA, snRNA, or tRNA. The positions of these modifications can be studied with RNA digestion followed by traditional liquid chromatography separation coupled with mass spectrometry or thin-layer chromatography due to their high abundances. The limit to these methods is their sensitivity; they cannot be applied to map modifications in low abundant mRNAs and lncRNAs, most of which appear to play critical roles in regulating gene expression. In certain embodiments, the methods of the current disclosure provide new opportunities to investigate distributions of not only mRNA and lncRNA modifications but also dynamic modifications on tRNA, snRNA, and rRNA that could not be effectively probed transcriptome-wide in the past. In certain embodiments, the methods and compositions of the disclosure can be readily applied to any class of RNA. In certain embodiments, the methods and compositions of the disclosure are applied to mRNA and/or lncRNA. In specific embodiments, the ability to identify all adenine modifications at single-base resolution will significantly advance the frontier of epitranscriptomics and enable transcriptome-wide investigations that associate genomic variations and mutations with health and diseases (e.g., animal health and diseases). In certain embodiments, technologies provided herein enable transcriptome-wide investigations that associate genomic variations and mutations with human health and diseases. In certain embodiments, the methods and compositions of the disclosure are applied to DNA, such as but not limited to genomic DNA. In some embodiments, the ability to identify all adenine modifications at single-base resolution will significantly advance the frontier of DNA regulation and enable genome-wide investigations that associate genomic variations and mutations with health and diseases (e.g., animal health and diseases). In certain embodiments, technologies provided herein enable genome-wide investigations that associate genomic variations and mutations with human health and diseases. [0118] In some embodiments, nucleic acids comprise, consist essentially of, or consist of single stranded DNA (ssDNA). In some embodiments, nucleic acids comprise, consist essentially of, or consist of double stranded DNA (dsDNA). In some embodiments, nucleic acids comprise, consist essentially of, or consist of DNA. In some embodiments, nucleic acids

137741474.1 - 34 - comprise, consist essentially of, or consists of single stranded RNA (ssRNA). In some embodiments, nucleic acids comprise, consist essentially of, or consist of double stranded RNA (dsRNA). In some embodiments, nucleic acids comprise, consist essentially of, or consist of RNA. In some embodiments, nucleic acids comprise, consist essentially of, or consist of DNA- RNA hybrids. [0119] Mammalian mRNA and lncRNA can be modified at tens of thousands of sites. Many of these modifications are conserved in almost all eukaryotes. m ⁶ A is the most prevalent internal modification in eukaryotic mRNA with ~3 per mRNA in mammalian cells. This modification plays essential and broad roles in cell differentiation, cell development, and numerous other cellular processes. The frequency of the other modifications range from 0.2-1 per mRNA in mammalian cells; some of them have been shown to have significant functional implications. [0120] N6-methyladenosine (m ⁶ A), the most prevalent internal mRNA modification in higher eukaryotes, depicts a regulatory network extensively involved in physiological and pathological processes (see e.g., Frye, M. et al., 2016; Peer, E. et al., 2017; Nachtergaele, S. & He, C.2018; and Jiang, X. et al., 2021). m6A alters mRNA processing, structure, translation, and decay without changing the genetic code (see e.g., He, P.C. & He, C. 2021). At the molecular level, the regulatory mechanism governed by m ⁶ A can be highly heterogeneous; functional outcomes of m ⁶ A modifications vary significantly across different transcripts, different regions in the same transcript, and different cell types (see e.g., He, P.C. & He, C. 2021). Comprehensive and quantitative mapping of m6A, aimed at elucidating the multitude of roles served by the modification, remains challenging, especially with limited input materials. [0121] m6A-seq (see e.g., Dominissini, D. et al., 2012) and MeRIP-seq (see e.g., Meyer, K.D. et al., 2012), the most widely used m ⁶ A-mapping methods, capture m ⁶ A-containing transcripts by antibody-mediated immunoprecipitation and detect m ⁶ A at a resolution of 100- 200 nucleotides (nt). The enrichment process requires bulk input materials: a typical m6A-seq or MeRIP-seq workflow starts with mRNA extraction from millions of cells, precluding their application to samples of limited quantities. Although additional solutions have been proposed towards transcriptome-wide m ⁶ A profiling, including miCLIP (see e.g., Linder, B. et al., 2015), MAZTER-seq (see e.g., Garcia-Campos, M.A. et al., 2019), m6A-REF-seq (see e.g., Zhang, Z. et al., 2019), m6A-SEAL (see e.g., Wang, Y. et al., 2020), metabolic labeling-enabled m6A sequencing (see e.g., Shu, X. et al., 2020), DART-seq (see e.g., Meyer, K.D. et al., 2019), and m6A-SAC-seq (see e.g., Hu, L. et al., 2022), their applications are limited by input amounts,

137741474.1 - 35 - antibody specificities, crosslinking efficiencies, non-quantitative readouts, predefined or biased sequence contexts, overexpression of effector proteins, and/or complicated workflows. [0122] Furthermore, the m ⁶ A field lacks a simple and/or cost-efficient method that can quantify the modification level at individual m ⁶ A sites to connect the methylation density, on for example pluripotency transcription factors in stem cells, with transcript abundances and/or development stage. Existing methods rely on oligonucleotide probes, which anneal to individual transcripts to enable m ⁶ A-dependent biochemical readout (see e.g., Wang, Y. et al., 2020; Liu, N. et al., 2013; and Xiao, Y. et al., 2018). However, in general, these methods demand large input materials (e.g., micrograms of total RNA, or millions of cells) and the input requirement scales with the number of m ⁶ A sites subject to evaluation. As a result, in general, site-specific m ⁶ A quantification has so far only been demonstrated for abundant RNA species in cultured cell lines. The probe annealing process also faces specificity challenges, especially when targeting transcripts of low abundance, leading to inaccurate quantification. A method that allows facile detection of individual m ⁶ A sites with stoichiometry information would bridge this critical gap in epitranscriptomic research. Provided herein are such technologies. [0123] In some embodiments, evolved TadA-assisted N6-methyladenine sequencing (eTAM-seq), an enzyme-assisted sequencing technology for quantitative, base-resolution profiling of m ⁶ A and/or 6mA, facilitates detection of individual m ⁶ A and/or 6mA sites with stoichiometry information. In some embodiments, eTAM-seq functions by global adenine deamination, enabling detection of m ⁶ A and/or 6mA as persistent A. In some embodiments, adenine-to-inosine (I) conversion rates of up to or greater than 98% are achieved. In some embodiments, a hyperactive TadA variant is utilized to catalyze A-to-I conversion. As described herein, in some embodiments with eTAM-seq, the m ⁶ A profile in the whole transcriptomes of cells (e.g., HeLa and mouse embryonic stem cells (mESCs)) is quantified. In some embodiments, deep sequencing-free, site-specific m ⁶ A quantification with as few as 10 cells, an input demand orders of magnitude lower than existing quantitative profiling methods, is achieved. In some embodiments, eTAM-seq enables faithful detection and quantification of m ⁶ A and/or 6mA with limited nucleic acid input, launching a robust solution to deciphering the epitranscriptome and/or DNA regulation. A. N ⁶ -methyladenosine (m ⁶ A) and N ⁶ -deoxyadenosine methylation (6mA) [0124] m ⁶ A occurs at a high frequency in RNA (e.g. mRNA or lncRNA). It is also reversible and dynamically regulated. The m ⁶ A modification appears to affect almost every phase of mRNA metabolism and function, thereby impacting diverse biological processes.

137741474.1 - 36 - Therefore, m ⁶ A studies so far embody the concept of “epitranscriptome”; its functional significance and implementation are exerted by three groups of proteins: “writers” that install, “erasers” that remove, and “readers” that bind or recognize m ⁶ A in order to determine the cellular fate of the modified mRNA/lncRNA. [0125] In mammals, m ⁶ A is installed by a three-protein core complex comprised of two catalytic subunits, METTL3/METTL14 and an accessory factor WTAP. Depletion of METTL3 homologs readily leads to developmental arrest or defects in gametogenesis in yeast, flies, and plants. In zebra fish, knockdown of METTL3 leads to smaller head, eyes, and brain ventricle, and curved notochord. The phenotypes in mammals are more severe. Both methyltransferases METTL3 and METTL14 are essential in mammals. m ⁶ A is a critical regulator in the differentiation of mouse embryonic stem cells (mESCs). [0126] m ⁶ A on mRNA can be reversed by two RNA demethylases, FTO and ALKBH5. Defects of FTO and AlkBH5 lead to altered metabolism, neural development retardation, and compromised spermatogenesis. A common variant of the FTO gene (an intron mutation) has been shown to generate a predisposition to obesity. Knockout mouse models revealed that FTO is important to development: most knockout mice die at the embryo state or within the first month of birth; those that survived tended to lose body weight and were smaller compared to the control mice. A mutation of the human FTO coding region has also been linked to mental retardation. The Alkbh5 knockout male mice exhibit significant spermatogenesis defects with compromised fertility. The fact that FTO and ALKBH5 show noticeable but very different phenotypes in humans or mice strongly indicates that reversible m ⁶ A RNA methylation plays important roles in biological regulation. [0127] m ⁶ A is recognized by “reader” proteins to exhibit biological functions, just like the interplay between DNA cytosine-methylation and methyl-CpG-binding proteins that regulate gene expression through binding to methylated cytosines. Applicants have identified several m ⁶ A-specific binding proteins in humans that belong to the YTH family: YTHDF1, YTHDF2, and YTHDC1. All these proteins bind the m ⁶ A-containing RNA selectively over unmethylated RNA through direct accommodation of the methyl group in their structures. Functional characterizations revealed that YTHDF2 affects cytoplasmic localization and mediates the decay of methylated mRNA, YTHDF1 promotes translation of methylated mRNA through facilitating translation initiation, and YTHDC1 affects the nuclear export of methylated mRNA. At the organismal level, knockout of Ythdc1 or Ythdf2 is embryonically lethal in mouse.

137741474.1 - 37 - [0128] m ⁶ A methylation can significantly affect the mRNA and lncRNA structure transcriptome-wide. The m ⁶ A effect on mRNA/lncRNA structure, termed “m ⁶ A-switch,” can dramatically affect protein-RNA interactions to impact mRNA abundance and alternative splicing of the methylated RNA. Therefore, m ⁶ A exerts its functions not only through being directly “read,” but also through RNA structural remodeling. [0129] N ⁶ -deoxyadenosine methylation (6mA; aka 2’-deoxyl-N6-methyladenosine or N6- methyldeoxyadenosine) is a prevalent DNA modification in prokaryotes (see e.g., Sanchez- Romero M.A. & Casadesus, J.2020) and some unicellular eukaryotes, such as Chlamydomonas reinhardtii and Tetrahymena thermophila (see e.g., Fu, Y. et al., 2015; and Wang, Y.2017). In bacteria, N ⁶ -methyldeoxyadenosine regulates DNA replication, repair, and defense (see e.g., Sanchez-Romero M.A. & Casadesus, J.2020). The function of 6mA in unicellular eukaryotes, despite their defined positioning in nucleosome-flanked linker regions, is less understood. In higher eukaryotes, 6mA has been reported to be present at low abundances (0.1-10 ppm) (see e.g., O’Brown, Z.K. et al., 2019; Liu, X., et al., 2021; and Lyu, C. et al., 2022), frequently approaching the detection limit of ultra-high-performance liquid chromatography-quadruple mass spectrometry (UHPLC-MS/MS). The low abundances and the lack of sensitive profiling methods have collectively led to the ongoing debate on the 6mA landscape in higher eukaryotes (see e.g., Kong Y., et al., 2023). [0130] The controversies among different studies can be partially attributed to the limited sensitivity and accuracy of the existing detection methods. For example, liquid chromatography-mass spectrometry cannot distinguish eukaryotic 6mA from bacterial DNA contamination. Antibody-based methods, including dot blot and DNA immunoprecipitation, can be impacted by confounding factors beyond 6mA, such as contamination of m ⁶ A in RNA, local sequences and secondary structures (see e.g., Douvlataniotis, K. et al., 2020). Both single- molecule real-time (SMRT) (see e.g., Flusberg, B.A. et al., 2010; and Fang, G. et al., 2012), and nanopore (see e.g., Clarke, J. et al., 2009; and Tourancheau, A. et al., 2021) sequencing methods detect 6mA quantitatively at single-base resolution, with SMRT sequencing having been reported to offer superior signal-to-noise ratios. Nevertheless, as 6mA cannot be reliably copied in vitro, as SMRT and nanopore detection of 6mA is incompatible with DNA amplification, thus these technologies are not readily applicable to samples comprising limited amounts of nucleic acids. [0131] As described herein and in the authors academic publications, evolved TadA- assisted N ⁶ -methyladenine sequencing (eTAM-seq) can be successfully used to sequence m ⁶ A in mRNA (see e.g., Xiao, YL., Liu, S., Ge, R. et al. Transcriptome-wide profiling and

137741474.1 - 38 - quantification of N6-methyladenosine by enzyme-assisted adenosine deamination. Nat Biotechnol 41, 993–1003 (2023), which is incorporated herein in its entirety), the inventors reasoned that the same principles could be applied to map 6mA in DNA. As described herein, in some embodiments, the inventors found that certain TadA mutants (e.g., TadA8r mutant; SEQ ID NO: 25) showed higher efficiencies for deaminating dA in DNA relative to rA deamination in RNA, but that in either case, 6mA or m ⁶ A remained intact. B. Other Adenine Modifications [0132] The methods and compositions of the disclosure are useful in the detection of adenine modifications (e.g., adenosine and/or deoxyadenosine). It is contemplated that the methods and compositions may be useful for detecting and/or quantifying any one or more of the adenine modifications listed below. In some embodiments, eTAM-seq is adapted to map and quantify other adenine base modifications, wherein the aimed modification can be selectively removed to generate a proper control.

137741474.1 - 39 - [0133] To summarize, mammalian nucleic acids, such as mRNA and lncRNA contain many internal modifications with abundances ranging from 0.2-3 modified nucleotides per mRNA. This range of abundance suggests the presence of hundreds to tens of thousands of modified sites for each modification type in mammalian transcriptomes. Further, m ⁶ A and m ¹ A

137741474.1 - 40 - are known to be reversible and undergo dynamic regulation. m ⁶ A is the most abundant and has been best studied with broad and fundamental roles uncovered so far. Other modifications could provide additional tuning of mRNA metabolism and function. The lack of highly sensitive, selective, and robust sequencing approaches for adenine modifications presents current technology barriers that significantly hinder biological investigations. Development of single-base resolution and highly sensitive methods will be required in order to move the field forward and also to enable new discoveries on the functions of RNA modifications and their associations with human diseases. II. Enzyme-catalyzed deamination [0134] The current disclosure provides methods and compositions comprising enzyme- mediated deamination for base-resolution sequencing of modified adenines (e.g. m ⁶ A, 6mA, etc.) in nucleic acids, such as RNA and/or DNA. The current disclosure is based on a method that converts only unmodified A in RNA or DNA into a different base, leaving modified A untouched, and thereby allowing differentiation of A from m ⁶ A in sequencing (FIG.1A). In certain embodiments, provided herein are enzyme-assisted (e.g., deaminase assisted) technologies for the identification of adenine modifications in a target nucleic acid (e.g., DNA and/or RNA). [0135] The nucleotide as well as the protein, polypeptide, and peptide sequences for a number of genes described herein have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov/) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art. [0136] It is contemplated that in some compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein). A. tRNA adenosine deaminase (TadA) [0137] In some embodiments, provided herein are methods that achieve at least about 95%, 96%, 97%, 98%, or 99% A-to-I conversion rates. In some embodiments, provided herein are

137741474.1 - 41 - methods that achieve at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any range derivable therein, A- to-I conversion rates. In some embodiments, methods provided herein comprise contacting a target nucleic acid species with an adenine deaminase enzyme exactly 1, 2, 3, 4, or 5 times. In some embodiments, methods provided herein comprise contacting a target RNA species with an adenosine deaminase enzyme exactly 1, 2, 3, 4, or 5 times. In some embodiments, methods provided herein comprise contacting a target DNA species with an adenosine deaminase enzyme that can deaminate adenine exactly 1, 2, 3, 4, or 5 times. In some embodiments, methods provided herein comprise contacting a target RNA species with a adenosine deaminase enzyme for less than or equal to: 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25, 0.2, 0.15, or 0.1 hours, or any range derivable herein. In some embodiments, methods provided herein comprise contacting a target DNA species with an adenosine deaminase enzyme for less than or equal to: 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25, 0.2, 0.15, or 0.1 hours, or any range derivable herein. [0138] As detailed in the Examples of the application, TadA enzymes that deaminate A to inosine (I) in nucleic acids (e.g., DNA and/or RNA) can be utilized in methods described herein. In some embodiments, the TadA enzymes suitable for use in technologies described herein are active against nucleotide species including DNA and/or RNA species, and possesses lower activity towards m ⁶ A and/or m6A compared to unmodified adenines A. Therefore, in some embodiments, after selective deamination of all A in the transcriptome and/or a subset thereof, and subsequent RT-PCR followed by sequencing, only m ⁶ A will be read as A. Additionally or alternatively, in some embodiments, after selective deamination of all A in the genome and/or a subset thereof, and subsequent sequencing, only 6mA will be read as A. [0139] In some embodiments, provided herein are methods that achieve at least about 95%, 96%, 97%, 98%, or 99% A-to-I conversion rates, while maintaining less than or equal to about or exactly 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.001%, 0.0002%, or 0.0001% (or any range derivable therein) false positive rates. In some embodiments, provided herein are methods that achieve at least about 95%, 96%, 97%, 98%, or 99% A-to-I conversion rates, while maintaining less than or equal to about or exactly 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.001%, 0.0002%, or 0.0001% (or any range derivable therein) false negative rates. [0140] The sequences associated with each of the GenBank Accession numbers is herein incorporated by reference for all purposes. In some embodiments, the TadA enzyme comprises TadA8.20. In some embodiments, the TadA enzyme comprises pyx033I, pyx047a, pyx047c,

137741474.1 - 42 - pyx047d, pyx047e, pyx047f, pyx047g, pyx047i, or pyx047k. In some embodiments, the TadA enzyme comprises 088a, 088c, 088d, 088e, or 088f. In some embodiments, a TadA enzyme comprises TadA8r (TadAR5.2). In certain embodiments, a TadA enzyme comprises one or more of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, S146C, D147R, R152P, Q154R, E155V, I156F, K157N, K161N, T166I, and/or D167N mutations relative to wild type TadA (SEQ ID NO: 1). [0141] In some embodiments, the TadA enzyme comprises an amino acid sequence with 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity to any one of SEQ ID NOs: 1 to 33, or 105-113. [0142] In certain embodiments, a TadA comprises one or more amino acid substitutions relative to wild type TadA (SEQ ID NO: 1), wherein the one or more amino acid substitutions comprises a substitution at amino acid 23, 27, 36, 47, 48, 51, 76, 82, 106, 108, 109, 110, 111, 114, 119, 122, 123, 126, 127, 146, 147, 152, 154, 155, 156, 157, 161, 166, 167, or a combination thereof. [0143] In certain embodiments, TadA comprises one or more amino acid substitutions relative to wild type TadA (SEQ ID NO: 1), wherein the one or more amino acid substitutions comprises a W23R, E27D, H36L, R47K, P48A, R51H, R51L, I76F, I76Y, V82S, A106V, D108G, D108N, A109S, K110R, T111H, T111R, A114V, D119N, H122R, H122N, H123Y, M126I, N127K, S146C, D147R, F149Y, R152P, Q154R, E155V, I156F, K157N, K161N, T166I, and/or D167N substitution. [0144] In some embodiments, a TadA comprises a K110R substitution relative to wild type TadA. In some embodiments, a TadA comprises a T111H substitution relative to wild type TadA. In some embodiments, a TadA comprises a T111R substitution relative to wild type TadA. In some embodiments, a TadA comprises a A114V substitution relative to wild type TadA. In some embodiments, a TadA comprises a M126I substitution relative to wild type TadA. In some embodiments, a TadA comprises a N127K substitution relative to wild type TadA. In some embodiments, a TadA comprises a W23R substitution relative to wild type TadA. In some embodiments, a TadA comprises a E27D substitution relative to wild type TadA. In some embodiments, a TadA comprises a H36L substitution relative to wild type TadA. In some embodiments, a TadA comprises a P48A substitution relative to wild type TadA. In some embodiments, a TadA comprises a R51H substitution relative to wild type TadA. In some embodiments, a TadA comprises a R51L substitution relative to wild type

