Method and kit for detecting n6-methyladenine

Abstract

The present application relates to the field of molecular biology, and in particular to the field of nucleic acid detection and analysis. In particular, provided in the present application is a method for detecting N6-methyladenine in a nucleic acid molecule. In addition, further provided in the present application is a kit, wherein the kit can be used in implementing the method of the present application.

Description

Methods and kits for the detection of N6-methyladenine Technical Field

[0001] This application relates to the field of molecular biology, particularly the field of nucleic acid detection and analysis. Specifically, this application provides a method for detecting N6-methyladenine in nucleic acid molecules. Furthermore, this application provides a kit for implementing the method of this application. Background Technology

[0002] N6-methyladenosine (m6A) is the most prevalent and widely studied chemical modification in eukaryotic mRNA, playing a crucial role in various biological processes and giving rise to the field of epitranscriptomics. The ubiquity, dynamics, and functions of m6A are controlled by its regulatory effectors, including methyltransferases, demethylases, and binding proteins (referred to as "writers," "erasers," and "readers," respectively). The identification of these effectors has spurred rapid research into m6A, elucidating its key roles in RNA metabolism, such as alternative splicing, localization, translation efficiency, and stability. Meanwhile, recent advances in understanding the biological consequences of m6A in various physiological and pathological processes have opened new avenues for research in epitranscriptomics.

[0003] Since 2012, advances in transcriptome-wide sequencing methods for m6A detection have greatly facilitated the elucidation of the functional roles and regulatory mechanisms of m6A modifications. While these methods provide detailed descriptions of the methylation landscape and its dynamics across diverse biological environments, significant limitations remain. Antibody-dependent methods, such as m6A-seq, MeRIP, and miCLIP, are highly dependent on antibody specificity and require large amounts of input RNA. Enzyme-dependent techniques, including DART-seq, MAZTER-seq, and m6A-REF-seq, can produce false positives or false negatives due to inconsistent enzyme activity. Similarly, enzyme-assisted chemical labeling methods, such as m6A-SEAL-seq and m6A-SAC-seq, are susceptible to influences from RNA structure and sequence. In general, these technologies all have limitations, most importantly, they share the common drawback of not being able to provide absolute quantitative detection of m6A modifications. Third-generation sequencing methods, such as nanopore sequencing, can directly detect m6A at the single-molecule level, but may face challenges such as high error rates, complex data analysis, and high costs. Summary of the Invention

[0004] The advent of GLORI and eTAM-seq technologies has significantly expanded the scope of m6A quantification analysis across the transcriptome. These quantitative methods greatly enhance the ability to elucidate the impact of dynamic changes in m6A on stimulus response, cellular response, and pathological states. Both methods distinguish m6A from unmethylated adenosine (A) through selective deamination, converting unmethylated A to inosine (I), while m6A remains unchanged. eTAM-seq uses a highly active tRNA adenosine deaminase variant (TadA), whose specificity may be affected by local RNA structure and sequence. Unlike eTAM-seq, GLORI utilizes a glyoxal-catalyzed, nitrite-mediated chemical reaction to achieve efficient, specific, and unbiased deamination of A, providing more accurate m6A detection and quantification. Despite the technological advantages of GLORI, the inventors of this application have found that GLORI (referred to herein as GLORI 1.0) causes severe RNA degradation, limiting its application in low-volume input RNA. For example, GLORI 1.0 can measure the m6A methyl group of approximately 100,000 cells; however, biological research often only obtains a limited number of cells (one to several orders of magnitude less), which is beyond the capabilities of the existing GLORI method (GLORI 1.0).

[0005] Through extensive research, the inventors of this application have developed two novel, mild and ultra-fast GLORI technologies (GLORI 2.0 and GLORI 3.0), which utilize a novel one-step deamination reaction to achieve high A to I conversion while maintaining RNA integrity. Experimental verification shows that GLORI 2.0 significantly improves the sensitivity and robustness of m6A quantification in transcriptome-wide and site-specific detection, especially for low-abundance transcripts. GLORI 3.0, building upon GLORI 2.0, introduces a reverse transcription (RT) silencing vector RNA to minimize RNA loss and achieve m6A quantification under ultra-low RNA input. In this application, the inventors successfully quantified the m6A methyl group sequence in the cytoplasm and synaptic regions of the dorsal hippocampus of a single mouse using GLORI 3.0.