137741474.1 - 43 - TadA. In some embodiments, a TadA comprises a I76F substitution relative to wild type TadA. In some embodiments, a TadA comprises a I76Y substitution relative to wild type TadA. In some embodiments, a TadA comprises a V82S substitution relative to wild type TadA. In some embodiments, a TadA comprises a A106V substitution relative to wild type TadA. In some embodiments, a TadA comprises a D108G substitution relative to wild type TadA. In some embodiments, a TadA comprises a D108N substitution relative to wild type TadA. In some embodiments, a TadA comprises a A109S substitution relative to wild type TadA. In some embodiments, a TadA comprises a D119N substitution relative to wild type TadA. In some embodiments, a TadA comprises a H122R substitution relative to wild type TadA. In some embodiments, a TadA comprises a H122N substitution relative to wild type TadA. In some embodiments, a TadA comprises a H123Y substitution relative to wild type TadA. In some embodiments, a TadA comprises a M126I substitution relative to wild type TadA. In some embodiments, a TadA comprises a S146C substitution relative to wild type TadA. In some embodiments, a TadA comprises a D147R substitution relative to wild type TadA. In some embodiments, a TadA comprises a F149Y substitution relative to wild type TadA. In some embodiments, a TadA comprises a R152P substitution relative to wild type TadA. In some embodiments, a TadA comprises a Q154R substitution relative to wild type TadA. In some embodiments, a TadA comprises a E155V substitution relative to wild type TadA. In some embodiments, a TadA comprises a I156F substitution relative to wild type TadA. In some embodiments, a TadA comprises a K157N substitution relative to wild type TadA. In some embodiments, a TadA comprises a K161N substitution relative to wild type TadA. In some embodiments, a TadA comprises a T166I substitution relative to wild type TadA. In some embodiments, a TadA comprises a D167N substitution relative to wild type TadA.  [0145] In some embodiments, one or more TadA substitutions comprise or consist of D108G and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, D108G, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, I76F, D108G, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, D108G, K110R, H122R, M126I, N127K, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, D108G, K110R, H122R, M126I, and N127K, substitutions. In some embodiments, one or more TadA substitutions comprise or consist of E27D, P48A, R51H, I76F, D108G, K110R, H122R, M126I, N127K, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of E27D, R47K, P48A, R51H, I76F, D108G, K110R, H122R,

137741474.1 - 44 - M126I, N127K, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of E27D, P48A, R51H, D108G, K110R, A114V, H122R, M126I, and N127K substitutions. In some embodiments, one or more TadA substitutions comprise or consist of E27D, R47K, P48A, R51H, I76F, D108G, K110R, A114V, H122R, M126I, and N127K substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, and I156F substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H122R, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, and I156F substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, S146C, D147R, R152P, Q154R, E155V, and I156F substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, I156F, and K157N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, I156F, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, I156F, and T166I substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, I156F, and D167N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of W23R, H36L, R47K, P48A, R51L, I76F, V82S, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, S146C, D147R, R152P, Q154R, E155V, and I156F substitutions. In some embodiments, one or more TadA substitutions comprise or consist of W23R, H36L, R47K, P48A, R51L, I76F, V82S, A106V, D108G, K110R, T111H, D119N, H122N, H123Y, M126I, N127K, S146C, D147R, R152P, Q154R, E155V, and I156F substitutions. In some embodiments, one or more TadA substitutions comprise or consist of W23R, H36L, R47K, P48A, R51L, I76Y, V82S, A106V, D108G, K110R, T111H, A114V, D119N, H123Y, M126I, N127K, S146C, D147R, R152P, Q154R, E155V, and I156F substitutions. In some embodiments, one or more TadA substitutions comprise or consist of W23R, H36L, R47K, P48A, R51L, I76F, V82S, A106V, D108G, A109S, K110R, T111H, A114V, D119N, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, and I156F substitutions. In some

137741474.1 - 45 - embodiments, one or more TadA substitutions comprise or consist of W23R, R47K, P48A, R51L, I76Y, V82S, A106V, D108G, A109S, K110R, T111H, A114V, D119N, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, and I156F substitutions. In some embodiments, one or more TadA substitutions comprise or consist of W23R, H36L, R47K, P48A, R51L, I76Y, V82S, A106V, D108G, K110R, T111H, A114V, D119N, H123Y, M126I, N127K, S146C, D147R, R152P, Q154R, E155V, I156F, K157N, K161N, T166I, and D167N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of W23R, H36L, R47K, P48A, R51L, I76Y, V82S, A106V, D108G, K110R, T111H, A114V, D119N, H123Y, M126I, N127K, S146C, D147R, R152P, Q154R, E155V, I156F, T166I, and D167N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, D108G, M126I, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, D108G, N127K, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, I76F, D108G, K110R, N127K, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, D108G, K110R, M126I, N127K, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of D108G, K110R, N127K, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, and I156F substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, S146C, D147R, R152P, Q154R, E155V, and I156F substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, I156F, and K157N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, I156F, and K161N substitutions. In some embodiments, one or more TadA substitutions comprise or consist of P48A, R51H, I76F, A106V, D108G, K110R, T111H, D119N, H123Y, M126I, N127K, D147R, R152P, Q154R, E155V, I156F, and T166I substitutions. [0146] In certain aspects the size of a protein or polypeptide (wild-type or modified) may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,

137741474.1 - 46 - 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues or nucleic acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). [0147] The polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous to at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 or more contiguous amino acids or nucleic acids, or any range derivable therein, of SEQ ID NOs: 1 to 33, or 105-113. In specific aspects, the peptide or polypeptide is or is based on a human sequence. In certain aspects, the peptide or polypeptide is not naturally occurring and/or is in a combination of peptides or polypeptides. [0148] The polypeptides of the disclosure may include at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,

137741474.1 - 47 - 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 substitutions (or any range derivable therein). [0149] In some aspects, the polypeptide comprises one or more substitutions at one or more amino acid positions selected from amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and/or 200 of any of SEQ ID NOs: 1 to 33, or 105-113, wherein each substitution is independently chosen from an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine; and wherein the polypeptide is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) sequence identity to one of SEQ ID NOs: 1 to 33, or 105-113. [0150] In some aspects, the protein or polypeptide may comprise amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,

137741474.1 - 48 - 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) of SEQ ID NOS: 1 to 33, or 105-113. [0151] In some aspects, the protein or polypeptide may comprise amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) of SEQ ID NOs: 1 to 33, or 105-113 and have or have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) sequence identity to one of SEQ ID NOs: 1 to 33, or 105-113. [0152] In some aspects, the protein, polypeptide, or nucleic acid may comprise, comprise at least, or comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) contiguous amino acids or nucleic acids of SEQ ID NOs: 1 to 33, or 105-113. [0153] In some aspects, the polypeptide, protein, or nucleic acid may comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,

137741474.1 - 49 - 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) contiguous amino acids of SEQ ID NOs: 1 to 33, or 105-113 that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous to one of SEQ ID NOs: 1 to 33, or 105-113. [0154] In some aspects there is a nucleic acid molecule or polypeptide starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 of any of SEQ ID NOs: 1 to 33, or 105-113 and comprising at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) contiguous amino acids or nucleotides of any of SEQ ID NOs: 1 to 33, or 105-113.

137741474.1 - 50 - [0155] In some embodiments, the TadA enzyme has the sequence associated with any one of SEQ ID NOs: 1 to 33, or 105-113. SEQ ID NO: 1 – Wild type TadA MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLH HP GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 1). SEQ ID NO: 2 – TadA enzyme 7.10 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH YP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 2). SEQ ID NO: 3 – TadA enzyme 8.20 (“TadA8.20”) MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MA LRQGGLVMQNYRLYDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH HP GMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD (SEQ ID NO: 3). SEQ ID NO: 4 – TadA enzyme 8e MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLN YP GMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 4). SEQ ID NO: 5 – TadA enzyme pyx033I MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGAKTGAAGSLMDVLH HP GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 5). SEQ ID NO: 6 – TadA enzyme pyx047a MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGAKTGAAGSLMDVLH HP GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 6). SEQ ID NO: 7 – TadA enzyme pyx047c MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGRHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGAKTGAAGSLMDVLH HP GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 7). SEQ ID NO: 8 – TadA enzyme pyx047d MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGRHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGAKTGAAGSLMDVLH HP GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 8).

137741474.1 - 51 - SEQ ID NO: 9 – TadA enzyme pyx047e MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGRHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGAKTGAAGSLMDVLH HP GINHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 9). SEQ ID NO: 10 – TadA enzyme pyx047f MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGRHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGAKTGAAGSLMDVLH HP GMKHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 10). SEQ ID NO: 11 – TadA enzyme pyx047g MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGRHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGARTGAAGSLMDVLH HP GMKHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 11). SEQ ID NO: 12 – TadA enzyme pyx047i MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGARTGAAGSLMDVLH HP GIKHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 12). SEQ ID NO: 13 – TadA enzyme pyx047k MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGARTGAAGSLMDVLH HP GMKHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 13). SEQ ID NO: 14 – TadA enzyme R2.0 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGARTGAAGSLMDVLR HP GIKHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 14). SEQ ID NO: 15 – TadA enzyme R2.1 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGARTGAAGSLMDVLR HP GIKHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 15). SEQ ID NO: 16 – TadA enzyme R3.0 MSEVEFSHEYWMRHALTLAKRAWDERDVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGARTGAAGSLMDVLR HP GIKHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 16).

137741474.1 - 52 - SEQ ID NO: 17 – TadA enzyme R3.1 MSEVEFSHEYWMRHALTLAKRAWDERDVPVGAVLVHNNRVIGEGWNKAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGARTGAAGSLMDVLR HP GIKHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 17). SEQ ID NO: 18 – TadA enzyme R3.2 MSEVEFSHEYWMRHALTLAKRAWDERDVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGARTGAVGSLMDVLR HP GIKHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 18). SEQ ID NO: 19 – TadA enzyme R3.3 MSEVEFSHEYWMRHALTLAKRAWDERDVPVGAVLVHNNRVIGEGWNKAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGARTGAVGSLMDVLR HP GIKHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 19). SEQ ID NO: 20 – TadA enzyme 088a (R4.0) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAAGSLMNVLH YP GIKHRVEITEGILADECAALLSRFFRMPRRVFKAQKKAQSSTD (SEQ ID NO: 20). SEQ ID NO: 21 – TadA enzyme R4.1 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAAGSLMNVLR YP GIKHRVEITEGILADECAALLSRFFRMPRRVFKAQKKAQSSTD (SEQ ID NO: 21). SEQ ID NO: 22 – TadA enzyme R4.6 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAAGSLMNVLH YP GIKHRVEITEGILADECAALLSRFFRMPRRVFKAQKKAQSSTN (SEQ ID NO: 22). SEQ ID NO: 23 – TadA enzyme R5.0 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNKAIGLHDPTAHAEI MA LRQGGLVMQNYRLFDATLYSTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAAGSLMNVLH YP GIKHRVEITEGILADECAALLCRFFRMPRRVFKAQKKAQSSTD (SEQ ID NO: 23). SEQ ID NO: 24 – TadA enzyme R5.1 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNKAIGLHDPTAHAEI MA LRQGGLVMQNYRLFDATLYSTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAAGSLMNVLN YP GIKHRVEITEGILADECAALLCRFFRMPRRVFKAQKKAQSSTD (SEQ ID NO: 24).

137741474.1 - 53 - SEQ ID NO: 25 – TadA enzyme R5.2 (“TadA8r”) MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNKAIGLHDPTAHAEI MA LRQGGLVMQNYRLYDATLYSTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAVGSLMNVLH YP GIKHRVEITEGILADECAALLCRFFRMPRRVFKAQKKAQSSTD (SEQ ID NO: 25). SEQ ID NO: 26 – TadA enzyme R5.3 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNKAIGLHDPTAHAEI MA LRQGGLVMQNYRLFDATLYSTLEPCVMCAGAMIHSRIGRVVFGVRGSRHGAVGSLMNVLH YP GIKHRVEITEGILADECAALLSRFFRMPRRVFKAQKKAQSSTD (SEQ ID NO: 26). SEQ ID NO: 27 – TadA enzyme R5.4 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVHNNRVIGEGWNKAIGLHDPTAHAEI MA LRQGGLVMQNYRLYDATLYSTLEPCVMCAGAMIHSRIGRVVFGVRGSRHGAVGSLMNVLH YP GIKHRVEITEGILADECAALLSRFFRMPRRVFKAQKKAQSSTD (SEQ ID NO: 27). SEQ ID NO: 28 – TadA enzyme R5.5 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNKAIGLHDPTAHAEI MA LRQGGLVMQNYRLYDATLYSTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAVGSLMNVLH YP GIKHRVEITEGILADECAALLCRFFRMPRRVFNAQKNAQSSIN (SEQ ID NO: 28). SEQ ID NO: 29 – TadA enzyme R5.6 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNKAIGLHDPTAHAEI MA LRQGGLVMQNYRLYDATLYSTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAVGSLMNVLH YP GIKHRVEITEGILADECAALLCRFFRMPRRVFKAQKKAQSSIN (SEQ ID NO: 29). SEQ ID NO: 30 – TadA enzyme 088c MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAAGSLMNVLH YP GIKHRVEITEGILADECAALLCRFFRMPRRVFKAQKKAQSSTD (SEQ ID NO: 30). SEQ ID NO: 31 – TadA enzyme 088d MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAAGSLMNVLH YP GIKHRVEITEGILADECAALLSRFFRMPRRVFNAQKKAQSSTD (SEQ ID NO: 31). SEQ ID NO: 32 – TadA enzyme 088e MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAAGSLMNVLH YP GIKHRVEITEGILADECAALLSRFFRMPRRVFKAQKNAQSSTD (SEQ ID NO: 32).

137741474.1 - 54 - SEQ ID NO: 33 – TadA enzyme 088f MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGVRGARHGAAGSLMNVLH YP GIKHRVEITEGILADECAALLSRFFRMPRRVFKAQKKAQSSID (SEQ ID NO: 33). SEQ ID NO: 105 – TadA enzyme 047b MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEI MA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGARTGAAGSLMDVLH HP GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 105). SEQ ID NO: 106 – TadA enzyme 047h MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRAIGRHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGARTGAAGSLMDVLH HP GIKHRVEITEGILADECAALLSDFFRMRRQEIKAQKNAQSSTD (SEQ ID NO: 106). SEQ ID NO: 107 – TadA enzyme 047j MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGHHDPTAHAEI MA LRQGGLVMQNYRLFDATLYVTLEPCVMCAGAMIHSRIGRVVFGARGAKTGAAGSLMDVLH HP GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 107). SEQ ID NO: 108 – TadA enzyme 098a MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVHNNRVIGEGWNRAIGLHDPTAHAEI MA LRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMDVLH YP GMNHRVEITEGILADECAALLCRFYRMPRQVFNAQKKAQSSID (SEQ ID NO: 108). SEQ ID NO: 109 – TadA enzyme 098b MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVHNNRVIGEGWNRAIGLHDPTAHAEI MA LRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMDVLH YP GMNHRVEITEGILADECAALLCRFYRMPRRVFNAQKKAQSSID (SEQ ID NO: 109). SEQ ID NO: 110 – TadA enzyme 098c MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVHNNRVIGEGWNRAIGLHDPTAHAEI MA LRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMDVLH YP GMNHRVEITEGILADECAALLCRFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 110). SEQ ID NO: 111 – TadA enzyme 098d MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MA LRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMDVLH YP GMNHRVEITEGILADECAALLCRFYRMPRQVFNAQKKAQSSID (SEQ ID NO: 112).

137741474.1 - 55 - SEQ ID NO: 112 – TadA enzyme 098e MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MA LRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMDVLH YP GMNHRVEITEGILADECAALLCRFYRMPRQVFNAQKNAQSSID (SEQ ID NO: 112). SEQ ID NO: 113 – TadA enzyme 098f MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MA LRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMDVLH YP GMNHRVEITEGILADECAALLCRFYRMPRRVFNAQKNAQSSIN (SEQ ID NO: 113). B. Protein preparation [0156] A variety of proteins can be purified using methods known in the art. Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or a microbial culture. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps exploit differences in protein size, physico-chemical properties and binding affinity. [0157] Evaluating purification yield. The most general method to monitor the purification process is by running a SDS-PAGE of the different steps. This method only gives a rough measure of the amounts of different proteins in the mixture, and it is not able to distinguish between proteins with similar molecular weight. If the protein has a distinguishing spectroscopic feature or an enzymatic activity, this property can be used to detect and quantify the specific protein, and thus to select the fractions of the separation, that contains the protein. If antibodies against the protein are available then western blotting and ELISA can specifically detect and quantify the amount of desired protein. Some proteins function as receptors and can be detected during purification steps by a ligand binding assay, often using a radioactive ligand. [0158] In order to evaluate the process of multistep purification, the amount of the specific protein has to be compared to the amount of total protein. The latter can be determined by the Bradford total protein assay or by absorbance of light at 280 nm, however some reagents used during the purification process may interfere with the quantification. For example, imidazole (commonly used for purification of polyhistidine-tagged recombinant proteins) is an amino acid analogue and at low concentrations will interfere with the bicinchoninic acid (BCA) assay

137741474.1 - 56 - for total protein quantification. Impurities in low-grade imidazole will also absorb at 280 nm, resulting in an inaccurate reading of protein concentration from UV absorbance. [0159] Another method to be considered is Surface Plasmon Resonance (SPR). SPR can detect binding of label free molecules on the surface of a chip. If the desired protein is an antibody, binding can be translated to directly to the activity of the protein. One can express the active concentration of the protein as the percent of the total protein. SPR can be a powerful method for quickly determining protein activity and overall yield. It is a powerful technology that requires an instrument to perform. [0160] Methods of protein purification. The methods used in protein purification can roughly be divided into analytical and preparative methods. The distinction is not exact, but the deciding factor is the amount of protein that can practically be purified with that method. Analytical methods aim to detect and identify a protein in a mixture, whereas preparative methods aim to produce large quantities of the protein for other purposes, such as structural biology or industrial use. [0161] Depending on the source, the protein has to be brought into solution by breaking the tissue or cells containing it. There are several methods to achieve this: Repeated freezing and thawing, sonication, homogenization by high pressure, filtration (either via cellulose-based depth filters or cross-flow filtration), or permeabilization by organic solvents. The method of choice depends on how fragile the protein is and how sturdy the cells are. After this extraction process soluble proteins will be in the solvent, and can be separated from cell membranes, DNA etc. by centrifugation. The extraction process also extracts proteases, which will start digesting the proteins in the solution. If the protein is sensitive to proteolysis, it is usually desirable to proceed quickly, and keep the extract cooled, to slow down proteolysis. [0162] In bulk protein purification, a common first step to isolate proteins is precipitation with ammonium sulfate (NH4)2SO4. This is performed by adding increasing amounts of ammonium sulfate and collecting the different fractions of precipitate protein. One advantage of this method is that it can be performed inexpensively with very large volumes. [0163] The first proteins to be purified are water-soluble proteins. Purification of integral membrane proteins requires disruption of the cell membrane in order to isolate any one particular protein from others that are in the same membrane compartment. Sometimes a particular membrane fraction can be isolated first, such as isolating mitochondria from cells before purifying a protein located in a mitochondrial membrane. A detergent such as sodium dodecyl sulfate (SDS) can be used to dissolve cell membranes and keep membrane proteins in solution during purification; however, because SDS causes denaturation, milder detergents

137741474.1 - 57 - such as Triton X-100 or CHAPS can be used to retain the protein's native conformation during complete purification. [0164] Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying masses or densities suspended in a liquid. When a vessel (typically a tube or bottle) containing a mixture of proteins or other particulate matter, such as bacterial cells, is rotated at high speeds, the angular momentum yields an outward force to each particle that is proportional to its mass. The tendency of a given particle to move through the liquid because of this force is offset by the resistance the liquid exerts on the particle. The net effect of "spinning" the sample in a centrifuge is that massive, small, and dense particles move outward faster than less massive particles or particles with more "drag" in the liquid. When suspensions of particles are "spun" in a centrifuge, a "pellet" may form at the bottom of the vessel that is enriched for the most massive particles with low drag in the liquid. Non-compacted particles still remaining mostly in the liquid are called the "supernatant" and can be removed from the vessel to separate the supernatant from the pellet. The rate of centrifugation is specified by the angular acceleration applied to the sample, typically measured in comparison to the g. If samples are centrifuged long enough, the particles in the vessel will reach equilibrium wherein the particles accumulate specifically at a point in the vessel where their buoyant density is balanced with centrifugal force. Such an "equilibrium" centrifugation can allow extensive purification of a given particle. [0165] Sucrose gradient centrifugation is a linear concentration gradient of sugar (typically sucrose, glycerol, or a silica based density gradient media, like Percoll™) is generated in a tube such that the highest concentration is on the bottom and lowest on top. A protein sample is then layered on top of the gradient and spun at high speeds in an ultracentrifuge. This causes heavy macromolecules to migrate towards the bottom of the tube faster than lighter material. After separating the protein/particles, the gradient is then fractionated and collected. [0166] Usually a protein purification protocol contains one or more chromatographic steps. The basic procedure in chromatography is to flow the solution containing the protein through a column packed with various materials. Different proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the protein from the column. Usually proteins are detected as they are coming off the column by their absorbance at 280 nm. Many different chromatographic methods exist: [0167] Chromatography can be used to separate protein in solution or denaturing conditions by using porous gels. This technique is known as size exclusion chromatography. The principle is that smaller molecules have to traverse a larger volume in a porous matrix.