[0006] In summary, the updated GLORI versions provided in this application, GLORI 2.0 and GLORI 3.0, enable the widespread application of m6A quantification in epitranscriptomics research.

[0007] N6-methyladenine detection method

[0008] Therefore, in one aspect, this application provides a method for detecting N6-methyladenine in nucleic acid molecules, comprising the following steps:

[0009] (1) Provide a sample to be tested, wherein the sample to be tested contains the nucleic acid molecule to be tested;

[0010] (2) In the presence of carbonyl compounds, the nucleic acid molecules to be tested react with nitrite, causing the adenine in the nucleic acid molecules to be tested to be converted into hypoxanthine;

[0011] (3) Detect the nucleic acid molecules obtained in the previous step;

[0012] Prior to step (2) (e.g., before the nucleic acid molecule to be tested reacts with nitrite), the amino group of guanine in the nucleic acid molecule to be tested is not protected.

[0013] It will be readily understood by those skilled in the art that the statement "the amino group of guanine is not protected" is intended to describe a state in which the guanine in the nucleic acid molecule to be tested is not masked for chemical reactivity (e.g., the amino group of guanine is in a reactive state). In some embodiments, the statement "the amino group of guanine is not protected" is intended to indicate that the guanine in the nucleic acid molecule to be tested has not formed an adduct with a carbonyl compound.

[0014] In some embodiments, in step (2), the nucleic acid molecule to be tested is reacted with nitrite in an acidic environment (e.g., a weakly acidic environment, such as at pH 4.0-6.5, 4.0-4.5, 4.0-6.0, 4.5-6.0, or 5.0-6.0) and in the presence of the carbonyl compound.

[0015] Those skilled in the art will readily understand that the provision of an acidic environment in the reaction system can be achieved through a variety of methods commonly used in the art. In some embodiments of this application, the acidic environment (e.g., a weakly acidic environment) required for step (2) is provided by using acids (including but not limited to boric acid, hydrochloric acid, and acetic acid); for example, the pH of the reaction system in step (2) is adjusted to 4.0-6.5, 4.0-4.5, 4.0-6.0, 4.5-6.0, or 5.0-6.0 by using acids (including but not limited to boric acid, hydrochloric acid, and acetic acid).

[0016] Based on the disclosure of this application, those skilled in the art will readily understand that the technical solutions provided in this application (GLORI 2.0, GLORI 3.0) omit the additional guanosine protection step compared to GLORI 1.0, thus effectively reducing RNA degradation. In GLORI 1.0, before contacting RNA with nitrite to undergo a nitrosation reaction to achieve Ato I conversion, the guanosine in the RNA to be treated is additionally protected (e.g., by reacting a carbonyl compound with the guanosine to form a guanosine adduct). Subsequently, the guanosine-protected sample RNA is contacted with nitrite and a carbonyl compound to achieve Ato I conversion in the sample RNA. Therefore, it can be understood that in GLORI 1.0, before the nitrosation reaction for A to I conversion, the guanosine in the sample RNA molecule is in a protected state (e.g., existing in the form of an adduct). The technical solutions of this application (GLORI 2.0, GLORI 3.0) omit the additional guanosine protection step. Without protecting the guanosine, the sample RNA molecules are contacted with nitrite and carbonyl compounds to carry out A to I conversion. Therefore, it can be understood that in the technical solutions of this application (GLORI 2.0, GLORI 3.0), the guanosine in the sample RNA molecules is in an unprotected state before the nitrosation reaction of A to I conversion.

[0017] It should also be noted that, since this application uses carbonyl compounds to catalyze the A to I conversion reaction, carbonyl compounds are present in the reaction system of step (2) of this application. Therefore, in addition to the A to I conversion in the nucleic acid molecule to be tested, some guanosine of the nucleic acid molecule to be tested may also form guanosine adducts with carbonyl compounds in the presence of carbonyl compounds (for example, in an embodiment where glyoxal is present in the reaction system, some guanosine of the nucleic acid molecule to be tested will form N-type adducts with glyoxal). 1 N 2 -Dihydroxyguanosine). Therefore, after step (2), it is significantly advantageous to restore any guanosine adducts that may be present in the product nucleic acid molecule to guanosine.

[0018] In some embodiments, after step (2) and before step (3), the method further includes converting the guanosine adduct in the nucleic acid molecule of the product of step (2) into guanosine.

[0019] In some embodiments, the nucleic acid molecule to be tested is RNA, DNA, or a DNA / RNA hybrid. In some embodiments, the method includes converting the guanosine adduct in the nucleic acid molecule of the product from step (2) to guanosine by heat treatment under alkaline conditions. In some embodiments, the guanosine adduct in the nucleic acid molecule of the product from step (2) is converted to guanosine by heat treatment in a formamide-containing triethylamine acetate (TEAA) buffer at pH 8-9 or in a dimethyl sulfoxide (DMSO)-containing phosphate buffer at pH 7.1-8. In some embodiments, the guanosine adduct in the nucleic acid molecule of the product from step (2) is converted to guanosine by heat treatment in a 45-50 v / v% formamide-containing triethylamine acetate (TEAA) buffer at pH 8-9 or in a 40-60 v / v% dimethyl sulfoxide (DMSO)-containing phosphate buffer at pH 7.1-8.

[0020] In some embodiments, the heat treatment is: a) treating the nucleic acid molecules of the product from step (2) at a temperature of 80–95°C for 5–30 min (e.g., 5–10 min), or b) treating the nucleic acid molecules of the product from step (2) at a temperature of 60–70°C for 1–3 h (e.g., 1.5–2.5 h, about 2 h).

[0021] In some embodiments, the guanosine adduct in the nucleic acid molecule of the product from step (2) is converted to guanosine by heat treatment as described in a), in a triethylamine acetate (TEAA) buffer containing approximately 47.5 v / v% formamide at a pH of approximately 8.6, or...

[0022] The guanosine adduct in the nucleic acid molecule of the product from step (2) is converted to guanosine by heat treatment as described in b) in a phosphate buffer containing about 50 v / v% dimethyl sulfoxide (DMSO) at a pH of about 7.4.

[0023] In some embodiments, the nucleic acid molecule to be tested is RNA or a DNA / RNA hybrid. In some embodiments, the guanosine adduct in the nucleic acid molecule of the product from step (2) is converted to guanosine under alkaline conditions. In some embodiments, the guanosine adduct in the nucleic acid molecule of the product from step (2) is converted to guanosine by heat treatment under alkaline conditions. In some embodiments, the guanosine adduct in the nucleic acid molecule of the product from step (2) is converted to guanosine by heat treatment in a formamide-containing triethylamine acetate (TEAA) buffer at pH 8-9. In some embodiments, a first reaction is carried out by heat treatment in a formamide-containing triethylamine acetate (TEAA) buffer at pH 8-9, and a second reaction is carried out by heat treatment in a dimethyl sulfoxide (DMSO)-containing phosphate buffer at pH 7.1-8 to convert the guanosine adduct in the nucleic acid molecule of the product from step (2) to guanosine. In some embodiments, a primary reaction is carried out by heat treatment in a triethylamine acetate (TEAA) buffer containing 45-50 v / v% formamide at pH 8-9, and a secondary reaction is carried out by heat treatment in a phosphate buffer containing 40-60 v / v% dimethyl sulfoxide (DMSO) at pH 7.1-8, to convert the guanosine adduct in the nucleic acid molecule of the product from step (2) into guanosine.

[0024] In some embodiments, the heat treatment is:

[0025] a) Treat the nucleic acid molecules produced in step (2) at a temperature of 80–95°C for 5–30 min (e.g., 5–10 min), or,

[0026] b) Treat the nucleic acid molecules produced in step (2) at a temperature of 60–70°C for 1–3 h (e.g., 1.5–2.5 h, about 2 h).

[0027] In some embodiments, the guanosine adduct in the nucleic acid molecule of the product of step (2) is converted to guanosine by heat treatment as described in a) in a triethylamine acetate (TEAA) buffer containing about 47.5 v / v% formamide at a pH of about 8.6.