137741474.1 - 58 - Consequentially, proteins of a certain range in size will require a variable volume of eluent (solvent) before being collected at the other end of the column of gel. [0168] In the context of protein purification, the eluant is usually pooled in different test tubes. All test tubes containing no measurable trace of the protein to purify are discarded. The remaining solution is thus made of the protein to purify and any other similarly-sized proteins. [0169] Ion exchange chromatography separates compounds according to the nature and degree of their ionic charge. The column to be used is selected according to its type and strength of charge. Anion exchange resins have a positive charge and are used to retain and separate negatively charged compounds, while cation exchange resins have a negative charge and are used to separate positively charged molecules. Before the separation begins a buffer is pumped through the column to equilibrate the opposing charged ions. Upon injection of the sample, solute molecules will exchange with the buffer ions as each competes for the binding sites on the resin. The length of retention for each solute depends upon the strength of its charge. The most weakly charged compounds will elute first, followed by those with successively stronger charges. Because of the nature of the separating mechanism, pH, buffer type, buffer concentration, and temperature all play important roles in controlling the separation. [0170] Affinity Chromatography is a separation technique based upon molecular conformation, which frequently utilizes application specific resins. These resins have ligands attached to their surfaces which are specific for the compounds to be separated. Most frequently, these ligands function in a fashion similar to that of antibody-antigen interactions. This "lock and key" fit between the ligand and its target compound makes it highly specific, frequently generating a single peak, while all else in the sample is unretained. In some embodiments, the affinity chromatography comprises maltose-binding protein (MBP). In some embodiments, a tRNA-specific adenosine deaminase enzyme is conjugated and/or fused to an MBP protein. In some embodiments, a fusion and/or conjugation can be at the N terminus and/or C terminus of a tRNA-specific adenosine deaminase enzyme. In some embodiments, a fusion and/or conjugation can modify enzymatic activity and/or improve enzyme solubility. [0171] Many membrane proteins are glycoproteins and can be purified by lectin affinity chromatography. Detergent-solubilized proteins can be allowed to bind to a chromatography resin that has been modified to have a covalently attached lectin. Proteins that do not bind to the lectin are washed away and then specifically bound glycoproteins can be eluted by adding a high concentration of a sugar that competes with the bound glycoproteins at the lectin binding site. Some lectins have high affinity binding to oligosaccharides of glycoproteins that is hard to compete with sugars, and bound glycoproteins need to be released by denaturing the lectin.

137741474.1 - 59 - [0172] A common technique involves engineering a sequence of 6 to 8 histidines into the N- or C-terminal of the protein. The polyhistidine binds strongly to divalent metal ions such as nickel and cobalt. The protein can be passed through a column containing immobilized nickel ions, which binds the polyhistidine tag. All untagged proteins pass through the column. The protein can be eluted with imidazole, which competes with the polyhistidine tag for binding to the column, or by a decrease in pH (typically to 4.5), which decreases the affinity of the tag for the resin. While this procedure is generally used for the purification of recombinant proteins with an engineered affinity tag (such as a 6xHis tag or Clontech's HAT tag), it can also be used for natural proteins with an inherent affinity for divalent cations. [0173] Immunoaffinity chromatography uses the specific binding of an antibody to the target protein to selectively purify the protein. The procedure involves immobilizing an antibody to a column material, which then selectively binds the protein, while everything else flows through. The protein can be eluted by changing the pH or the salinity. Because this method does not involve engineering in a tag, it can be used for proteins from natural sources. [0174] Another way to tag proteins is to engineer an antigen peptide tag onto the protein, and then purify the protein on a column or by incubating with a loose resin that is coated with an immobilized antibody. This particular procedure is known as immunoprecipitation. Immunoprecipitation is quite capable of generating an extremely specific interaction which usually results in binding only the desired protein. The purified tagged proteins can then easily be separated from the other proteins in solution and later eluted back into clean solution. Tags can be cleaved by use of a protease. This often involves engineering a protease cleavage site between the tag and the protein. [0175] High performance liquid chromatography or high pressure liquid chromatography (HPLC) is a form of chromatography applying high pressure to drive the solutes through the column faster. This means that the diffusion is limited and the resolution is improved. The most common form is "reversed phase" HPLC, where the column material is hydrophobic. The proteins are eluted by a gradient of increasing amounts of an organic solvent, such as acetonitrile. The proteins elute according to their hydrophobicity. After purification by HPLC the protein is in a solution that only contains volatile compounds, and can easily be lyophilized. HPLC purification frequently results in denaturation of the purified proteins and is thus not applicable to proteins that do not spontaneously refold. [0176] At the end of a protein purification, the protein often has to be concentrated. Different methods exist. If the solution doesn't contain any other soluble component than the

137741474.1 - 60 - protein in question the protein can be lyophilized (dried). This is commonly done after an HPLC run. This simply removes all volatile component leaving the proteins behind. [0177] Ultrafiltration concentrates a protein solution using selective permeable membranes. The function of the membrane is to let the water and small molecules pass through while retaining the protein. The solution is forced against the membrane by mechanical pump or gas pressure or centrifugation. [0178] Gel electrophoresis is a common laboratory technique that can be used both as preparative and analytical method. The principle of electrophoresis relies on the movement of a charged ion in an electric field. In practice, the proteins are denatured in a solution containing a detergent (SDS). In these conditions, the proteins are unfolded and coated with negatively charged detergent molecules. The proteins in SDS-PAGE are separated on the sole basis of their size. [0179] In analytical methods, the protein migrate as bands based on size. Each band can be detected using stains such as Coomassie blue dye or silver stain. Preparative methods to purify large amounts of protein, require the extraction of the protein from the electrophoretic gel. This extraction may involve excision of the gel containing a band, or eluting the band directly off the gel as it runs off the end of the gel. [0180] In the context of a purification strategy, denaturing condition electrophoresis provides an improved resolution over size exclusion chromatography, but does not scale to large quantity of proteins in a sample as well as the late chromatography columns. [0181] Methods of the disclosure may involve purification of proteins by any combination of methods known in the art and/or discussed herein. In some embodiments, the protein is purified by a combination of one or more of affinity chromatography, ion exchange chromatograph, and gel filtration chromatography. In some embodiments, the affinity chromatography is anti-FLAG. In some embodiments, the ion exchange chromatography is heparin. III. Assays Utilizing Adenine modification [0182] Nucleic acid analysis and evaluation includes various methods of amplifying, fragmenting, and/or hybridizing nucleic acids that have or have not been modified. [0183] Methodologies are available for large scale sequence analysis. In certain aspects, the methods described exploit these genomic analysis methodologies and adapt them for uses incorporating the methodologies described herein. In certain instances the methods can be used to perform high resolution adenine modification analysis on modified adenines in nucleic acids,

137741474.1 - 61 - (e.g., RNA and/or DNA). Therefore, in some embodiments, methods are directed to analysis of the adenine modification status of a nucleic acid sample, comprising one or more of the steps: (a) contacting the target nucleic acid with an adenosine deaminase enzyme to generate a target nucleic acid with deaminated adenine, (b) sequencing the target nucleic acid with deaminated adenine; wherein the modified adenine is detected when the nucleotide sequence is adenine. In some embodiments, methods are directed to analysis of the adenosine modification status of a RNA sample, comprising one or more of the steps: (a) contacting the target RNA with an adenosine deaminase enzyme to generate a target RNA with deaminated adenosines, (b) sequencing the target RNA with deaminated adenosines; wherein the modified adenosine is detected when the nucleotide sequence is adenosine. In some embodiments, methods are directed to analysis of the adenine modification status of a DNA sample, comprising one or more of the steps: (a) generating single stranded DNA (ssDNA) from the DNA sample, (b) contacting the target DNA with an adenosine deaminase enzyme to generate a target DNA with deaminated adenine, (c) sequencing the target DNA with deaminated adenine; wherein the modified adenine is detected when the nucleotide sequence is adenine. [0184] In some embodiments, the method further comprises steps such as sequencing a control nucleic acid (e.g., RNA and/or DNA); comparing the sequence of the target nucleic acid (e.g., DNA and/or RNA) with deaminated adenines to the sequence of the control nucleic acid; generating a nucleic acid strand that is complementary with the target and/or control nucleic acid and hybridizing the complementary nucleic acid strand with the target nucleic acid; and comparing the known sequence of the target nucleic acid with the sequence of the target nucleic acid with deaminated adenines. [0185] In some embodiments, the method further comprises steps such as sequencing a control RNA; comparing the sequence of the target RNA with deaminated adenosines to the sequence of the control RNA; generating a nucleic acid strand that is complementary with the target and/or control RNA and hybridizing the complementary nucleic acid strand with the target RNA; and comparing the known sequence of the target RNA with the sequence of the target RNA with deaminated adenosines. [0186] In some embodiments, the method further comprises steps such as sequencing a control DNA; comparing the sequence of the target DNA with deaminated adenines to the sequence of the control DNA; generating a nucleic acid strand that is complementary with the target and/or control DNA and hybridizing the complementary nucleic acid strand with the target DNA; and comparing the known sequence of the target DNA with the sequence of the target DNA with deaminated adenines.

137741474.1 - 62 - [0187] In certain embodiments, TadA mediated A-to-I conversion can be performed on nucleic acids, such as RNA and/or DNA that has reduced secondary and/or tertiary structure. In certain embodiments, RNA and/or DNA secondary structure and/or tertiary structure can be modified by controlling an assay temperature. In certain embodiments, TadA mediated A-to-I conversion is performed at a temperature of about 30 ^o C, about 31 ^o C, about 32 ^o C, about 33 ^o C, about 34 ^o C, about 35 ^o C, about 36 ^o C, about 37 ^o C, about 38 ^o C, about 39 ^o C, about 40 ^o C, about 41 ^o C, about 42 ^o C, about 43 ^o C, about 44 ^o C, about 45 ^o C, about 46 ^o C, about 47 ^o C, about 48 ^o C, about 49 ^o C, about 50 ^o C, about 51 ^o C, about 52 ^o C, about 53 ^o C, about 54 ^o C, or about 55 ^o C, or any range derivable therein. In certain embodiments, multiple rounds of TadA mediated A-to-I conversion can be performed in sequential order. In certain embodiments, TadA mediated A-to-I conversion can be performed for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 170 minutes, or about 180 minutes, about 190 minutes, about 200 minutes, about 210 minutes, about 220 minutes, about 230 minutes, about 240 minutes, or any range derivable therein. In certain embodiments, TadA mediated A-to-I conversion can be performed for less than or equal to about 48 hours, 36 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1.5 hours, 1 hours, 0.5 hours, or 0.25 hours, or any range derivable therein. In certain embodiments, TadA mediated A-to-I conversion can be performed for less than about 20 minutes. In certain embodiments, TadA mediated A-to-I conversion can be performed for greater than about 240 minutes. In certain embodiments, an initial TadA mediated A-to-I conversion incubation period is at a higher temperature than one or more subsequent TadA mediated A-to-I conversion incubation period(s). In certain embodiments, a first TadA incubation period is about 1 h or about 2 h, and at least a second TadA incubation period is about 1 h or about 2 h. [0188] In certain embodiments, TadA mediated A-to-I conversion is performed at a controlled pH. In certain embodiments, a controlled pH can be between about 5.5 and about 8.5. In certain embodiments, TadA mediated A-to-I conversion is performed at a pH of about 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, or 8.0, or any range derivable therein. In certain embodiments, TadA mediated A-to-I conversion is performed at a pH that is near neutral. In certain embodiments, TadA mediated A-to-I conversion is performed at a pH of about or exactly 7.5.

137741474.1 - 63 - [0189] In certain embodiments, TadA mediated A-to-I conversion can be performed at a TadA enzyme concentration of about 1 µM to about 50 µM, including any range derivable therein. In certain embodiments, a TadA enzyme concentration is about less than about 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 µM. A. Nucleic acid (e.g., RNA and/or DNA) isolation [0190] Nucleic acid) may be isolated from an organism of interest, including, but not limited to eukaryotic organisms and prokaryotic organisms, preferably mammalian organisms, such as humans. Nucleic acids may be isolated from cells grown in vitro or from cells in vivo. Nucleic acids may be isolated from tissues such as bone, blood, liver, heart, etc. Furthermore, the nucleic acids may be isolated and fractionated by techniques that separate different nucleic acid types. In certain embodiments, nucleic acids comprise, consist essentially of, or consist of DNA molecules. In certain embodiments, target nucleic acids comprise, consist essentially of, or consist of DNA molecules. In certain embodiments, nucleic acids comprise, consist essentially of, or consist of RNA molecules. In certain embodiments, target nucleic acids comprise, consist essentially of, or consist of RNA molecules. Therefore, in some embodiments, RNA is purified, and the purified RNA comprises at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% of a particular RNA such as mRNA, lncRNA, non-coding RNA, microRNA, pri-microRNA, pre-piRNA, rRNA, tRNA, snoRNA, or snRNA. [0191] In certain embodiments, nucleic acids are not isolated from an organism of interest (e.g., a subject). In certain embodiments, nucleic acids are isolated from an organism of interest (e.g., a subject). In certain embodiments, nucleic acids are not isolated from a sample obtained from an organism of interest. In certain embodiments, nucleic acids are isolated from a sample obtained from an organism of interest. In some embodiments, nucleic acids may be detected in situ. In some embodiments, nucleic acids may be detected in a subject and/or in a sample obtained from a subject. In some embodiments, nucleic acids are treated using methods described herein and nucleotide modifications are identified by in situ hybridization. In some embodiments, in situ hybridization comprises fluorescent in situ hybridization (FISH). [0192] In some embodiments, nucleic acids are enriched for (e.g., pulled down) prior to contact with a TadA enzyme. In some embodiments, nucleic acids are enriched for using antibody targeting m ⁶ A and/or 6mA. In some embodiments, nucleic acid enrichment can increase sensitivity down to less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or lower than 1% m ⁶ A and/or 6mA to A ratios.

137741474.1 - 64 - [0193] In some embodiments, nucleic acids are denatured to single strands prior to contact with a TadA enzyme. In some embodiments, nucleic acids are denatured using NaOH, DMSO, high pH, salt, and/or heat. In some embodiments, nucleic acids are denatured using NaOH. In some embodiments, nucleic acids are prevented from renaturation prior to contact with a TadA enzyme. In some embodiments, nucleic acids are prevented from renaturation using DMSO, heat, high pH, and/or formamide. In some embodiments, nucleic acids are prevented from renaturation using DMSO. [0194] In some embodiments, technologies provided herein comprise m ⁶ A detection and/or quantification with less than or equal to 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, or any range derivable therein, picograms (pg) total RNA and/or DNA. [0195] In some embodiments, technologies provided herein comprise m ⁶ A detection and/or quantification with less than or equal to 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, or any range derivable therein, picograms (pg) total mRNA and/or DNA. [0196] In some embodiments, technologies provided herein comprise m ⁶ A detection and/or quantification from less than or equal to 100,000, 50,000, 20,000, 10,000, 5,000, 2,500, 2,000, 1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, or any range derivable, cells. In some embodiments, technologies provided herein comprise m ⁶ A detection and/or quantification from cell free RNA and/or DNA samples. B. Sequencing [0197] The sequencing may be done by known methods of sequencing nucleic acids. In certain embodiments, the target nucleic acids molecules are sequenced using any suitable sequencing technique known in the art. In some embodiments, the target nucleic acids molecules are sequenced by Sanger sequencing. In one example, the sequencing is single- molecule sequencing-by-synthesis. Single-molecule sequencing is shown for example in U.S. Pat. Nos.: 7,169,560, 6,818,395, 7,282,337, the contents of each of these references is incorporated by reference herein in its entirety. Other examples of sequencing nucleic acids may include Maxam-Gilbert techniques, Sanger type techniques, Sequencing by Synthesis methods (SBS), Sequencing by Hybridization (SBH), Sequencing by Ligation (SBL),

137741474.1 - 65 - Sequencing by Incorporation (SBI) techniques, massively parallel signature sequencing (MPSS), polony sequencing techniques, nanopore, waveguide and other single molecule detection techniques, reversible terminator techniques, or other sequencing technique now know or may be developed in the future. [0198] In one embodiment, the sequencing is Illumina sequencing. Illumina sequencing is based on the amplification of DNA on a solid surface using fold-back PCR and anchored primers. Genomic DNA is fragmented, and adapters are added to the 5' and 3' ends of the fragments. DNA fragments that are attached to the surface of flow cell channels are extended and bridge amplified. The fragments become double stranded, and the double stranded molecules are denatured. Multiple cycles of the solid-phase amplification followed by denaturation can create several million clusters of approximately 1,000 copies of single- stranded DNA molecules of the same template in each channel of the flow cell. Primers, DNA polymerase and four fluorophore-labeled, reversibly terminating nucleotides are used to perform sequential sequencing. After nucleotide incorporation, a laser is used to excite the fluorophores, and an image is captured and the identity of the first base is recorded. The 3' terminators and fluorophores from each incorporated base are removed and the incorporation, detection and identification steps are repeated. [0199] In another embodiment, Ion Torrent sequencing can be used. (See, e.g., U.S. patent application numbers 2009/0026082, 2009/0127589, 2010/0035252, 2010/0137143, 2010/0188073, 2010/0197507, 2010/0282617, 2010/0300559), 2010/0300895, 2010/0301398, and 2010/0304982), the content of each of which is incorporated by reference herein in its entirety.) Oligonucleotide adaptors are ligated to the ends of target nucleic acid molecules. The adaptors serve as primers for amplification and sequencing of the fragments. The fragments can be attached to a surface and is attached at a resolution such that the fragments are individually resolvable. Addition of one or more nucleotides releases a proton (H+), which signal detected and recorded in a sequencing instrument. The signal strength is proportional to the number of nucleotides incorporated. [0200] In some embodiments, sequencing the target RNA comprises creating a complementary DNA (cDNA) from the target RNA. In some embodiments, sequencing the target RNA comprises reverse transcription. In some embodiments, sequencing the target RNA comprises contacting the target RNA with an enzyme capable of transcribing DNA using the target RNA as a template (e.g. reverse transcriptase). In some embodiments, a cDNA of the target RNA is sequenced. The sequence of the cDNA is determined, and the cDNA sequence is used to determine the sequence of the target RNA. In some embodiments, the target RNA is

137741474.1 - 66 - determined to have a modified adenosine at the corresponding position of the cDNA that is determined by sequencing to be thymine. [0201] In some embodiments, sequencing the target nucleic acid (e.g., RNA and/or DNA) comprises amplification of nucleic acids. Amplification can be done by techniques known in the art, such as but not limited to PCR, that uses primers, polymerase, deoxynucleoside triphosphates, buffers, and bivalent and monovalent cations in a reaction that generates copies of a target nucleic acid sequence from a single or few copies of the target nucleic acid sequence. [0202] In certain embodiments, the reading of the sequenced target nucleic acid is quantitative and reflective of the proportion of modified adenines in the target nucleic acid. C. Controls [0203] In some embodiments, the methods described herein further comprise control samples. In some embodiments comprising, intrinsically present nucleotide modifications (e.g., inosine in DNA and/or RNA), such as but not limited to products of spontaneous hydrolytic or nitrosative stress and/or RNA editing products should not influence the in vitro deamination (A has already been converted to I). However, in some situations, an accurate assignment of the methylation fraction at each modification site could be affected if the same site is also intrinsically deaminated to some extent. In addition, in some embodiments, the adenosine deaminase enzyme may exhibit certain sequence and/or structure biases that need to be corrected. Therefore, in some embodiments, a control sample with minimum methylation may be useful and/or necessary to correct for these factors. In some embodiments, an adenosine deaminase enzyme does not exhibit significant sequence and/or structure biases. As such, in some embodiments, a control sample with minimum methylation is not required to correct for these factors. [0204] In some embodiments, the control sample is a control transcript with minimum adenine modification. In some embodiments, the control sample is created with in vitro transcription (IVT), is otherwise synthesized in vitro. In some embodiments, the control sample can be created from cellular nucleic acids (e.g., mRNA, and/or DNA, etc.) that are isolated and subjected to a demethylase that is specific for the adenine modification. For example, the m ⁶ A modification can be demethylated by way of ALKBH5-catalyzed or FTO-catalyzed m ⁶ A demethylation. ALKBH5 and FTO are the two most well-known m ⁶ A RNA demethylases, however, additional m ⁶ A RNA demethylases are contemplated herein and may be used in aspects of the present disclosure. Additionally, in some embodiments additional and/or