[0028] In some embodiments, a primary reaction is carried out in a triethylamine acetate (TEAA) buffer containing about 47.5 v / v% formamide at a pH of about 8.6 by the heat treatment described in a), and a secondary reaction is carried out in a phosphate buffer containing about 50 v / v% dimethyl sulfoxide (DMSO) at a pH of about 7.4 by the heat treatment described in b), to convert the guanosine adduct in the nucleic acid molecule of the product of step (2) into guanosine.

[0029] In some embodiments, the nucleic acid molecule to be tested is DNA or a DNA / RNA hybrid. In some embodiments, the guanosine adduct in the nucleic acid molecule of the product from step (2) is converted to guanosine under alkaline conditions. In some embodiments, the guanosine adduct in the nucleic acid molecule of the product from step (2) is converted to guanosine by heat treatment under alkaline conditions. In some embodiments, the guanosine adduct in the nucleic acid molecule of the product from step (2) is converted to guanosine by heat treatment in a formamide-containing triethylamine acetate (TEAA) buffer at pH 8-9. In some embodiments, the guanosine adduct in the nucleic acid molecule of the product from step (2) is converted to guanosine by heat treatment in a formamide-containing triethylamine acetate (TEAA) buffer at pH 8-9 at 45-50 v / v% formamide. In some embodiments, the heat treatment is: treating the nucleic acid molecule of the product from step (2) at a temperature of 80-95°C for 5-30 min (e.g., 5-10 min). In some embodiments, the guanosine adduct in the nucleic acid molecule of the product of step (2) is converted to guanosine by heat treatment as described in a) in a triethylamine acetate (TEAA) buffer containing about 47.5 v / v% formamide at a pH of about 8.6.

[0030] In some embodiments, the carbonyl compound is selected from compounds of formula I and any combination thereof.

[0031] Where R1 is an aldehyde group, C 1-3 Alkyl (e.g., methyl, ethyl, or n-propyl) or halogen-substituted C 1-3 Alkyl groups (e.g., trichloromethyl, trifluoromethyl), where R2 is H or C. 1-3 Alkyl (e.g., methyl, ethyl, or n-propyl), phenyl, or HC(=O)-CH(Br)-; or

[0032] R1 and R2 form cyclic structures with the carbonyl groups to which they are attached, such as ninhydrin.

[0033] In some embodiments, the carbonyl compound is selected from: glyoxal, 2,3-butanedione, ninhydrin, 2-bromomalondialdehyde, acetone aldehyde, trichloroacetaldehyde, phenylglyoxal, and any combination thereof.

[0034] In some embodiments, the carbonyl compound is selected from: glyoxal, 2-bromomalondialdehyde, 2,3-butanedione, phenylglyoxal, trichloroacetaldehyde, ninhydrin, and any combination thereof.

[0035] In some embodiments, the carbonyl compound is glyoxal.

[0036] In some implementations, in step (2), the nucleic acid molecule to be tested is reacted with nitrite in a solution containing the carbonyl compound.

[0037] In some embodiments, the solution is water, an aqueous solution of p-toluenesulfonic acid, an aqueous solution of phosphoric acid, 2-(N-morpholine)ethanesulfonic acid (MES) buffer, sodium acetate buffer, 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, piperazine-1,4-diethanesulfonic acid (PIPES) buffer, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPPS) buffer, or tris(hydroxymethyl)aminomethane (TRIS) buffer, or any combination thereof.

[0038] In some embodiments, the solution is an aqueous solution of p-toluenesulfonic acid, an aqueous solution of phosphoric acid, a 2-(N-morpholine)ethanesulfonic acid (MES) buffer, or a sodium acetate buffer.

[0039] In some embodiments, the solution is a 2-(N-morpholine)ethanesulfonic acid (MES) buffer.

[0040] In some embodiments, the solution is a 2-(N-morpholine)ethanesulfonic acid (MES) buffer, wherein the final concentration of 2-(N-morpholine)ethanesulfonic acid (MES) is 10-750 mM (e.g., 10-500 mM, 10-100 mM, 10-60 mM, 10-50 mM, 20-100 mM, 20-60 mM, 20-50 mM, e.g., about 40 mM).