137741474.1 - 67 - alternative DNA demethylases are contemplated and may be used in aspects of the present disclosure. [0205] In some embodiments, technologies described herein comprise a control sample. In some embodiments, to construct a control sample, the nucleic acid preparation comprising the target nucleic acid may be separated into multiple, such as at least, at most, or exactly 2, 3, 4, 5, 6, or more portions (or any derivable range therein). In some embodiments, one or more portion may be subjected to modification-specific demethylation to remove the modification on the nucleic acid. In some embodiments, one portion is contacted with FTO to catalyze m ⁶ A demethylation to remove most m ⁶ A on RNA and/or DNA. In some embodiments, mRNA samples will be subjected to the enzyme-mediated deamination, RT-PCR amplification, and high-throughput sequencing. In some embodiments, DNA samples will be subjected to the enzyme-mediated deamination, PCR amplification, and high-throughput sequencing. In some embodiments, because the modified adenine is resistant to deamination, it will be read as A in the sample without modification-specific demethylation (e.g. FTO treatment). In some embodiments, the demethylation (e.g. FTO-treated) control m ⁶ A is converted to A, which is deaminated to inosine (I) in the deamination step (an inosine can be read as G by most sequencing technologies). In some embodiments, a comparison of two parallel sequencing data will accurately reveal specific modification sites (i.e. m ⁶ A when FTO is used as the demethylase) at base resolution and eliminate potential nucleic acid editing and/or damage at the modification site (unmodified A-to-I) and potential biases of the deamination step. D. Analysis [0206] In some embodiments, methods described herein comprise analysis of RNA and/or DNA sequences and/or RNA and/or DNA sequencing results. In certain embodiments, the analysis comprises the removal of adapter sequences, addition and/or reading of barcodes (e.g., random and/or known, added to the 3ʹ and/or 5ʹ end of a nucleic acid molecule), mapping to a genome, retainment of only uniquely mapped reads, and/or filtering for high-quality nucleic acids in which poorly processed nucleic acid fragments are removed (e.g., RNA and/or DNA fragments with >50% unconverted adenines). In certain embodiments, the analysis comprises estimation of conversion rates for nucleic acids and/or control samples, calculation of apparent methylation levels using a maximum likelihood estimator based on a binomial model, estimation of deaminase accessibility for each A site, fitting of a linear model between site accessibility and total counts of A, G, A+G at an individual A site (e.g., to shrink accessibility estimates and/or reduce estimation bias), training using an appropriate number of sample sites

137741474.1 - 68 - (e.g., 2,000 randomly sampled sites with 10-fold cross-validation prior to being applied to predict site accessibility), adjustment of apparent methylation levels calculated from nucleic acids data with site accessibility, and/or obtainment of the final estimated methylation levels. In some embodiments, RNA analysis is as described herein, e.g., in the Methods and/or Examples. In some embodiments, analysis of nucleic acids is as described herein, e.g., in the Methods and/or Examples. IV. Kits [0207] The present disclosure additionally provides kits for detecting modified adenines in a target nucleic acid. Each kit may also include additional components that are useful for amplifying the nucleic acid, or sequencing the nucleic acid, or other applications of the present disclosure as described herein. The kit may optionally provide additional components that are useful in the procedure. These optional components include buffers, capture reagents, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information. The kit may also include reagents for RNA and/or DNA isolation and/or purification. V. Diagnosis and Methods of Treatment [0208] The present disclosure additionally provides methods of detecting, methods of measuring, methods of diagnosing, and/or methods of ameliorating and/or treating diseases associated with RNA and/or DNA methylation. In some embodiments, a disease associated with RNA methylation is a cancerous disease. In some embodiments, a disease associated with DNA methylation is an infectious disease. In some embodiments, provided herein are methods of detecting, diagnosing, predicting, ameliorating, and/or treating cancer and/or infections. In some embodiments, provided herein are methods of detecting, diagnosing, predicting, ameliorating, and/or treating Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, provided herein are methods of detecting, diagnosing, predicting, ameliorating, and/or treating colorectal cancer (CRC). [0209] In certain embodiments, provided herein are target biomarkers suitable for use in detecting, diagnosing, and/or predicting a disease state. In some embodiments, a target biomarker is a primary microRNA (pri-miRNA). In some embodiments, a target biomarker is a pre-cursor-miRNA (pre-miRNA). In some embodiments, a target biomarker is mature miRNA. In some embodiments, pri-miRNAs contain m ⁶ A modifications upstream to a pre- miRNA, which can promote miRNA maturation, for example, by recruiting microprocessor

137741474.1 - 69 - and dicing complexes such as DGCR8 (see e.g., Ma, S. et al., 2019, and Han, X. et al., 2021). In some embodiments, high METTL3 level, which can be found in many types of tumors (see e.g., Ma, S. et al., 2019, and Li, J. & Gregory, R. I., 2021), can result in hypermethylation of pri-miRNA, causing overexpression of mature miRNAs and downstream dysregulation. As a non-limiting example, in pancreatic ductal adenocarcinoma (PDAC), METTL3 upregulation was found to be related to cigarette smoking, resulting in overexpression of miR-25-3p, which suppresses PHLPP2 and subsequently activates oncogenic AKT-p70S6K signaling (see e.g., Zhang, J. et al., 2019). In another non-limiting example, in colorectal cancer (CRC), METTL3 upregulation leads to overexpression of miR-1246, which targets the expression of anti- oncogene SPRED2 and further reverses the inhibition of MAPK pathway (see e.g., Peng, W. et al., 2019). Additionally, beyond pri-miRNA, it has been found that in CRC, miR-17-5p was also hypermethylated in its mature form (see e.g., Konno, M. et al., 2019). [0210] In some embodiments, a primary microRNA suitable for use in methods of detecting, methods of measuring, methods of diagnosing, and/or methods of ameliorating and/or treating a disease associated with RNA methylation comprises or is pri-miR-25 (SEQ ID NO: 114; bolded is the GGACU m ⁶ A motif (see e.g., Zhang, J. et al., 2019); italicized is the pre-miR-25 sequence; and italicized and underlined is the miR-25-3p sequence). In some embodiments, a pre-miR-25 sequence comprises or is pre-miR-25 (SEQ ID NO: 115; italicized in SEQ ID NO: 114). In some embodiments, an miRNA sequence comprises or is miR-25-3p (SEQ ID NO: 116; italicized and underlined in SEQ ID NO: 114). SEQ ID NO: 114 – Pri-miR-25 sequence CAGUGGCGUUCAAAAGGGUCUGGUCUCCCUCACAGGACAGCUGAACUCCGGGACUGGCCA GU GUUGAGAGGCGGAGACUUGGGCAAUUGCUGGACGCUGCCCUGGGCAUUGCACUUGUCUCG GU CUGACAGUGCCGGCCCAACACUGCGGAUGCUGGGGGGAGGGGGGAUUCCACUCCUGUUUU GU GAGUAGGCGACCCAUGGGCUGCCCAGCCUUAAAGCCAGAACAAGGGUGU (SEQ ID NO: 114). SEQ ID NO: 115 – pre-miR-25 sequence GGCCAGUGUUGAGAGGCGGAGACUUGGGCAAUUGCUGGACGCUGCCCUGGGCAUUGCACU UG UCUCGGUCUGACAGUGCCGGCC (SEQ ID NO: 115). SEQ ID NO: 116 – miR-25-3p sequence CAUUGCACUUGUCUCGGUCUGA (SEQ ID NO: 116). [0211] In some embodiments, provided herein are methods of detecting, diagnosing, predicting, ameliorating, and/or treating a disease comprising measuring of RNA methylation. In certain embodiments, the efficacy of an aforementioned method can be compared to

137741474.1 - 70 - alternative methods utilized in the field. In certain embodiments, when compared to the detection of METTL3 expression levels or global RNA methylation levels, methods described herein (e.g., comprising eTAM-seq) can detect m ⁶ A on pri- and/or mature miRNA in a site- specific and/or site-quantitative method. In some embodiments, specificity and/or quantitative accuracy associated with methods described herein can provide more information specific to the certain disease type, for example but not limited to, a certain cancer type. In some embodiments, pri-miRNA m ⁶ A sites can be deduced by their relative position to a pre-miRNA region and/or by presence of a DRACH motif. In some embodiments, site detection and quantification by eTAM-seq can be verified by de novo sequencing and/or sequence specific sequencing. In some embodiments, following an optional verification, a biomarker could be readily detected by a sequence specific version of eTAM-seq. In some embodiments, a sequence specific version of eTAM-seq permits use of minimal sample amounts. In some embodiments, use of minimal sample amounts enables early diagnosis and low-cost screening of disease states, such as but not limited to, cancers. [0212] In some embodiments, provided herein are methods of detecting, diagnosing, predicting, ameliorating, and/or treating a disease comprising measuring of RNA methylation followed by disease state specific treatment. In some embodiments, inhibition of METTL3 function can reverse the overexpression of mature miRNAs (see e.g., Peng, W. et al., 2019). In some embodiments, small molecule METTL3 inhibitors function as a suitable treatment drug (see e.g., Li, J. & Gregory, R. I.2021). In some embodiments, after detection and/or verification of biomarker sites by eTAM-seq, Cas13b-ALKBH5/FTO fusion proteins that target specific sequences using CRISPR technology can be a preferred therapeutic and/or disease state ameliorating treatment (e.g., due to the potential precision of the associated intervention). In certain embodiments, this method of treatment may be suitable for various types of cancer cell lines and ex vivo tumor tissues (see e.g., Li, J. et al., 2020; Su, G., et al., 2022; and Wilson, C., et al., 2020). In certain embodiments, smaller Cas proteins (e.g., smaller Cas13 proteins) can be utilized (see e.g., Kannan, S. et al., 2022). VI. Examples [0213] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods described herein

137741474.1 - 71 - are presently representative of certain embodiments, are provided as an example, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art. Methods [0214] Unless otherwise stated, assays and experiments described in the following examples were performed as described herein. Cell culture [0215] Human HeLa cells and mouse embryonic stem cells (mESC) were purchased from ATCC. HeLa cells were grown in DMEM (Gibco, 11965092) media supplemented with 10% FBS (Gibco) and 1% 100× Pen/Strep (Gibco). WT (wild type), control knockout, and Mettl3 conditional knockout (cKO) mESCs were maintained in DMEM (Invitrogen) supplemented with 15% FBS (Gibco), 1% nucleosides (100×) (Millipore), 1 mM L-glutamine (Gibco), 1% nonessential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), 1,000 U/ml LIF (Millipore), 3 μM CHIR99021 (Stemcell), and 1 μM PD0325901 (Stemcell). All cells were cultured at 37 °C under 5.0% CO ₂ . [0216] Mettl3 cKO mES cell lines were generated following previously reported methods (see e.g., Liu et al., 2020). Briefly, mESCs derived from Mettl3 ^flox/flox mouse blastocyst were transfected with 200 ng PB-CAG-Puromycin-P2A-CreERT2 and 100 ng PBase by electroporation. After 24 h, electroporated cells were treated with 1 μg/ml Puromycin to generate stable Mettl3 ^flox/flox ; CreERT2 mES clones. To induce deletion, Mettl3 ^flox/flox ; CreERT2 ESC cells were treated with 1 μg/ml 4-hydroxytamoxifen (Sigma). These Mettl3 KO cells were cultured for 48 h before harvesting. Untreated Mettl3 ^flox/flox ; CreERT2 ESC cells were used as control (ctrl) mESCs. Poly-A RNA extraction [0217] Cells were cultured to 70-80% confluency, rinsed with 1× PBS (Gibco), and lysed by the direct addition of TRIzol reagent (Invitrogen). Total RNA was then collected following the manufacturer’s protocol. Poly A ⁺ RNA was extracted from purified total RNA using Dynabeads mRNA DIRECT Purification Kits (Invitrogen). Western blot [0218] Cells were lysed in RIPA lysis buffer (Pierce) supplemented with complete protease inhibitor cocktail (Takara). Lysates were boiled at 95 °C in NuPAGE LDS loading buffer (Invitrogen) for 10 min and then stored at –80 °C for use in the next step. A total of 30 μg

137741474.1 - 72 - protein per sample was loaded into 4-12% NuPAGE Bis-Tris gel and transferred to PVDF membranes (Life Technologies). Membranes were blocked in 5% milk PBST for 30 min at room temperature (RT), incubated in 1:1000 (v/v) dilution of anti-METTL3 antibody (abcam, ab195352) at 4 °C overnight, washed, and incubated in 1:5000 (v/v) dilution of goat anti rabbit igG-HRP (abcam, ab6721) for 1 h at RT. Membrane region lower than 50 kD were used as loading control and directly washed and incubated in 1:1000 (v/v) dilution of anti-GAPDH mAb-HRP (CST, 3683) for 1 h at RT. Protein bands were detected using SuperSignal West Dura Extended Duration Substrate kit (Thermo) and FluroChem R (Proteinsimple). Overexpression and purification of recombinant TadA8.20 protein [0219] Wild type TadA and TadA8.20 fused to an N-terminal hexahistidine-tagged maltose binding protein (6xHis-MBP) were cloned into a pET28a vector. A TEV protease cleavage site (ENLYFQ|G) was installed between MBP and TadA variants. Expression plasmids will be deposited to Addgene. [0220] BL21 Rosetta 2 (DE3) competent cells were transformed with the recombinant plasmids and grown on Luria broth (LB) agar plates supplemented with 50 ^g/mL kanamycin and 25 ^g/mL chloramphenicol. Successfully transformed bacteria were always cultured in the presence of 50 ^g/mL kanamycin and 25 ^g/mL chloramphenicol unless otherwise noted. Single colonies were inoculated into fresh LB medium and grown in an incubator shaker (37 ^o C, 220 rpm) for 12-18 h. A 10 mL saturated start culture was used to inoculate 1 L fresh medium. Bacteria were grown at 37 ^o C until OD ₆₀₀ reached 0.5. The culture was cooled down immediately to 4 ^o C and induced with 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). Bacteria were cultured at 16 ^o C for an additional 20 h before pelleting by centrifugation at 4,000 g. [0221] Bacterial pellets were lysed by sonication in buffer A (50 mM Tris, 500 mM NaCl, 10 mM ^-mercaptoethanol, and 10% (v/v) glycerol; pH 7.5). Lysed bacteria were clarified by centrifugation at 4°C, 23,000 g. The supernatant was loaded onto a Ni-NTA Superflow Cartridge (Qiagen, 30761), washed with 30 mL of buffer A supplemented with 50 mM imidazole, and eluted with a gradient of imidazole from 50 mM to 500 mM in buffer A. [0222] The eluted protein was incubated with TEV protease and dialyzed in buffer A at 4 ^o C overnight. The protein mixture was reloaded onto a Ni-NTA Superflow Cartridge, washed with buffer B (50 mM Tris, 1 M NaCl, 10 mM ^-mercaptoethanol, and 10% (v/v) glycerol; pH 8.0), and eluted by buffer B supplemented with 50 mM imidazole. Finally, MBP-free TadA8.20 was purified by size-exclusion chromatography (ENRICH™ SEC 65010 x 300 mm Column,

137741474.1 - 73 - Bio-Rad, 7801650) and concentrated to approximately 4 mg/mL. The column was balanced and eluted with buffer C (50 mM Tris, 200 mM NaCl, 10 mM ^-mercaptoethanol, and 10% (v/v) glycerol; pH 7.5). Preparation of A- and m ⁶ A-bearing E. coli tRNA (Arg2, CGT) and RNA probes [0223] Double-stranded DNA templates carrying T7 promoter were prepared by primer extension with two single-stranded DNA oligos (Tables 6 and 7). Unmethylated and methylated E. coli tRNA (Arg2, CGT), RNA#1, and RNA#2 were synthesized by in vitro transcription using T7 RNA polymerase. ATP and N ⁶ -methyl-ATP (TriLink, N-1013) were supplied in the presence of UTP, CTP, and GTP to synthesize unmethylated and methylated RNA, respectively. RNA was purified by E.Z.N.A Micro RNA kits (Omega Bio-Tek, R7034) and quantified by NanoDrop One (Thermo Fisher Scientific). Spike-in probes synthesis [0224] Spike-in probes were synthesized and pooled according to a previously published protocol (see e.g., Hu, L. et al., 2022). Sequences for spike-in probes are listed in Table 11. Probes 1-8 were mixed in a ratio (w/w) of 20%, 15%, 10%, 5%, 5%, 10%, 15%, 20%, respectively. The final probe mixture was composed of 5 sets of UMI-labeled RNA oligos of 0/25/50/75/100% m ⁶ A. Preparation of IVT transcriptomes [0225] Modification-free control RNAs were prepared from HeLa and mESC total mRNA based on previously published protocols (see e.g., Zhang, Z. et al., 2021 and Hagemann-Jensen et al., 2020). With minor modifications. Oligo-dT(30)VN primer annealed to 100 ng of purified poly A ⁺ RNA at 65 ºC for 5 min. RNA was reverse transcribed in 20 ^L 1x RT buffer (Thermo Scientific, EP0753, containing 50 mM Tris-HCl, pH 8.3; 75 mM KCl, 3 mM MgCl ₂ , 10 mM DTT) in the presence of 40 pmol of 5Bio-T7-TSO (/5Biosg/ACTCTAATACGACTCACTATAGGGAGAGGGCrGrGrG) (SEQ ID NO: 118), 1 mM of GTP, 5% (w/v) PEG 8000, 0.5 mM each of dNTP, 5 mM of RNaseOUT (Invitrogen, 10777019), and 200 U of Maxima H- Reverse Transcriptase (Thermo Scientific, EP0753) under the following conditions: 42 ºC for 90 min, 10 cycles of [50 ºC for 2 min plus 42 ºC for 2 min], 85 ºC for 5 min. To the 20 ^L of RT reaction, 10 ^L of RNase H (NEB, M0297L), 70 ^L of RNase-free H2O, and 100 ^L of Ultra II Q5 Master Mix (NEB, M0544X) were added to make the second-strand synthesis mixture, which was incubated under the following conditions: 37 ºC for 15 min, 95 ºC for 1 min, 65 ºC for 10 min. The reaction was purified with 160 ^L (0.8x,

137741474.1 - 74 - v/v) of AMPure XP beads (Beckman Coulter, A63882) following the manufacturer’s directions. [0226] The purified and concentrated dsDNA was in vitro transcribed (IVT) in 1x T7 Reaction Buffer (NEB E2040S, containing 40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT, 2 mM spermidine, pH 7.9) with 10 mM of each NTP, and 2 ^L of T7 RNA Polymerase Mix (NEB E2040S) in 20 ^L volume at 37 ºC overnight. The IVT mixture was further treated with TURBO DNase (Invitrogen, AM2238) and purified by acid-phenol chloroform (Invitrogen, AM9722) extraction and ethanol precipitation to yield 2.5-10 ^g of IVT RNA. In vitro deamination of RNA probes by TadA8.20 [0227] All reactions were carried out in a deamination buffer (50 mM Tris, 25 mM KCl, 2.5 mM MgCl2, 2 mM dithiothreitol, and 10 % (v/v) glycerol; pH 7.5) in the presence of 10 U SUPERase•In™ RNase Inhibitor (Thermo Fisher Scientific, AM2694). RNA was always preheated to 95 ^o C for 3 min and immediately cooled down before use. [0228] To assay deaminase activity on the natural substrate E. coli tRNA, 200 ng RNA and 100 nM wild type TadA or TadA8.20 were incubated at 37 ^o C for 1 h. For a typical deamination assay on RNA probes, 10 ng RNA was incubated with 10 ^M TadA8.20 in 20 ^L deamination buffer at 37 °C for 3 h. All reactions were quenched by incubating at 95 °C for 10 min. Temperature and pH were adjusted to for in vitro deamination (FIG.7). Reverse transcription polymerase chain reaction (RT-PCR) [0229] To convert RNA into complementary DNA for sequencing purposes, a 2 ^L deamination reaction was aliquoted, to which 0.5 ^L of 50 ^M reverse transcription primer was supplied. Primer annealing was enabled by heating up the mixture to 95 ^o C for 3 min, cooling down at a ramping rate of 2 ^o C/s, and incubation at 25 ^o C for 2 min. To the reaction, 0.5 ^L of GoScript reverse transcriptase (Promega, A5003) was added together with 2 ^L of 5x GoScript RT buffer, 1 ^L of 25 mM MgCl2, 0.5 ^L of 10 mM dNTPs, and 3.5 ^L nuclease- free H ₂ O. The reverse transcription reaction was incubated at 42 ^o C for 1 h and then quenched at 65 ^o C for 20 min. [0230] To a 20 ^L PCR or quantitative PCR reaction (EvaGreen qPCR Master Mix, Biotium 31041), 0.1 ^L of the reverse transcription reaction was supplied as template. A typical PCR program includes initiation at 95 ^o C for 3 min; 30 cycles of amplification (denaturing at 95 ^o C for 10 s, annealing at 60 ^o C for 10 s followed by extension at 72 ^o C for 20 s); and final extension at 72 ^o C for 5 min. qPCR reactions were performed on a CFX96 ^TM Real-Time PCR System (Bio-Rad).

137741474.1 - 75 - [0231] Sequences of primers for reverse transcription, site-specific amplification, and Illumina adapter installation are listed in Tables 8-10. Overexpression and purification of recombinant FTO [0232] The human FTO gene was cloned into a pET28a vector and transformed into BL21(DE3) cells (NEB). Successfully transformed bacteria were cultured at 37 ºC in 2xYT broth Teknova) to an O.D. of 0.8-1.0. The culture was cooled to 16 ºC and supplemented with 0.1 mM IPTG (Sigma), 10 μM ZnSO ₄ (sigma), and 2 μM (NH ₄ ) ₂ Fe(SO ₄ ) ₂ (sigma). Bacteria were cultured overnight at 16 ºC following induction. [0233] Bacteria were collected via centrifugation and lysed in buffer D (300 mM NaCl (Fisher), 50 mM imidazole (Fisher), and 50 mM of Na ₂ HPO ₄ (Sigma); pH 8.0). The lysate was clarified by centrifugation and loaded onto a nickel column (Ni Sepharose 6 FF, Cytiva), washed with buffer E (150 mM NaCl, 25 mM imidazole, and 10 mM Tris-HCl (Invitrogen); pH 7.5), and eluted with buffer F (150 mM NaCl, 250 mM imidazole, and 10 mM Tris-HCl; pH 7.5). The eluate was loaded onto an anion-exchange column (SOURCE 15Q, Cytiva) and fractionated with 0-50% of Buffer G (1.5 M NaCl, 20 mM Tris-HCl; pH 7.5) over 30 min. The resulting protein was concentrated and buffer exchanged using a 10 kD MWCO filter (Cat. No. 28932296, Cytiva) before being flash frozen in 30% glycerol for future use. eTAM-seq library preparation [0234] 50 ng of purified poly A ⁺ RNA and 25 ng of IVT control RNA from HeLa or mES cells were depleted of poly A tails, end-repaired, and ligated to 3’ adapters following a previously published protocol (see e.g., Hu, L. et al., 2022). Briefly, RNA was annealed to 100 pmol of oligo-dT (Thermo Scientific, SO132) and digested by 5 U of RNase H (NEB, M0297L) at 37 °C for 30 min. Without purification, the RNA was fragmented in 1× Zinc fragmentation buffer (10 mM ZnCl2 and 10 mM Tris-HCl, pH 7.5) at 70 °C for 5 min. The fragmentation reaction was quenched by the addition of 10 mM EDTA, then treated with 50 U of T4 PNK (NEB, M0201L) at 37 °C for 1 h. The end-repaired RNA was purified by RNA Clean & Concentrator (Zymo) kits, mixed with 2% (w/w) of spike-in probes, and ligated to 20 pmol of 3’ adapter (/5rApp/AGATCGGAAGAGCGTCGTG/3Bio/) (SEQ ID NO: 34) using 400 U of T4 RNA ligase 2, truncated KQ (NEB, M0373L) at 25 °C for 2 h, then 16 °C overnight. Excess adapters were first digested by 5ʹ Deadenylase (NEB, M0331S) at 30 °C for 1 h, then by and RecJf (NEB, M0264L) at 37 °C for 1 h. [0235] The ligated poly A ⁺ RNA was divided into two halves and designated to the FTO- and FTO ⁺ groups. The FTO-, FTO ⁺ , and IVT groups were immobilized on Dynabeads MyOne Streptavidin C1 (Invitrogen, 65002). The FTO ⁺ group was demethylated by incubating with

137741474.1 - 76 - 200 pmol of FTO in 1× FTO reaction buffer (2 mM sodium ascorbate (Sigma), 65 μM ammonium iron(II) sulfate (Sigma), 0.3 mM α-ketoglutarate (Sigma), 0.1 mg/mL BSA (NEB), and 50 mM HEPES-KOH; pH 7.0) supplemented with 10% (v/v) of SUPERase•In RNase Inhibitor (Invitrogen, AM2696) at 37 ºC for 1 h. The beads were washed by resuspension in 0.1% PBST (1× PBS (Gibco) supplemented with 0.1% (v/v) tween 20 (Sigma)), 1x Binding/Wash buffer (1 M NaCl, 0.5 mM EDTA, 5 mM Tris-HCl; pH 7.5), and twice with 10 mM Tris HCl (pH 7.5), consecutively. [0236] The three RNA samples were then deaminated on beads with 200 pmol of TadA8.20 in the deamination buffer (50 mM Tris, 25 mM KCl, 2.5 mM MgCl ₂ , 2 mM dithiothreitol, and 10 % (v/v) glycerol; pH 7.5) supplemented with 10% (v/v) of SUPERase•In RNase Inhibitor (Invitrogen, AM2696) at 53 ºC for 1 h. This reaction was repeated twice at 44 °C, 1 h each, by draining the supernatant on a magnetic rack and resuspending the beads in fresh reaction mixtures, lasting 3 h in total. The beads were washed sequentially by resuspension in 0.1% PBST (v/v), 1x Binding/Wash buffer, and twice in 10 mM Tris HCl (pH 7.5). [0237] RNA was annealed to 2 pmol of RT primer (ACACGACGCTCTTCCGATCT) (SEQ ID NO: 35), at 70 °C for 2 min, then reverse transcribed in 1x RT buffer (Thermo Scientific, EP0753, containing 50 mM Tris-HCl, pH 8.3; 75 mM KCl, 3 mM MgCl ₂ , 10 mM DTT) with 5 mM of RNaseOUT (Invitrogen, 10777019) and 200 U of Maxima H- Reverse Transcriptase (Thermo Scientific, EP0753) at 50 °C for 1 h. The cDNA was released by boiling the beads in 0.5% (v/v) SDS for 10 min. The eluate was purified by DNA Clean & Concentrator (Zymo) kits. Purified cDNA was ligated to cDNA Adapter (/5Phos/NNNNNNAGATCGGAAGAGCACACGTCTG/3SpC3/) (SEQ ID NO: 36) using 30 U of T4 RNA ligase (NEB) at 25 ºC overnight, following a previously published protocol (see e.g., Hu, L. et al., 2022). The reaction was purified again by DNA Clean & Concentrator (Zymo) kits and then PCR amplified with NEBNext Ultra II Q5 Master Mix (NEB, M0544X) and NEBNext Unique Dual Index Primer for Illumina (NEB, E6440S), following the manufacturer’s directions. Typically, 10-11 cycles of PCR were carried out to generate enough DNA. The resulting library was purified by AMPure XP beads (Beckman Coulter, A63882) following the manufacturer’s directions and submitted for next-generation sequencing. Site-specific amplification and barcoding of HeLa mRNA and IVT samples [0238] Purified HeLa poly A ⁺ RNA (300 ng) was depleted of poly A tails, end-repaired, ligated to 3’ adapters, and immobilized on Dynabeads MyOne Streptavidin C1 (Invitrogen) as described in eTAM-seq library preparation. RNA was deaminated and reverse transcribed following a similar protocol for NGS library construction. Specifically, RNA samples were

137741474.1 - 77 - deaminated on beads with 200 pmol of TadA8.20 in the deamination buffer (50 mM Tris, 25 mM KCl, 2.5 mM MgCl2, 2 mM dithiothreitol, and 10 % (v/v) glycerol; pH 7.5) supplemented with 10% (v/v) of SUPERase•In RNase Inhibitor (Invitrogen) at 37 ºC for 1 h. This reaction was repeated twice at by draining the supernatant on a magnetic rack and resuspending the beads in fresh reaction mixtures, lasting 3 h in total. The beads were washed by resuspension in 0.1% PBST (v/v), 1x Binding/Wash buffer, and twice in 10 mM Tris HCl, pH 7.5, consecutively. cDNA was eluted by boiling the beads in DNase-free water (Invitrogen) and transferred into new tubes immediately. Sites of interest were PCR amplified from the eluted cDNA using transcript-specific primers. [0239] To demonstrate m ⁶ A quantification with limited input, the same protocol was applied to 50 ng, 5 ng, or 500 pg of HeLa total RNA. The resulting cDNA was split into two halves to amply ACTB and EIF2A fragments. [0240] Both rounds of PCR were set up using EvaGreen qPCR Master Mix.1 ^st round PCR was carried out at a 20 ^L scale with cDNA generated from different amounts of mRNA or total RNA, and 0.5 ^M forward and reverse primers. Primers were designed to recognize sequences post deamination, leaving out 10-20 nt sequences surrounding the target m ⁶ A sites. PCR reactions were analyzed by agarose gel electrophoresis. 1 ^L of PCR products were subjected to enzymatic cleanup (Exonuclease I and Shrimp Alkaline Phosphatase, New England BioLabs, M0293 and M0371) and Sanger sequencing. Gel purification was performed for PCR reactions that did not yield bright and single bands. [0241] 2 ^nd round PCR was carried out only for small-scale amplicon deep sequencing. To a 20 ^L reaction, 1 ^L of the 1 ^st round PCR reaction was supplied as the template together with 0.5 ^M Illumina P7 and P5 index primers. Barcoded PCR products were pooled and gel purified using QIAquick Gel Extraction Kit (Qiagen, 28706) before being subjected to next- generation sequencing on an Illumina MiSeq Instrument. eTAM-seq data pre-processing [0242] Adapters were removed from raw eTAM-seq data by Cutadapt v1.18 (see e.g., martin, M. 2011). The 6-nt random barcodes at the 5’ end of R2 were extracted by the subcommand extract from UMI-tools (see e.g., Smith, T. et al., 2017) v1.1.1. R2 reads longer than 39 nt were used for further analysis. For HeLa samples, reads were first mapped to human rRNA sequences using HISAT-3N (see e.g., Zhang, Y. et al., 2021) v2.2.1 (--time --base- change A,G --no-spliced-alignment --no-softclip --norc --no-unal --rna-strandness F). The remaining non-rRNA reads were mapped to the human genome (hg38) and the GENCODE

137741474.1 - 78 - v27 gene annotation (--time --base-change A,G --repeat --repeat-limit 1000 --bowtie2-dp 0 -- no-unal --rna-strandness F). For mESC samples, reads were first mapped to mouse rRNA sequences using HISAT-3N v2.2.1, with the remaining non-rRNA reads mapped to the mouse genome (mm10) and the GENCODE vM25 gene annotation. We used the same HSAT-3N parameters for HeLa and mESC data. Only uniquely mapped reads were kept and deduplicated by the subcommand dedup from UMI-tools. To apply the statistical models for m ⁶ A detection and quantification, we used high-quality datasets in which RNA fragments poorly processed by TadA8.20 (> 50% unconverted A) were eliminated. Custom scripts were developed to count converted and unconverted As across the whole genome. MeRIP-seq data processing [0243] Adapters in HeLa MeRIP-seq data (GSE46705 (see e.g., Liu, J. et al., 2014)) were removed by Cutadapt v1.18 (see e.g., Martin, M. 2011). Adapter-free reads were mapped to the human genome (hg38) using HISAT2 v 2.1.0 (see e.g., Kim, D. et al., 2015) with default parameters. Aligned sequences were divided into different strands and m ⁶ A peaks were called using MACS2 v2.1.1 (see e.g., Zhang, Y. et al., 2008) with the following parameters: -f BAM -B --SPMR --nomodel --tsize 50 --extsize 150 --keep-dup all. Peaks meeting the cutoff (fold enrichment > 2 and p-value < 0.05) were kept for further analysis. For mESC MeRIP-seq data, called peaks were downloaded from supplementary files of GSE151028 (see e.g., Zhang, Z. et al., 2021). Common peaks from two replicates were kept for further analysis. Identification of endogenous RNA-editing sites [0244] RNA-seq data of two HeLa biological replicates (ENCSR000CPR) were downloaded from the ENCODE project website (https://www.encodeproject.org/). Data processing follows pipelines described in previous reports (see e.g., Ramaswami, G. et al., 2012; Lo Giudice, C. et al., 2020; and Cuddleston, W. H. et al., 2022) with minor adjustments. Briefly, the first 5 nt bases of all raw reads were clipped. The trimmed reads were mapped to rRNA using Bowtie2 v2.4.5 (see e.g., Langmead, B. et al., 2012), with remaining reads mapped to small RNAs. Reads that did not map to rRNA and small RNAs were then mapped to the genome (hg38) by STAR v2.7.9a (see e.g., Dobin, A. et al., 2013). Reads that failed to map in proper pair or did not map into primary alignment were discarded. PCR duplicates were also removed at the stage. The remaining reads from two replicates were merged and subject to mutation calling from the pileup format data. During mutation calling, the following filters were applied to identify high-quality and high-confidence RNA-editing sites: (1) only sites with read mapping quality ^ 20 and base quality ^ 25 were considered; (2) sites mapped to

137741474.1 - 79 - common genomic variants in dbSNP (v151) and gnomAD (v3.1.1) with allele frequency > 5% were discarded; (3) sites within 5 bp of known splice junctions were removed; (4) Alu sites were called with read coverage ^ 10 and mutation rate ^ 5%; (5) non-Alu sites were called with variant reads ^ 4 and mutation rate ^ 10%; (6) non-Alu sites in simple repeats according to the Repeat Masker annotation were discarded; (7) non-Alu sites were subject to the BLAT correction according to previous studies (see e.g., Ramaswami, G. et al., 2012; and Lo Giudice, C. et al., 2020). m ⁶ A detection and quantification from eTAM-seq data [0245] Detection with IVT controls. Sample-specific conversion rates for mRNA and IVT samples were estimated using the majority of A sites based on the hypothesis that most A sites are unmethylated and accessible to TadA8.20. The sample-specific conversion rate was plugged in to normalize the A and G counts observed at individual A sites in the mRNA sample, and their apparent methylation levels using a maximum likelihood estimator based on a binomial model was then calculated. Meanwhile, deaminase accessibility for each A site was estimated using adjusted A and G counts reported by the IVT sample. A linear model between site accessibility and total counts of A, G, A+G observed at individual A sites to shrink accessibility estimates and reduce estimation bias was estimated. The model was trained using 2,000 randomly sampled sites with 10-fold cross-validation prior to being applied to predict site accessibility. The apparent methylation levels calculated from mRNA data with site accessibility were adjusted and the final estimated methylation levels were obtained (true methylation levels). Methylation sites were defined as A sites that 1) have ^ 10 read counts in both mRNA and IVT samples; 2) passed Fisher tests with a false discovery rate (FDR) < 0.05; and 3) showed exposed methylation levels (estimated methylation level * site accessibility) ^ 10%. [0246] Detection with FTO controls. Sample-specific conversion rates similar to “detection with IVT controls” were estimated. As FTO partially demethylated m ⁶ A, an upper bound for FTO efficiency in FTO-treated samples was also estimated. Assuming a binomial model for both mRNA and FTO-treated mRNA, site accessibility and methylation levels were jointly estimated using a maximum likelihood approach. Methylation sites are defined with the same criteria as described in “detection with IVT controls”. [0247] All functions were implemented in R. Statistical models are detailed methods “statistical models.”

137741474.1 - 80 - Analysis of the impact of m ⁶ A on mRNA stability [0248] The half-lives of HeLa mRNA were profiled previously via actinomycin D- mediated transcription inhibition followed by RNA-seq at different time points (GSE49339) (see e.g., Wang, X. et al., 2014). Adapters were removed from RNA-seq data by Cutadapt v1.18 (see e.g., Martin, M.2011). Reads were then mapped to the human genome (hg38) using HISAT2 v 2.1.0 (see e.g., Kim, D. et al., 2015) with default parameters. Raw reads of each gene were counted by featureCounts from Subread v1.6.4 (see e.g., Liao, Y. et al., 2013) and normalized by sequencing depth and gene length using the transcripts per million (TPM) method. The fraction of the spike-in RNA in total sequenced RNA is calculated using the following equation: ^^ = ^^⋅ ^^⋅ ^^/ ^^, where ^^ is the dilution factor of spike-in added to each RNA sample, ^^ is the volume (in microliter) of diluted spike-in RNA, ^^ is the concentration (in attomole per microliter) of each spike-in, and ^^ is the mass of total RNA in each sample (in microgram). The TPM values for External RNA Controls Consortium (ERCC) spike-in were correlated to their amounts (in attomole) to build a linear regression model in R 3.5.1, y = ax + b, in which y is the amount of the spike-in RNA (in log2 form), and x is the TPM value (in log2 form). The best-fit dose-response curve in each sample was used to estimate the amount of mRNA (in attomole) for each gene, which was subsequently applied to calculate mRNA half-lives following a previously described protocol (see e.g., Chen, C.Y. et al., 2008). [0249] Methylation loads were calculated by integrating all methylation signals detected in given mRNA. For example, a transcript with three m ⁶ A sites of 0.3, 0.7, and 1.0 methylation corresponds to a methylation load of 0.3 + 0.7 + 1.0 = 2.0. Methylated transcripts were then ranked by their methylation loads. Low-, medium-, and high-methylation bins were defined by transcript ranks: top 1/3 as high, middle 1/3 as medium, and bottom 1/3 as low. An additional bin was included to cover methylation-free transcripts. Only mRNA carrying at least one eTAM-seq evaluated site were considered. Cumulative analysis was performed to probe the correlation between mRNA half-lives and their total methylation loads. Statistical models [0250] All functions are implemented in R. Statistical models are detailed herein in “Model for an untreated mRNA sample with an IVT control” and “Model for an untreated mRNA sample with an FTO+ control”. Assay conditions to maximize A-to-I conversion yields [0251] The Inventors hypothesized that efficiency of TadA8.20-mediated A-to-I conversion could be improved by 1) reducing RNA secondary structure; and/or 2) increasing

137741474.1 - 81 - enzyme efficiency. This hypothesis was tested from two directions: assay temperature and pH. Higher temperatures may denature RNA, thereby exposing previously inaccessible regions. Meanwhile, more hydroxide nucleophile, the key intermediate for the deamination reaction, may arise with higher pH and consequently lead to higher A-to-I conversion efficiency. Related results are provided in FIG.7. [0252] Conversion efficiency was improved by both higher temperature and higher pH, with temperature making a more significant contribution. Two confounding factors were impacting the assay as the temperature was raised: 1) resolution of RNA secondary structure; and 2) impaired and eventually inactivated enzyme. The inventors determined that TadA8.20 stayed robust up to 44 ^o C and quickly lost activity at 55 °C. Meanwhile, when the assay was carried out at 53 ^o C, significantly elevated G signals in regions resistant to A-to-I conversion at 37 ^o C were observed, a fact that may be attributed to resolved RNA secondary structure. Higher pH also led to an increase in A-to-I conversion, albeit to a lesser extent. As RNA is less stable at higher pH, a close-to-neutral pH (pH 7.5) was maintained. An exemplary assay condition comprised: 1 h incubation at 53 ^o C (for hard-to-convert regions) followed by 2 h treatment at 44 ^o C with freshly supplemented enzyme (e.g., for global A-to-I conversion with TadA8.20). Model for an untreated mRNA sample with an IVT control [0253] Model Annotations: introduced were the following global parameters: k1: Conversion rate for the untreated m6A sample k2: Conversion rate for the IVT control sample [0254] Look at each site i: ^ _i : True A proportion, i.e., the underlying proportion of A in total A (including A and m6A) without any treatment ωi: Site-specific accessibility [0255] Expected proportion of nucleotide A at each site i can be represented as: [0256] Expected proportion of nucleotide G at each site i can be represented as:

137741474.1 - 82 - [0257] Ideally, if then can be used to estimate the abundance of m6A. However, in reality, the conversion rates, and site accessibility are not all 100%. . Thus there is a need to estimate conversion rates and site- specific accessibility to quantify the actual methylation level. [0258] Estimating conversion rates: majority of A sites are accessible and un-methylated. For un-methylated and fully accessible A sites, we have: [0259] Let represent observed G rates, respectively, for each site i, calculated: [0260] where i belong to sites after removing 10% outliers. Finally estimate conversion rates by: [0261] Estimating apparent methylation level: Denoting apparent methylation level using . Assuming the sampling of G following a binomial model, use the untreated mRNA sample to quantify apparent methylation level for each site. [0262] Estimating site-specific accessibility: For un-methylated sites, estimate their accessibilities by:

137741474.1 - 83 - [0263] Then fit a model between initial accessibility estimates and observed counts to further remove unwanted variation. Additionally; introduce the following variables to present observed counts in the IVT control: [0264] The following linear model (with intercept) was estimated using randomly sampled 2,000 data points with 10-fold cross-validation. [0265] The accessibility for each site is predicted as, [0266] Calibrating β estimates using site-specific accessibility to obtain methylation level. The methylation level was calibrated using is apparent methylation level obtained previously. [0267] Calling methylated sites. The final methylation sites are defined as the follow: A) Total number of read counts greater than 10 in both IVT and untreated mRNA samples; B) Fisher test between IVT and untreated mRNA with adjusted p-values no greater than 0.05; C) At least 10% exposed methylation level. Model for an untreated mRNA sample with an FTO+ control [0268] Model Annotations: In FTO- sample, m6A sites stay as m6A, un-methylated A will be converted into G. In FTO+ sample, m6A sites that react with FTO will be converted into G, m6A sites that do not react with FTO will stay methylation, un-methylated A will be converted into G. Assume the following global parameters: k1: Conversion rate for FTO- sample k ₂ : Conversion rate for FTO+ sample

137741474.1 - 84 - γ: FTO efficiency in FTO+ sample [0269] Look at each site i: ^ _i : True A proportion, i.e., the underlying proportion of A in total A (including A and m6A) without any treatment ωi: Site-specific accessibility. [0270] Expected proportion of nucleotide A at each site i can be represented as: [0271] Expected proportion of nucleotide G at each site i can be represented as: [0272] Ideally if k ₁ = k ₂ = α = ω _i = 1, R _iG+ = 1, we can use R _iG+ - R _iG- = 1 - ^ _i to estimate the abundance of m6A. However, in reality, the conversion rates, FTO efficiency and site accessibility are not all 100%, is very negative, one can observe a reversed trend. [0273] Estimating conversion rates: Majority of A sites are accessible and un-methylated. For un-methylated A sites with full accessibility: [0274] Let represent observed G rates, respectively, for each site i, will calculate: [0275] where i belong to sites after removing 10% outliers. Finally, estimate conversion rates by:

137741474.1 - 85 - [0276] Estimating FTO efficiency from FTO+ sample. Denote apparent methylation level or exposed methylation using . For highly methylated sites, where βi is close to 0, rate of nucleotide G in FTO+ reduces to: . [0277] Assuming the sampling of G following a binomial model, using the FTO- sample to quantify apparent methylation level for each site. Selected sites with an estimated methylation rate from FTO- greater than 0.95 and observed G rate in FTO+ greater than 0.25 (excluding low accessibility) to obtain an estimated FTO efficiency, i.e., . [0278] Jointly estimating site-specific m6A methylation level ^ _i and site-specific accessibility ωi. [0279] Assuming reads for FTO- and FTO+ samples distribute according to the following binomial distributions, respectively: [0280] The probability model for FTO- and FTO+ can be written, and its log likelihood is as follows: [0281] The MLE of ^i and ωi can then be calculated by optimizing the above log likelihood function using BFGS algorithm implemented in R function optim(), i.e., . [0282] Calling methylated sites. Final methylation sites are defined as the following: A) Total number of read counts greater than 10 in both FTO+ and FTO- samples; B) Fisher test between FTO+ and FTO- with adjusted p-values no greater than 0.05; C) At least 10% exposed methylation level.

137741474.1 - 86 - Estimating site accessibility using IVT controls [0283] The inventors proposed that accessibility of a given A site stayed the same in HeLa and IVT samples because of sequence context, which prompts formation of secondary structures, and is consistent in both samples. In IVT samples, lower deamination rates can only arise if a site is partially blocked, whereas in mRNA samples, both the presence of m ⁶ A and compromised accessibility contribute to lower deamination rates. We therefore propose the following relationships and equations: G%(mRNA) ^ G%(IVT); Presence of m ⁶ A = (G%(mRNA) < G%(IVT) with statistical significance); Apparent methylation level = 1 – G%(mRNA); Accessibility of an A site = G%(IVT); and True methylation level = 1– G%(mRNA)/G%(IVT). [0284] The accessibility parameter calculated based on the A and G counts observed in IVT samples is applied to the apparent methylation signals detected in mRNA and output the true methylation levels. The statistical model is detailed above in “Statistical models” and “model for an untreated mRNA sample with an IVT control,” and the following simplified worksheet showcases the workflow. [0285] Note that the methylation level estimation can be more error prone at sites of low accessibility as only accessible A/m ⁶ A produces eTAM-seq signals. Define exposed methylation level = true methylation level * site accessibility. Exposed methylation levels are only used to define the cutoff for high-confidence m ⁶ A sites, with the intention to exclude hits of extremely low methylation and accessibility. “Methylation levels” in this study always refer to true methylation levels. Table 1 – True methylation level

137741474.1 - 87 - Comparison of three biological replicates of eTAM-seq (HeLa/IVT) using merged IVT controls [0286] As sequence context, the primary determinant of site accessibility, remained the same in RNA samples collected from the same genetic background, the inventors envisioned that in vitro transcribed RNA should behave consistently across biological replicates for site accessibility estimation. To this end, the inventors merged HeLa-IVT1-3 for a more comprehensive control transcriptomes. The three biological replicates of eTAM-seq (HeLa-1- 3) were processed using this merged control transcriptome and called out m6A sites with exposed methylation levels ≥ 10%. As the merged IVT control covers significantly more sites, the inventors detected many more m6A sites in each biological sample: 42,135, 42,029, and 42,119 for HeLa-1, HeLa-2, and HeLa-3 respectively, of which 34,321 were persistent across the three replicates (FIG.12). Only 3,330 (8%), 3,161 (8%), and 3,481 (8%) sites were unique to HeLa-1, HeLa-2, and HeLa-3, respectively. The majority of hits, 36,474 for HeLa-1 (87%), 36,435 for HeLa-2 (87%), and 36,129 for HeLa-3 (86%), emerge in DRACH motifs. [0287] When eTAM-seq (HeLa/IVT-1), eTAM-seq (HeLa/IVT-2), and eTAM-seq (HeLa/IVT-3) results were analyzed separately, it was found that eTAM-seq (HeLa/IVT-1) and eTAM-seq (HeLa/IVT-2) had more unique hits than eTAM-seq (HeLa/IVT-3) (FIG. 2A), likely due to deeper sampling by HeLa-IVT1 and HeLa-IVT2. Therefore, the inventors posited that some of these unique hits arose due to heterogeneity in sampling. To support this hypothesis, overlapping deep sequencing of eTAM-seq (HeLa/IVT-1) with eTAM-seq (HeLa/IVT-2) and eTAM-seq (HeLa/IVT-3) was conducted. In this case, only 961 (4.9%) and 546 (3.6%) hits were unique to eTAM-seq (HeLa/IVT-2) and eTAM-seq (HeLa/IVT-3), respectively. Collectively, a portion of the replicate-unique hits can be attributed to heterogeneity in sampling, rather than false positive detection by eTAM-seq. Site-specific m ⁶ A quantification by amplicon deep sequencing [0288] Raw next-generation sequencing data were merged by Pear v0.9.8. before mapping to the target sequences using bwa-mem2 v2.2.1. Mapped reads were sorted and indexed using samtools v1.14. Bases mapped to individual A sites were counted using pysamstats v1.1.1. A and G counts were extracted for individual A sites covered by R2 using re.finditer. Methylation levels are reported as apparent A fractions.

137741474.1 - 88 - Site-specific m ⁶ A quantification by eTAM-Sanger [0289] The A and G fractions observed at all A sites were quantified by EditR (see e.g., Kluesner, M.G. et al., 2018). Methylation levels were reported as apparent A fractions. Extraction of endogenous A-to-I editing sites, and analysis of the impact of endogenous A-to-I editing on eTAM-seq [0290] RNA editing is a natural process widely occurring in eukaryotes. The most common type of RNA editing is A-to-I conversion mediated by adenosine deaminases acting on RNA (ADARs) (see e.g., Bass, B.L., 2002) As endogenous A-to-I editing shifts the distribution of A and G in RNA-seq at genomic A sites, the impact of this phenomenon on eTAM-seq was analyzed. RNA-seq data from two HeLa biological replicates (ENCSR000CPR) from the ENCODE project website (https://www.encodeproject.org/) were utilized for RNA-editing site identification. Two samples were combined for mutation calling and stringent quality control and multiple filters were applied to obtain high-confidence RNA-editing sites (see FIG.15A for detailed processing steps). A total of 29,052 RNA-editing sites were identified, with 77.3% (22,472) being A-to-I editing. The majority of RNA-editing sites (79.7%) were found in Alu elements. In sites mapped to non-Alu elements, 43% located in introns, 30% located in 3’ UTRs, 13% in IGRs, 10% in ncRNA, 3% in CDS, and 1% in 5’ UTRs (FIG. 15B). Overall, the genomic distribution of RNA-editing sites was distinct from that of m6A sites detected by eTAM-seq. Only three sites overlapped between RNA-editing sites and eTAM-seq hits (FIG. 15C). It can therefore be concluded that the fidelity of eTAM-seq is not compromised by endogenous RNA-editing events. Biological replicates of eTAM-seq (HeLa/FTO) [0291] Three biological samples of HeLa mRNA were treated by FTO (FIG. 18). Both untreated and FTO-treated mRNA samples were further processed by TadA8.20, resulting in three biological replicates – eTAM-seq (HeLa/FTO-1), eTAM-seq (HeLa/FTO-2), and eTAM- seq (HeLa/FTO-3). The three replicates were processed separately and m6A sites with exposed methylation levels ≥ 10% were called out. In sum, 21,728, 20,337, and 15,789 m6A sites from individual replicates were identified, 19,646 (90%), 18,542 (91%), and 14,407 (91%) of which were in DRACH motifs. Of the identified hits, 13,147 (61-83%) were common to three replicates and showed highly consistent methylation levels (Pearson’s r = 0.96-0.97). Only 3,699, 2,492, and 950 sites were unique to eTAM-seq (HeLa/FTO-1), eTAM-seq (HeLa/FTO- 2), and eTAM-seq (HeLa/FTO-3), respectively.

137741474.1 - 89 - [0292] In addition to comparing methylation levels reported by different biological replicates, correlative analysis on methylation levels estimated using different control transcriptomes was also performed. Cross comparison of 6 samples (HeLa/IVT-1, HeLa/IVT- 2, HeLa/IVT-3, HeLa/FTO-1, HeLa/FTO-2, and HeLa/FTO-3) showed that the estimated methylation levels in the same biological sample were almost identical when referenced to different controls (Pearson’s r = 0.99-1.00). Moreover, methylation levels in different biological samples estimated using different controls were highly correlated (Pearson’s r > 0.96), confirming the consistency of m6A landscape in HeLa cells and the high reproducibility of eTAM-seq. Biological replicates of eTAM-seq on mESCs [0293] Two batches of mESCs were harvested and IVT and FTO-treated samples were prepared separately. Two biological replicates were obtained accordingly with both IVT and FTO controls: mESC/IVT-1, mESC/IVT-2; mESC/FTO-1, mESC/FTO-2 (FIG. 22). In sum, 24,676 and 26,756 m6A sites in eTAM-seq (mESC/IVT-1) and eTAM-seq (mESC/IVT-2) were detected, respectively, 20,727 of which were shared. Many replicate-unique hits can be attributed to sequencing depth that inevitably varies among samples. Similar to what was observed for HeLa samples, the majority of hits were detected in DRACH motifs (21,524, 87% for mESC/IVT-1; 21508, 80% for mESC/IVT-2). The methylation levels reported by two biological replicates were highly consistent (Pearson’s r = 0.95), suggesting that 1) IVT functions consistently across replicates for accessibility estimation; and 2) the m6A deposition machinery favors a similar category of target sites in the same cell type, at least at the bulk level. [0294] Two IVT samples were then merged and a control transcriptome covering more A sites was obtained. With this control transcriptome, many more m6A sites were detected: 46,377 in mESC-1, 39,824, 86% in DRACH; 43,170 in mESC-2, 34,035, 79% in DRACH. 35,080 of these hits overlapped (76% of mESC-1, 81% of mESC-2). [0295] eTAM-seq (mESC/FTO-1) and eTAM-seq (mESC/FTO-2) samples were processed separately, and 28,788 (26,122 in DRACH, 91%) and 18,577 (16,210 in DRACH, 87%) m6A sites were identified, respectively. Similar to what was observed in eTAM-seq (mESC/IVT), the majority of these sites overlapped across two replicates (16,076, 56% of mESC/FTO-1 and 87% of mESC/FTO-2) with highly consistent methylation levels (Pearson’s r = 0.95). mESC samples also behaved consistently when different controls, IVT or FTO, were applied. To simplify the results, the comparison between eTAM-seq (mESC/IVT-1) and eTAM-seq (mESC/FTO-1) was presented. Very similar results were obtained when eTAM-seq

137741474.1 - 90 - (mESC/IVT-2) and eTAM-seq (mESC/FTO-2) were compared, or eTAM-seq (mESC/IVT-1) and eTAM-seq (mESC/FTO-2); and eTAM-seq (mESC/IVT-2) and eTAM-seq (mESC/FTO- 1) were cross compared. Collectively, two biological replicates of eTAM-seq for mESCs were carried out, confirming the consistency of m6A deposition in mESCs and the robustness of eTAM-seq. Table 2 – TadA8.20-enabled A-to-I conversion in different sequence contexts as reported by non-methylated RNA probes.

137741474.1 - 91 -

137741474.1 - 92 -

137741474.1 - 93 - ^o _{i )} s ¹ - ^t _{e a} ^p _{a 5 2 2 6 8 5 3 3 7 7 6 1 2 -} m _n R U ^e _r ^r ₍ m ² ₅ ² ₁ ^{2 5 4 0 9 0 5 1 7 4 1} ₄ a _{7 8 5 1 7 6 2 6 5 2 5 r} ^{9 a} _p a - _g ^c i _l ⁾ i ^{1 1} _p ^{) e 2 3 1 1} _p ^{) e 2 3 1} _p n ^{p s} _{e p p e p e} ^{1 e} _e ^{2 3 1} s ^{e e e d e} _p ^e _p ^e _p ^e _p ^e _p ^e _p ^e _p ^e _p ^e _p ^e _p e R _{t r r ( r r r r} ^d _{( r r r r} ^d _{( r r} ^e _{r c} ^o _{r t} p ^{n d} _{e n} ^{m a} _t ^a A N A N A N A N ₊ A O _{+ + +} ^{N g} _{e R R R R} O O O _{T T T T R n} ^{r i} _{c T} m m m m ^T _F ^T _F ^T _F ^T _F ^V _I ^V _I ^V _I ^V _I m n _e ^u y q ^{t e} _o ^{d 3} S ⁿ _{e –} G ^e l _i ^e _p ^d l ^{e d e d e d e d e d e d e d e d e d e d e} _lt ^t p W ^y _{i p} l t W ^y _{i p} l t W ^y _{i p} l t W ^y _{i p} l t W ^y _{i p} l t W ^y _{i p} l t W ^y _{i p} l t W ^y _{i p} l t W ^y _{i p} l t W ^y _{i p} l _{i p e o t} W ^y _t W ^y _t M ^k _{c 3 e} ^l _b ¹ _. a _{a a a a a a a a a a a a C} ⁴ _{7 T l} ^l _e ^L _e ^L _e ^L _e ^L _e ^{L L L L L L L L S} _E ⁴ ₁ ⁴ C H H H H _e H _e H _e H _e H _e H _e H _e H _e H m ₇ ⁷³ 1 M _{r (} m _{2 1 5 2 4 2 1 1 1 1 2 2 ) e} ^{g u} _q A _n ^{i 4} _{9 0 5 4 4} i _{s 1} ⁹ ₈ ² ₄ ⁴ ₅ ⁰ ₇ ⁶ ₄ ³ ₁ ³ ₀ ⁵ ₆ ⁶ ₀ ⁴ ₈ ⁵ _{2 n} ^d _a N _p R ^p _a ⁶ ₂ ⁹ m ⁷ ₇ ¹ 7 ⁸ ₉ ⁵ 7 ⁰ ₄ ⁹ 9 ⁵ ₀ ³ ₈ ⁴ ₅ ³ ₈ ⁰ ₅ ⁵ ₉ ⁸ ₅ ¹ _{3 -} U ^e _r ^r _{( 8} ⁵ ₁ ⁷ ₁ ³ ₈ ⁴ ₁ ⁵ ₁ ⁶ ₄ ⁹ ₈ ³ _{1 5} ⁹ a - ci _l ^{) ) ) p} _e ¹ _{p p p} e _p ^{1 e} _p ¹ _p ^{1 e} _p ¹ _p ^{e e} _e ² _p ¹ _p ¹ _p ^e _e ² _p ^{1 1 e} _e ² ( _r ^e _r ^e _{r (} ^e _{p r} ^e _{p r} ^e _p R _{t r} ^e _{r r} ^e _{r r} ^{d e d} _r ^d ₍ ^e _{r t} ⁿ _e m _t ^a ₊ A N ₊ A N A A _{+ + + e} ^r O _R O _R ^{N N} O O O _{T T T T} ^T _F m ^T _{R R F} m m m ^T _F ^T _F ^T _F ^V _I ^V _I ^V _I y ^t l l o ³ _l ³ _{l r} ^t _c ³ _{l r} ^{t n} _{t e} ^{e t} _{e o t} ^{t k} _{e o t} ^t _c k _{e o} ^d l k _i ^e _p ^d l _i ^e _p ^d l _i ^e _p ^d l _i ^{e d} l _i ^{e d} l _i ^{e d} l _i ^{e d} l _i ^{e d} l _i ^e W ^y _t W ^y _t W ^y _{p p p p p p} G _p M _c M _c M _{c t} W ^y _t W ^y _t W ^y _t W ^y _t W ^y _t W ^y _{t l} ^l _{C C C C C} ¹ _. ⁴ _{7 e} ^S _E ^S _E ^S _E ^S _{C C C C C C C E} ^S _E ^S _E ^S _E ^S _E ^S _E ^S _E ^S _E ^S _E ⁴ ₁ ⁴ C m m m m m m m m m m m m ₇ ⁷³ 1 Table 4 – Selected m ⁶ A sites for site-specific, deep sequencing-free methylation quantification. Table 5 – Site-specific quantification of m ⁶ A by eTAM-Sanger sequencing, eTAM- amplicon deep sequencing, and transcriptome-wide eTAM-seq.

137741474.1 - 96 - Table 6 – Sequences of RNA oligos used in in vitro deamination assays. Table 7 – Sequences of DNA oligos used to prepare dsDNA templates for in vitro transcription.

137741474.1 - 97 - Table 8 – Sequences of DNA oligos used for reverse transcription of RNA probes. Table 9 – Sequences of DNA oligos used to amplify target loci of HeLa mRNA and IVT RNA.

137741474.1 - 98 -

137741474.1 - 99 - Table 10 – Sequences of DNA oligos for installation of Illumina adapters. Table 11 – Sequences of spike-in RNA probes.

137741474.1 - 100 - Example 1: Evolved TadA-assisted N6-methyladenine sequencing (eTAM-seq) of RNA [0296] The sequencing platform described herein was inspired by the concept of bisulfite sequencing for methylation detection in DNA (see e.g., Frommer, M. et al., 1992), in which all unmethylated C is converted into U without impacting 5-methylcytosine. The inventors envisioned global deamination of A but not m6A (FIG.1A) – all unmethylated A is converted into I; I base-pairs with C and is read as G by reverse transcriptases. Persistent A corresponds to m6A. [0297] The search for global A deamination routes focused on enzymatic approaches that supported high efficiency and mild reaction conditions. The inventors hypothesized that laboratory-evolved hyperactive (deoxy)adenosine deaminases, unlike naturally occurring enzymes whose activity is tamed (see e.g., Walkley, C.R. & Li, J.B.2017), may facilitate robust global A deamination. Screening a panel of enzymes, it was determined that TadA8.20 (see e.g., Gaudelli, N.M. et al., 2017; Gaudelli, N.M. et al., 2020; and Grunewald, J. et al., 2019) (FIG.6A), an E. coli tRNA adenosine deaminase (TadA) variant evolved to function robustly on DNA with minimal context dependence, fit the desired criteria, e.g., when a single A or m6A is placed in different sequence contexts, TadA8.20 deaminated A close to completion without acting on m6A (FIGs. 1B-1E and FIG. 6B). TadA8.20 preserved RNA integrity as RNA pre- and post-enzymatic treatment produced the same amount of complementary DNA (cDNA) during reverse transcription (FIG.6C). [0298] As deamination efficiency was critical for faithful detection of m ⁶ A, the inventors screened a series of assay conditions and found that temperature elevation from 37 °C to 44 °C or 53 °C improved A-to-I conversion rates, particularly in regions resistant to deamination at 37 °C (see e.g., methods section “assay conditions to maximize A-to-I conversion yields).

137741474.1 - 101 - TadA8.20 purified in different batches functioned consistently in mediating A-to-I conversion (see e.g., FIG.8). [0299] TadA8.20 was incorporated into an RNA-seq workflow, and an evolved TadA- assisted N6-methyladenine sequencing (eTAM-seq) methodology was developed. The methodology was assessed for efficiency and context dependence of the enzyme using synthetic RNA probes with A/m6A flanked by two Ns (N = A, C, G, or U). TadA8.20 attained a global A-to-I conversion rate of 99%, close to the efficiency offered by bisulfite treatment of C in RNA (see e.g., Kint, S. et al., 2018), and rejected m6A completely (FIG.1F). Importantly, TadA8.20 efficiently deaminates A in all DRACH sequences (D = A, G, or U; R = A or G; H = A, C, or U), the consensus motif hosting m6A modifications in eukaryotes (Table 2). [0300] Whether global A deamination enforced by TadA8.20 enables quantitative detection of m6A was then investigated. Additional RNA probes that contain 25%, 50%, and 75% m6A at the N flanked position (NNA/m6ANN) were synthesized, and labeled with unique molecular identifiers (UMIs). After incubation with TadA8.20, 1.69 ^0.03%, 26.1 ^0.6%, 46.5 ^0.3%, 73.9 ^0.5%, and 98.7 ^0.1% A in probes hosting 0%, 25%, 50%, 75%, and 100% m6A were detected, respectively (FIG.1G and FIG.7). Since the persistent A ratio correlates linearly to the extent of m6A in synthetic RNA probes (r ² = 1.00, FIG. 1G), it is concluded that eTAM-seq quantifies m6A at the site of interest. Collectively, these results demonstrated that TadA8.20 is mild, robust, selective, and insensitive to sequence contexts, paving the road for quantitative and base-resolution detection of m6A in biological samples. Example 2: eTAM-seq enables base-resolution detection of m6A in mammalian RNA [0301] eTAM-seq was applied to 50 ng of mRNA extracted from HeLa cells. To reduce secondary structures and to facilitate downstream sequencing, HeLa mRNA was processed into ~150 nt fragments prior to incubation with TadA8.20. Capillary gel electrophoresis of RNA incubated with TadA8.20 for 3 h indicated that the size distribution of RNA remained unchanged with no noticeable sample loss (FIG. 1H). Fragmented mRNA treated with or without TadA8.20 behaved similarly during library construction and RNA-seq, suggesting that the increased GC content (see e.g., Benjamini, Y. & Speed, T.P.2012), a consequence of global A deamination, poses minimal impact on cDNA synthesis, amplification, and sequencing. [0302] Following adapter removal, reads were mapped to the transcriptome allowing both A and G matched to genomic A sites. Given the reduced complexity of the transcriptome post- TadA8.20 treatment, conservative measures to ensure mapping accuracy were utilized, for example, only reads ^ 40 nt were accepted, and reads that could be mapped to more than one

137741474.1 - 102 - genomic locus were discarded (Table 3). Importantly, mRNA abundances reported by eTAM- seq were consistent with a published RNA-seq dataset (see e.g., Liu, J. et al., 2014) (Pearson’s r = 0.84-0.85, FIG. 10A), indicating that eTAM-seq sustained gene expression information captured by canonical RNA-seq. [0303] The conversion rate of a given A in the HeLa transcriptome was highly reproducible across biological replicates (Pearson’s r = 0.99, FIG.1I, FIG.10B, and FIG.10C), suggesting that deamination efficiency was governed by intrinsic properties of RNA rather than random factors introduced during sample preparation. [0304] Local secondary structures may shield a subset of A bases from TadA8.20, resulting in false positive persistent A signals in eTAM-seq, similar to bisulfite treatment-resistant cytosines in double-stranded RNA (see e.g., Hussain, S. et al., 2013). To account for this potential, the inventors developed a statistical model to determine the extent to which a given A site is shielded from TadA8.20 using a modification-free HeLa transcriptome prepared via in vitro transcription (IVT; Fig.11A and methods section “site accessibility”) (see e.g., Zhang, Z. et al., 2021). Sequence context, and consequently secondary structures and accessibility to TadA8.20, were consistent in HeLa and IVT samples. Persistent A signals that arise to the same extent in HeLa and modification-free IVT samples likely represent partially shielded unmethylated A sites. In the provided statistical model, “0” indicates a site fully blocked to TadA8.20 and “1” indicates a site fully accessible. The accessibility parameter to the apparent methylation levels detected by eTAM-seq was applied and the true methylation levels were determined (methods section “model for an untreated mRNA sample with an IVT control”). The accessibility adjustment sustained persistent A signals arising from m ⁶ A, but eliminated those caused by secondary structures, thereby improving the fidelity of eTAM-seq. [0305] Reads mapped to ribosomal RNA (rRNA) were analyzed first because human rRNA not only hosts two known m ⁶ A sites but is also more structured than mRNA (see e.g., Piekna- Przybylska, D. et al., 2008). ~1% rRNA reads in HeLa mRNA was detected (Table 3), a level typical for RNA purified by enriching polyadenylated sequences (see e.g., Herbert, Z.T. et al., 2018). A lower global A-to-I conversion rate in rRNA (85%) was observed, consistent with the hypothesis that highly structured RNA is more resistant to deamination. Nevertheless, HeLa and IVT samples produced the same levels of persistent A signals at these sites (FIG. 11B), which were therefore recognized as less accessible unmethylated A sites. Two well- characterized m6A sites in human rRNA, at position 1832 in 18S rRNA30, 31 and position 4220 in 28S rRNA32, 33, are cleanly detected by eTAM-seq (FIG.1J).

137741474.1 - 103 - [0306] eTAM-seq detects A chemically modified at the N ⁶ position. N ⁶ ,N ⁶ - dimethyladenosine (m ⁶ 2A) (see e.g., Poldermans, B. et al., 1979; and Lafontaine, D. et al., 1995), similar to m ⁶ A, is resistant to TadA8.20-catalyzed deamination and two conserved m ⁶ ₂ A sites in human 18S rRNA (see e.g., Zorbas, C. et al., 2015), positions 1850 and 1851, were detected by eTAM-seq (FIG.11C). On the other hand, 2’-O-methyladenosine (Am) is sensitive to TadA8.20 and will not generate persistent A signals in eTAM-seq (FIG. 11D). m ⁶ ₂ A, however, is extremely rare in mRNA and unlikely to make a significant contribution to eTAM- seq signals. N ⁶ ,2’-O-dimethyladenosine (m ⁶ Am), another A modification bearing a methyl group at the N ⁶ position, is located at the first transcribed nucleotide adjacent to the cap of ~10% of mammalian mRNA (see e.g., Wei, C. et al., 1975; and Wei, J. et al., 2018). The terminal location of m ⁶ Am may lower its sequencing coverage. Moreover, m ⁶ Am was quantified to be 0.027% of A in fragmented and ligated mRNA, 95% lower than that of m ⁶ A (0.55% of A, FIG. 11E). Together, these results supported the conclusion that eTAM-seq predominantly detects m ⁶ A in mammalian mRNA. Example 3: m6A profiling and quantification in the HeLa transcriptome [0307] m6A in the HeLa transcriptome was then profiled. To assess reproducibility of eTAM-seq, three biological replicates were conducted: replicate 1 (HeLa-1 and HeLa-IVT1), replicate 2 (HeLa-2 and HeLa-IVT2), and replicate 3 (HeLa-3 and HeLa-IVT3). The three replicates were processed separately and m6A sites with exposed methylation levels ≥ 10% (methylation level * site accessibility ≥ 10%, methods section “site accessibility”) were called out. This cutoff was chosen to remove sites of extremely low methylation and accessibility. 18,712, 19,439, and 15,159 m6A sites from replicate 1, 2 and 3, respectively, were identified. Of which 16,376 (88%), 16,600 (85%), and 13,151 (87%) were found in DRACH motifs (FIG. 2A, and FIG.12). As DRACH motifs host ~70% of m ⁶ A sites (see e.g., Linder, B. et al., 2015), and only ~7% of all A sites, in mammalian transcriptomes, the observed hit distribution among DRACH and non-DRACH sequences supports the robustness of eTAM-seq. Of the identified hits, only 2,607 (14%), 2,719 (14%), and 661 (4%) were unique to individual replicates (FIG. 2A). Sites common to three replicates showed highly consistent methylation levels (Pearson’s r = 0.96, FIG.2B, FIG 12, and methods section “comparison of three biological replicates”), confirming the reproducibility of eTAM-seq. [0308] Next, replicate-1 was sequenced at a deep level to better uncover the m ⁶ A landscape in HeLa cells.80,941 m ⁶ A sites in HeLa mRNA were detected, of which 12,454 sites (15%) showed methylation levels greater than 90% (FIG. 13A). Methylated sites were strongly

137741474.1 - 104 - enriched in DRACH sequences (FIG.2C), highlighting the strong motif preference of the m ⁶ A writer complex. m ⁶ A constitutes 0.41% of all A subject to evaluation (FIG. 2D). Given that the m ⁶ A fraction in the HeLa transcriptome was determined to be 0.2-0.6% by liquid chromatography/mass spectrometry (see e.g., Wang, X. et al., 2014) (0.55% in this work, FIG. 11E), these results suggest the conclusion that eTAM-seq captured the majority of m ⁶ A sites in the input RNA. [0309] m ⁶ A sites with exposed methylation levels ^ 10% (69,834) were extracted for further analysis.34,049 (49%) of these m ⁶ A sites overlap with a published MeRIP-seq dataset (see e.g., Liu, J. et al., 2014) and account for 8,398 (52%) of peaks detected by MeRIP-seq (FIG.13B). When considering sites with ^ 200 counts and ^ 90% methylation, 89% of eTAM- seq hits were co-discovered by MeRIP-seq (FIG.2E. It is noted that these numbers may not directly translate to other studies as MeRIP-seq can be affected by antibody specificity, immunoprecipitation workflow, and sequencing depth (see e.g., MyIntyre, A.B.R., et al., 2020), whereas eTAM-seq may detect additional m ⁶ A sites if more genomic A sites are effectively sampled. m ⁶ A sites were enriched around stop codons with significant distribution across 5’ untranslated regions (UTRs), coding sequences (CDS), and 3’ UTRs ( FIG. 2F), consistent with the transcriptome-wide m ⁶ A distribution reported by orthogonal detection methods (see e.g., Dominissini, D. et al., 2012; and Meyer, K.D. et al., 2012). A clear DRACH motif emerged in sequences surrounding m ⁶ A (FIG. 2G, and FIG. 13D). Further sequence context dissection of these m ⁶ A sites revealed 14.7% from GGACU, 11.8% from GAACU, and 10.5% from AGACU, a distribution fully corroborated by miCLIP (see e.g., Linder, B. et al., 2015) (FIG.2G, and FIG.13D). eTAM-seq hits were also overlapped with m ⁶ A-SAC-seq (see e.g., Hu, L. et al., 2022, and Ge, R. et al., 2022), a recently developed base-resolution m ⁶ A detection method. As m ⁶ A-SAC-seq is more sensitive to GA sequences, comparison described herein is limited to DGACH hits. eTAM-seq detects 36,993 m ⁶ A sites in DGACH, 27,135 (73%) of which are co-discovered by m ⁶ A-SAC-seq (27,135 divided by 34,790, 78%, FIG. 2H). [0310] Of all sequencing-captured A sites, 92% show accessibility ≥ 0.9 (FIG. 13E), indicating that the majority of A sites in the HeLa transcriptome are sensitive to eTAM-seq. Accessibility of methylated A sites, including those methylated to > 90%, was overwhelmingly skewed towards 1 (FIG. 13F), i.e., fully deaminated in the IVT control, indicating that persistent A signals observed at these sites in TadA8.20-treated mRNA were a result of methylation instead of incomplete deamination (FIG. 14). The inventors extracted from the

137741474.1 - 105 - HeLa transcriptome sites subject to A-to-I editing, which were almost mutually exclusive from eTAM-seq hits (FIG.15, and methods section “Extraction of endogenous A-to-I editing sites, and analysis of the impact of endogenous A-to-I editing on eTAM-seq”). [0311] In addition to the IVT transcriptome, the inventors employed an orthogonal control for eTAM-seq wherein the demethylase FTO was applied to HeLa mRNA to provide a demethylated transcriptome (FIG.16). FTO demethylated a large portion of m ⁶ A in spike-in RNA probes (FIG. 17), in line with previous reports (see e.g., Zhang, Z. et al., 2021). FTO treatment should only impact the deamination level of methylated A sites. Therefore, positions with significantly lower levels of persistent A following eTAM-seq in the FTO-treated sample (FTO+) compared with the original untreated sample (FTO–) were extracted as m ⁶ A sites (see methods “model for an untreated mRNA sample with an FTO+ control”). With this workflow, 47,840 m ⁶ A sites were identified, 40,096 (84%) of which showed exposed methylation levels ^ 10% (see methods “biological replicates of eTAM-seq (HeLa/FTO)”, and FIGs.18 and 19). Most hits (95%) were co-discovered with the HeLa/IVT dataset FIG.19C) and showed highly consistent methylation levels (Pearson’s r = 0.996, FIG.19D). Of the 40,096 m ⁶ A sites, 60% (24,044) overlapped with a published MeRIP-seq dataset (see e.g., Liu, J. et al., 2014), and covered 50% of MeRIP-seq peaks (FIGs. 19E and 19F). Taken together, these results suggested eTAM-seq functioned consistently in profiling m ⁶ A regardless of the mechanism through which the demethylated reference transcriptome (IVT or FTO demethylation) was prepared. [0312] The quantitative feature of eTAM-seq was then evaluated using five representative sites – MALAT1_2515, 2577, 2611 and TPT1_687, 703, the methylation levels of which have been previously determined to be 61%, 80%, 38%, 15%, and 1% by site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET) (see e.g., Liu, N. et al., 2013). eTAM-seq reported m ⁶ A fractions of 58%, 81%, and 51% for MALAT1_2515, 2577, and 2611, respectively (FIG.2I), in line with SCARLET results. However, much higher modification levels than previously reported for TPT1_687 and 703 – 42% and 77% (FIG. 2I) were observed. As results from the eTAM-seq (HeLa/IVT), eTAM-seq (HeLa/FTO), and m ⁶ A-SAC-seq datasets corroborated each other (FIG.20), these quantification results were found to be reliable. Discrepancies between the immediate study and the SCARLET study may be explained by 1) differences in HeLa cells and 2) inefficient/off-target probe annealing in SCARLET.

137741474.1 - 106 - [0313] eTAM-seq signals from 18 additional loci were extracted and overlayed with a published MeRIP-seq dataset (see e.g., Liu, N. et al., 2013) (FIG.2I and FIG. 21). In all 20 cases, including MALAT1 and TPT1, eTAM-seq peaks were found in or close to MeRIP-seq peaks. Of the 20 transcripts inspected, 19 hosted multiple m ⁶ A sites with several transcripts heavily methylated, such as ZBED5, MYC, and CXCR4, which bear 31, 22, and 23 m ⁶ A sites, respectively. These results, taken together, confirmed eTAM-seq was robust and reliable in capturing and quantifying m ⁶ A sites in the whole transcriptome. Example 4: Mapping m6A in the transcriptome of mouse embryonic stem cells [0314] eTAM-seq was then applied to the transcriptome of mouse embryonic stem cells to explore the involvement of m ⁶ A in mouse embryogenesis (see e.g., Guela, S. et al., 2015; Wang, Y. et al., 2014; and Batista, P.J. et al., 2014). Two biological replicates of eTAM-seq (mESC/IVT) were conducted, and detected 24,676 and 26,756 m ⁶ A sites. Among these, 20,727 were shared and showed consistent methylation levels (Pearson’s r = 0.95, methods section “biological replicates of eTAM-seq on mESCs,” and FIG.22). Replicate-1 was sequenced at a deeper level, resulting in 65,853 hits, 58,456 of which showed exposed methylation levels higher than 10% (FIG.3A, and FIG.23). m ⁶ A sites in mESCs again revealed a clear DRACH motif (84%) and were enriched around stop codons (FIG. 3B). 30,191 (52%) eTAM-seq- captured m ⁶ A sites overlapped with 6,097 (74%) MeRIP-seq peaks (see e.g., Zhang, Z. et al., 2021), wherein one MeRIP-seq peak covers 5.0 m ⁶ A sites averagely (median: 4, FIG.3C). A control mESC transcriptome was also prepared using FTO treatment. eTAM-seq (mESC/FTO) captured 47,966 sites with exposed methylation levels ≥ 10%.91% of eTAM-seq (mESC/FTO) hits overlapped with eTAM-seq (mESC/IVT) hits and report highly correlated methylation levels (Pearson’s r = 0.995, FIG.3D). [0315] Methylation of pluripotency transcription factors, Nanog, Sox2, and Klf4, is a hallmark of the naïve pluripotent state (see e.g., Guela, S. et al., 2015; and Batista, P.J. et al., 2014). and was clearly captured by eTAM-seq (FIG. 3E). In contrast, a single lowly methylated site persists across two eTAM-seq datasets in Oct4 (22.7% ±7.6%, FIG. 24), another key pluripotency transcription factor and a critical component of the Yamanaka cocktail (OSKM factors) (see e.g., Batista, P.J. et al., 2014; and Takahashi, K. & Yamanaka, S.2016). The absence of significant methylation in Oct4 was consistent with previous reports that the abundance of Oct4 mRNA was not significantly impacted by deleting Mettl3, the key m ⁶ A writer gene (see e.g., Guela, S. et al., 2015; and Batista, P.J. et al., 2014). The modification levels of the 11, 18, and 22 m ⁶ A sites in Nanog, Sox2, and Klf4, respectively, were quantified

137741474.1 - 107 - and 1, 7, and 7 of these sites were found to be methylated close to completion (> 80%, FIG. 3E), respectively. The density and stoichiometries of methylation in these transcripts have never been reported; therefore, the fidelity of eTAM-seq was evaluated by comparing the results with MeRIP-seq data (see e.g., Zhang, Z. et al., 2021). Positions and fractions of m ⁶ A detected by eTAM-seq were recapitulated in MeRIP-seq peak clusters (FIG.3E). Collectively, these results showed that eTAM-seq can be applied to mESCs for detection of m ⁶ A sites in mRNAs that are essential for embryogenesis. [0316] Next mESCs (Mettl3 ^flox/flox ) were generated. In these cells, Mettl3 was knocked out by small molecule-induced Cre recombination, and eTAM-seq was applied to mRNA isolated from Mettl3 knockout (Mettl3 KO) mESCs. As a control, mESCs wherein Cre-mediated Mettl3 KO was not induced (ctrl mESCs) were included, i.e., cells with intact m ⁶ A deposition machinery. Western blot confirmed strong depletion of METTL3 in Mettl3 KO mESCs (FIG. 25A). eTAM-seq captured 10,250 m ⁶ A sites in ctrl mESCs, 9,759 (95%) of which overlap with those identified in wildtype mESCs (FIG. 25B). In contrast, only 2,737 m ⁶ A sites were identified in Mettl3 KO mESCs (FIG. 25C). A moderate overlap was observed among m ⁶ A sites detected in ctrl and Mettl3 KO mESCs (1,922, 19% and 70% of ctrl and Mettl3 KO samples, respectively, FIG. 4A). m ⁶ A sites detected in ctrl mESCs were almost exclusively found in DRACH motifs (9,602, 94%), whereas 1,900 m ⁶ A sites (69%) arising in Mettl3 KO mESCs could be attributed to this consensus motif (FIG.4B). The median methylation levels in ctrl and Mettl3 KO mESCs were 55.6% and 20.0%, respectively (FIG. 4C). Mettl3 KO- specific m ⁶ A sites (815) showed low methylation levels (median: 17.8%) and DRACH occupancy (16%). Global reduction in methylation was clearly observed upon METTL3 depletion (FIGs. 4D and 4E). It can therefore be concluded that METTL3 is the dominant methyltransferase responsible for m ⁶ A installation on mRNA in mESCs. These results also unequivocally supported the fidelity of eTAM-seq. Example 5: m6A sites are unevenly distributed in the transcriptome and impact mRNA stability [0317] The inventors noted that m ⁶ A signals tended to cluster in the analyses of individual HeLa and mESC transcripts (FIG. 2I, FIG. 3E, and FIG. 21). To investigate whether such trends hold true across the HeLa transcriptome, permutation tests were performed under a null hypothesis that the distribution of m ⁶ A sites was uniform. The inventors sampled 10 times for possible locations of m ⁶ A (69,834) across all evaluated A sites 1) with no context constraint, or 2) forcing m ⁶ A-carrying 5-nt motifs to match the frequencies observed in eTAM-seq.

137741474.1 - 108 - Median gaps between two neighboring m ⁶ A sites under these two simulation criteria are 296 and 343 nt, larger than 62 nt observed in eTAM-seq (FIG.26), supporting the hypothesis that m ⁶ A has a tendency to cluster. [0318] Previous reports have linked m ⁶ A with mRNA degradation (see e.g., Wang, X. et al., 2014; and Lee, Y. et al., 2020). Thus, how the total methylation load on a transcript would impact the transcripts stability was examined (HeLa mRNA half-life dataset: GSE49339 (see e.g., Wang, X. et al., 2014)). Methylation levels for individual HeLa mRNA were summed and categorized into three groups – high, medium, and low methylation, each harboring 1,593 transcripts. A group of 1,758 unmethylated transcripts was included as a reference. The median half-lives for mRNA bearing high, medium, low, and no methylation were 4.8, 5.9, 6.7, and 6.8 h, respectively, in HeLa cells treated with control siRNA, and were extended to 8.2, 9.4, 8.9, and 9.4 h when METTL3 was knocked down (FIG. 4F), respectively. While highly methylated mRNA showed an average 29% reduction in half-life compared to unmethylated mRNA in control cells (4.8 versus 6.8 h), the difference largely diminished in cells with impaired methyltransferase activity, supporting a central role of m ⁶ A in mediating mRNA decay. [0319] YTHDF (YTH domain-containing family) proteins, a major family of m ⁶ A readers, interact with m ⁶ A-bearing transcripts to exert their regulatory function. Among them, YTHDF2 was reported to drive the degradation of m ⁶ A-modified transcripts (see e.g., Wang, X. et al., 2014; Wang, X. et al., 2015; Shi, H. et al., 2017; and Shi, H. et al., 2019). Indeed, the differences in half-life for mRNA of different methylation loads were largely reduced in cells treated with YTHDF2-targeting siRNA (FIG.27), confirming YTHDF2 as a core regulator for the stability of m ⁶ A-modified mRNA. Example 6: Site-specific, deep sequencing-free m6A quantification with limited input [0320] eTAM-seq offers a straightforward approach for site-specific detection and quantification of m ⁶ A, similar to bisulfite sequencing that has been widely applied in assessing DNA methylation at promoter and enhancer sites. A streamlined protocol for site-specific, deep sequencing-free m ⁶ A quantification was generated: a) fragmentation, b) global A deamination, c) reverse transcription, d) polymerase chain reaction, and e) Sanger sequencing (FIG.5A and FIG.28A). This eTAM-Sanger protocol was applied to 18 m ⁶ A sites (methylation level: 60- 99%, median: 83%) in HeLa transcripts of high, medium, and low abundances (Tables 4 and 5). EditR (see e.g., Kluesner, M.G. et al., 2018), a program developed to analyze base editing outcomes from Sanger sequencing data, was employed to quantify the A and G levels in Sanger

137741474.1 - 109 - traces (see methods sections “site-specific m ⁶ A quantification…”). To assess fidelity of m ⁶ A quantification by Sanger sequencing, small-scale deep sequencing was simultaneously utilized for analyses, with 10k reads allocated to each sample. [0321] Methylation levels quantified by whole-transcriptome eTAM-seq were used as references to evaluate eTAM-Sanger results. For all 18 inspected sites, eTAM-Sanger reported methylation levels within small deviations from whole-transcriptome eTAM-seq estimates (0.7%-20%, median deviation: 5.9%, FIG. 5B and FIGs. 28B-28F). Methylation stoichiometries were detected within ^ 10% for 15 of the 18 sites. It is noted that it was often challenging to quantify low levels of G signals in Sanger traces, which was a major source of error in eTAM-Sanger. Indeed, amplicon deep sequencing, which is more suited to quantifying mixed base signals, further improved the accuracy, delivering methylation estimates with a median deviation of 2.4% (0.3%-7.2%) from whole-transcriptome eTAM-seq results (FIG.5B and FIGs.28B-28F). These results supported the conclusion that eTAM-seq supports faithful m ⁶ A quantification regardless of the readout method, e.g., Sanger sequencing, amplicon deep sequencing, or whole-transcriptome sequencing. [0322] Control samples are not a prerequisite for site-specific m ⁶ A quantification. Methylation stoichiometries for JUNB_1352, GRWD1_1487, H2AFX_1331, and PPIB_823 were determined in the absence of IVT samples (FIG. 28E and 28F). Importantly, m ⁶ A quantification was achieved for low abundance mRNA, such as CLCN3 (Transcripts Per Million, TPM: 13, FIG. 5B) (see e.g., Liu, J. et al., 2014), confirming that, unlike probe- mediated detection mechanisms which are sensitive towards abundant transcripts, eTAM- Sanger quantified m ⁶ A in transcripts spanning a broad range of abundances (TPM 13-1,807). [0323] To assess the detection limit of eTAM-Sanger, two m6A-bearing sites in ACTB and EIF2A from diluted cDNA samples were amplified. PCR products from cDNA corresponding to 500 pg, 50 pg, and 5 pg starting mRNA for both sites (FIG.29) were successfully obtained, which produced Sanger traces almost identical to those started with 5 ng mRNA (FIG. 5C). eTAM-Sanger was then exploited for site-specific m6A quantification from total RNA. Although rRNA was not depleted in these samples, which is expected to compromise enzymatic treatment, reverse transcription, and target site amplification, short DNA fragments covering ACTB_1427 and EIF2A_994 with 25 ng, 2.5 ng, and 250 pg total RNA (FIG. 29) were successfully obtained, which corresponds to approximately 1,000, 100, and 10 cells, respectively. These amplicons generated Sanger traces similar to cDNA synthesized from 5 ng mRNA (FIG.5D). The methylation levels were quantified to be 70-78% for ACTB_1427 and

137741474.1 - 110 - 84-94% for EIF2A_994, within 7% and 9% differences from the levels reported by whole- transcriptome eTAM-seq. Collectively, these results showcase technologies suitable for reliable, deep sequencing-free m6A quantification with ultra-low sample input. Example 7: m ⁶ A quantification with TadA8r [0324] m ⁶ A modifications were quantitatively analyzed using TadA8r (SEQ ID NO: 25). As shown in FIG. 30A, TadA8r successfully deaminated non-modified adenosines in target RNA sequences (e.g., ACTB, SLC7A5, and JUNB transcripts) comprising m ⁶ A modifications. But, as shown in FIG.30B, TadA8r deaminated non-modified A, but not m ⁶ A at nucleotide 52 of exemplary in vitro deamination RNA2, aka Y1D, SEQ ID NOs: 39 and 160. Shown in FIG. 30C, TadA8r effectively deaminated adenosines in RNA that included relatively complex secondary structures, e.g., exemplary in vitro deamination RNA3, aka Y1G, SEQ ID NO: 40. In some conditions, TadA8r treatment resulted in lower levels of cytosine deamination relative to treatment other TadA enzymes (e.g., TadA8.20). As shown in FIG. 30D, TadA8r and TadA8.20 effectively deaminated adenosines in ACTB, CXCR4, SLC7A5, and JUNB target RNA molecules. [0325] In FIG.30E, the global TadA mediated deamination rate was determined on rRNA after 1X, 2X, and 3X TadA treatments (without FTO treatment) or FTO treatment followed by 3X TadA treatment. Each treatment was done utilizing TadA8r in 1X reaction buffer, followed by sequencing library creation using a Takara SMARTer v3 pico library construction kit. A filter to remove reads that had less than 50% deamination rates overall could be applied. In some conditions, filtering did not result in significant differences in global deamination rates, except for in samples that included FTO treatment, potentially due to sequencing bias introduced when reads were longer. In FIG.30F, the global deamination rate on mRNA after 1X, 2X, and 3X TadA treatments (without FTO treatment) or FTO treatment followed by 3X TadA treatment was determined. The first four treatments were performed using TadA8r in 1X reaction buffer, followed by sequencing library creation using a Takara SMARTer v3 pico library construction kit. A 50% deamination rate filter could be applied as described in Xiao, YL., Liu, S., Ge, R. et al. Transcriptome-wide profiling and quantification of N6- methyladenosine by enzyme-assisted adenosine deamination. Nat Biotechnol 41, 993–1003 (2023), which is incorporated herein by reference in its entirety; the 3X TadA Ligation library was prepared using said published protocol. [0326] As shown in FIGs. 31A-31D, TadA enzymes and associated methods described herein were found to be compatible with numerous commercial sequencing library construction

137741474.1 - 111 - kits. As shown herein, the inventors achieved global deamination rates in nucleic acids from biological samples of at least as high as 85% in rRNA and at least as high as 75% in mRNA after 1X TadA (e.g., TadA8r) treatment. The reactions were found reach completion within three or less treatment TadA contacting (e.g., treatment) repeats, with extremely high overall deamination rates (e.g., greater than or equal to 98%). Example 8: eTAM-seq in DNA [0327] Mapping 6mA in mammalian gDNA can be considered much more challenging than mapping m ⁶ A in mammalian RNA, although both methodologies can be based on deamination of adenine base (A) to inosine (I) to generate A-to-G mutation at unmodified A sites while modified sites, such as 6mA/m ⁶ A resists deamination and still read as A in sequencing. As the most abundant RNA modification in mammalian mRNA (m ⁶ A/A ratio = ~0.3-0.5%), there exists tens of thousands of m ⁶ A sites and many of them have been reported to occur at high fractions. In contrast, the 6mA present in higher eukaryotes has been reported to have much lower abundances (e.g., 6mA/A ratio 0.00001-0.001% ppm) (see e.g., O’Brown, Z.K. et al., 2019; Liu, X et la., 2021; and Lyu, C. et la., 2022; each of which are incorporated by reference herein in their entirety for the purposes described here) and therefore it is more likely to generate false positives and/or for said false positives to not be recognized as such. To accurately map the real 6mA sites present in higher eukaryotes gDNA, ultrahigh deamination efficiency is require to reduce false positives. To this end, the inventors investigated several evolved TadA mutants and found that the mutant TadA8r showed very high deamination efficiency for dA in DNA, higher than its efficiency for rA in RNA. [0328] The inventors utilized a synthetic 10 mer DNA oligo (TTTTTdATTTT) (SEQ ID NO: 165) as model to test the deamination efficiency and found that TadA8r treatment quantitatively converted dA to dI (FIG. 33A) while the corresponding 6mA (TTTTT6mATTTT) (SEQ ID NO: 161) oligo did not undergo any deamination (FIG. 32B). Further, the inventors found that the DNA adenine deamination could occur within 10 minutes of TadA8r treatment (FIG. 33A), in contrast, the deamination efficiency of rA in the corresponding 10 mer RNA oligo (UUUUUrAUUUU) (SEQ ID NO: 162) was slower, with no obvious deamination observed after 10 minutes, but with full deamination being observed within 16 hours (FIG.33B). This high deamination efficiency of dA in DNA can facilitate very high A-to-I conversion rates, which can significantly reduce the background and false positive rates. Provided in FIG. 34 is a schematic illustrating eTAM-seq in DNA. To further demonstrate the principle works well in longer DNA, the inventors synthesized a 100 bp DNA

137741474.1 - 112 - oligo containing multiple A sites and one 6mA site (SEQ ID NO: 163). After TadA8r treatment and PCR amplification, Sanger sequencing results showed that all the A sites were read as G while the 6mA sites remained to be read as A (FIGs.35-36), results which were also observed when TadA8.20 or TadA8e were assayed (FIG.37). [0329] To further evaluate the deamination efficiency, libraries were constructed in triplicate starting with fragmented lambda DNA from both DAM ⁺ and DAM- strains with the synthetic ssDNA oligo containing a 6mA modification as spike-in (SEQ ID NO: 163), the results showed non-biased A conversion at all motifs analyzed (FIGs.38-39, and 41). Provided in FIG.40 is a schematic depicting a statistical model for high confidence 6mA site detection in DNA. Given that deamination of dA by treatment of TadA8r worked on ssDNA, the inventors added NaOH to denature the dsDNA before TadA8r, and added DMSO in the reaction to prevent dsDNA renaturation. In addition, the inventors adopted a ramping temperature program to allow better denaturation of dsDNA to push the deamination reaction to go to completion as much as possible. After one or two rounds of TadA8r treatment and libraries construction (e.g., using a Swift kit), sequencing results showed two rounds of TadA8r treatment gave lower background than one round treatment, and control FTO treatment effectively demethylated the 6mA site in ssDNA such that the detected fraction at the known 6mA site decreased from 100% to <20%. Importantly, no obvious bias of the deamination efficiency along the sequence was observed (FIG.39). In addition, the inventors also analyzed all the A sites in lambda DNA. This data also showed very low unconverted rate (less than ~0.2%) efficiency on average (FIG.38), and the range of the unconverted rate along the lambda DNA sequence was between 0-4%, suggesting that no false positives were generated with a > 4% cut off . These results were highly reproducible across replicates, suggesting that DNA eTAM-seq methods were highly sensitive and robust. [0330] 6mA deposition in E. coli, as revealed by SMRT sequencing (see e.g., Fang, G. et al., 2012), is motif-driven and is binary, e.g., a site is either unmethylated or 100% methylated. To benchmark the performance of eTAM-seq, we chose DH5 ^ (NEB C2992I), a derivative of E. coli K-12 that carries two N ⁶ -adenine methyltransferases (see e.g. Kelleher, J.E. & Raleigh, E.A.1994), DNA adenine methyltransferase (Dam) responsible for methylating 5'-GATC, and EcoKI, a Type I restriction-modification enzyme that recognizes both 5'-AACN ₆ GTGC (SEQ ID NO: 166) and 5'-GCACN ₆ GTT (SEQ ID NO: 167) (see e.g., Vince, T., et al., 2003). To further evaluate the fidelity of eTAM-seq, the inventors included dam ^– /dcm ^– E. coli (NEB

137741474.1 - 113 - C2925I) in which all GATC sites remained unmethylated. The dam ⁺ and dam ^– strains are referred to herein as E. coli (dam ⁺ ) and E. coli (dam ^– ). [0331] Genomic DNA extracted from both strains were subjected to eTAM-seq analysis. For dam ⁺ samples, a very strong GATC motif was found in all replicates, consistent with the conserved motif reported for DAM ⁺ E. coli DNA. The overlap of the detected 6mA sites among three replicated reached 99% while that for the non-6mA sites reached 99.98%. Both 6mA and non-6mA sites were detected with minimal false positives (FIGs.45A-45B). A sites in E. coli (dam ⁺ ) segregated into two distinct populations based on their persistent A rates (Aprst) in eTAM-seq (FIG. 42): one grouping essentially at 0% and the other approaching 100%. Analysis of the TadA8r-resistant population revealed three sequence motifs: GATC, AACN6GTGC (SEQ ID NO: 166), and GCACN6GTT (SEQ ID NO: 167). Importantly, these putative 6mA sites covered all (or at least the vast majority of) GATC, AACN6GTGC, and GCACN ₆ GTT sites in E. coli (dam ⁺ ) genome, suggesting that these sequence motifs were both essential and sufficient for methylation deposition by Dam and EcoKI. Treatment with control FTO removed the segregation between these two groups (FIG.43), providing confirmation of accurate 6mA calling. [0332] The inventors then estimated the false discovery rate (FDR), false positive (FP), and false negative (FN) rates for eTAM-seq (FIGs. 42-44), and found them to be minimal, based on the premise that all N ⁶ -deoxyadenosine methylation in E. coli was confined to Dam- and EcoKI-target sites. Meanwhile, the inventors reanalyzed previously obtained SMRT sequencing data from E. coli C227-11 (FIG. 44) (see e.g., Fang, G. et al., 2012). With an optimized cutoff, SMRT sequencing delivered a false discovery rate (FDR) of 2.84%, fold scale higher than that of eTAM-seq FDR (FIG.42). Biological replicates of eTAM-seq showed 99% overlap and highly consistent methylation levels (FIG.45A-45B). Collectively, eTAM- seq faithfully detect N ⁶ -deoxyadenosine methylation in the E. coli genome. The persistent A signals in E. coli (dam ^– ) were markedly lower than those in E. coli (dam ⁺ ). Although A sites could still be sorted into two distinct populations based on their persistent A rates in eTAM- seq, the cluster converging towards 100% was much smaller and solely comprised AACN ₆ GTGC and GCACN ₆ GTT sites. Collectively, these results demonstrated that eTAM- seq was both sensitive and faithful in detection of DNA 6mA sites. Regarding the published 6mA data in E. coli using SMRT-seq, even though the false positive rate could be as low as 0.0005, this data still contained at least two major drawbacks: 1) the applied algorithm helped to lower False signal rate, but if looking at the true negative and true positive signals, they were less distinguishable, as the data presented here shows.2) SMRT-seq applied machine learning

137741474.1 - 114 - which was indeed effective for detecting 6mA at known motifs, but it lacks sensitivity at unknown motifs. In contrast, the methods provided herein were able to convert 6mA at the main motif GATC as well as at less popular motifs such as the two listed here, and thus enabled accurate genome-wide 6mA detection. [0333] Control FTO treatment was then applied to genomic DNA extracted from both E. coli strains (FIG.42). Owing to FTO-induced DNA degradation, the inventors limited TadA8r treatment in eTAM-seq to a single round instead of two to maintain library integrity. Consequently, the global deamination rate in FTO-treated DNA was slightly lower than in untreated samples during eTAM-seq. Nevertheless, this minor deficiency in global deamination should not impact 6mA detection and quantification, as only A sites deaminated to greater levels in FTO-treated samples were recognized as 6mA sites. [0334] FTO removed the majority of 6mA in the spike-in ssDNA oligo (FIG. 42), suggesting that the FTO-treated samples were effectively demethylated. Indeed, in eTAM-seq of FTO-treated E. coli (dam ⁺ ) samples, not only were unmodified A sites clustered near 0%, but the 6mA population also showed a drastic reduction in persistent A rates. The robust response to FTO corroborated the inventors assignment of 6mA sites. Most A sites in E. coli (dam ^– ) did not respond to FTO in eTAM-seq, with the exception of the EcoKI-target sites. Several A sites with >50% persistent A signals in eTAM-seq failed to respond to FTO, indicating that these were likely structured sites inaccessible to TadA8r, instead of genuine methylation sites. Upon exclusion of these false-positive signals identified through FTO treatment, the inventors further reduced the eTAM-seq FDR. These results underlined the value of utilizing controls, such as FTO, when performing 6mA detection/mapping. [0335] As described above, eTAM-seq can be utilized for quantitative detection of 6mA in prokaryotic samples (e.g., E. coli). Owing to the binary nature of methylation in E. coli, eTAM- seq signals were clearly separated, thus simplifying detection. Nevertheless, methylation may occur at less pronounced levels in other organisms. If benchmarked exclusively with high- methylation samples, eTAM-seq may not yield optimal results in these alternative scenarios. To address this concern, the inventors considered potential enrichment strategies (FIG.46) and prepared artificial E. coli genomes of varied methylation levels—20%, 10%, 5%, and 2.5%— by mixing genomic DNA from E. coli (dam ⁺ ) and E. coli (dam ^– ), and then benchmarked eTAM-seq using these artificial E. coli genomes as a mimic for DNA of moderate and low methylation levels. Given that E. coli (dam ⁺ ) and E. coli (dam ^– ) had different genetic backgrounds, the inventors masked regions of sequence variations from analysis. The inventors consider GATC sites (or GATC sites showing 100% persist A in unmixed DNA) as true

137741474.1 - 115 - methylation sites and other non-Dam, non-EcoKI A sites as unmethylated sites (FIG. 47). A statistical model was developed for 6mA detection that took into account read depths, background persistent A signals, and behaviors across biological replicates as well as the FTO control. Sensitivity was defined as the capture rate of true methylation sites (1 – FN) and fidelity as the frequency for correct classification of unmethylated sites (1 – FP). [0336] The median persistent A rates detected at true methylation sites in samples of 20% and 10% methylation were quantitatively measured using eTAM-seq (FIGs.48-49). Similar to other sequencing-based detection methods, more reads were needed to detect and quantify sites of moderate and low methylation sites in eTAM-seq. Nevertheless, the overall performance metrics for the 20% and 10% methylation samples were comparable to that of the genomic DNA derived from E. coli (dam ⁺ ) (100% methylation). Complete capture of 6mA was more challenging in samples of 5% and 2.5% methylation with moderate sequencing depths. Importantly, the FP rates remained low for samples of 5% and 2.5% methylation, as the statistical model rarely misidentified unmodified A sites as 6mA sites. The inventors therefore concluded that eTAM-seq can be extended to samples with moderate and low levels of DNA methylation. [0337] Increasing the sequencing depth can effectively reduce the FN rate (FIG. 50). Persistent A signals arising from 6mA sites that eluded detection showed a distribution distinct from that of unmodified A sites, indicating that additional optimization of the statistical model potentially could further reduce FN detection. Collectively, eTAM-seq quantitatively captured all true 6mA sites in samples of >10% methylation, and a majority of true 6mA sites in samples of >2.5% methylation. [0338] eTAM-seq can be streamlined with antibody enrichment, a particularly useful approach when detection takes precedence over quantification. As a demonstration, we immunoprecipitated E. coli genomes of 5% and 2.5% methylation using 6mA antibodies and subjected the resulting DNA to eTAM-seq (FIG.51). A significant fraction of true methylation sites approached full methylation (>95%), implying successful enrichment of 6mA-containing DNA. However, estimated methylation levels varied considerably across sites, indicating that site-specific enrichment was likely influenced by factors such as sequence context and proximity to other 6mA sites, which might better recruit antibodies due to a higher local density of 6mA. Despite these variations, persistent A rates remained low at unmethylated sites, suggesting that antibody enrichment did not compromise the fidelity of eTAM-seq. Significant enrichment often coincided with A-rich sequences, indicating potential non-specific binding of the 6mA antibody. This observation underscored the importance of careful inspection of 6mA

137741474.1 - 116 - immunoprecipitation data before calling 6mA status. Collectively, the inventors demonstrated that eTAM-seq, alone or when combined with 6mA immunoprecipitation, facilitated the detection of genuine 6mA sites in samples of 2.5% methylation while maintaining a low FDR. ********* [0339] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. All publications described herein are specifically incorporated by reference for all purposes. REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. 1. Frye, M., Jaffrey, S.R., Pan, T., Rechavi, G. & Suzuki, T. RNA modifications: what have we learned and where are we headed? Nat. Rev. Genet.17, 365-372 (2016). 2. Peer, E., Rechavi, G. & Dominissini, D. Epitranscriptomics: regulation of mRNA metabolism through modifications. Curr. Opin. Chem. Biol.41, 93-98 (2017). 3. Nachtergaele, S. & He, C. Chemical modifications in the life of an mRNA transcript. Annu. Rev. Genet.52, 349-372 (2018). 4. Jiang, X. et al. The role of m6A modification in the biological functions and diseases. Signal Transduct. Target. Ther.6, 74 (2021). 5. He, P.C. & He, C. m(6) A RNA methylation: from mechanisms to therapeutic potential. EMBO J.40, e105977 (2021).

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