[0041] In some embodiments, the pH of the solution is 4.0-6.5 (e.g., 4.0-4.5, 4.0-6.0, 4.5-6.0, 5.0-6.0).

[0042] In some embodiments, the total molar concentration of the carbonyl compound in the solution is greater than or equal to 50 mM.

[0043] In some embodiments, the total molar concentration of the carbonyl compound in the solution is greater than or equal to 1.0 M (e.g., 1.0-2.5 M, 1.0-3.0 M, 1.0-4.0 M, 1.0-5.0 M, 2.0-2.5 M, 2.0-3.0 M, 2.0-4.0 M, 2.0-5.0 M, for example, about 2.3 M).

[0044] In some embodiments, the solution contains glyoxal at a concentration greater than or equal to 50 mM, for example greater than or equal to 1.0 M (e.g., 1.0-2.5 M, 1.0-3.0 M, 1.0-4.0 M, 1.0-5.0 M, 2.0-2.5 M, 2.0-3.0 M, 2.0-4.0 M, 2.0-5.0 M, for example, about 2.3 M).

[0045] In some embodiments, the solution further contains an acid (e.g., boric acid, hydrochloric acid, acetic acid).

[0046] In some embodiments, the concentration of the acid is 5-800 mM (e.g., 50-800 mM, 50-500 mM, 50-100 mM, 100-800 mM, 100-500 mM, 500-800 mM, e.g., about 20 mM, about 50 mM, 100 mM, or about 500 mM).

[0047] In some embodiments, the solution contains 5-150 mM (e.g., 5-20 mM, 5-50 mM, 5-100 mM, 20-50 mM, 20-100 mM, 20-150 mM, e.g., about 20 mM or about 50 mM) of boric acid.

[0048] In some embodiments, the solution contains 50-500 mM (e.g., 50-300 mM, 50-100 mM, 100-300 mM, 100-500 mM, e.g., about 100 mM) of hydrochloric acid.

[0049] In some embodiments, the solution contains 50-800 mM (e.g., 50-500 mM, 100-800 mM, 100-500 mM, for example about 100 mM or 500 mM) of acetic acid.

[0050] In some embodiments, the nitrite is selected from sodium nitrite, potassium nitrite, and combinations thereof.

[0051] In some embodiments, in step (2), the concentration of the nitrite is 0.5-2.0M, for example 0.5-0.8M, 0.5-1.0M, 0.5-1.2M, 0.5-1.5M, 0.8-1.0M, 0.8-1.2M, 0.8-1.5M, 0.8-2.0M, for example about 0.75M or about 1.0M.

[0052] In some implementations, in step (2), the nucleic acid molecules to be tested are reacted with nitrite at 16-95°C (e.g., 16-30°C, 16-50°C, 16-65°C, 16-70°C, 16-80°C, 30-55°C, 30-50°C, 30-65°C, 30-70°C, 30-80°C, 30-95°C, 40-95°C, 40-80°C, 40-70°C, 40-65°C, 40-60°C, 40-55°C, 40-50°C, 45-60°C, 45-55°C, 45-50°C, 50-95°C, 5... React under temperature conditions of 0-80℃, 50-70℃, 50-65℃, 50-60℃, 50-55℃, 60-95℃, 60-80℃, 60-70℃, 60-65℃, 65-70℃, 65-80℃, 65-95℃, 70-75℃, 75-80℃, 70-95℃, 80-85℃, 85-90℃, 80-95℃, 90-95℃, for example, approximately 37℃, approximately 42℃, approximately 50℃, approximately 55℃, approximately 60℃, approximately 65℃, approximately 70℃, approximately 75℃, approximately 80℃, approximately 95℃).

[0053] In some implementations, in step (2), the nucleic acid molecules to be tested are reacted with nitrite at 16-95°C (e.g., 16-30°C, 16-50°C, 16-65°C, 16-70°C, 16-80°C, 30-55°C, 30-50°C, 30-65°C, 30-70°C, 30-80°C, 30-95°C, 40-95°C, 40-80°C, 40-70°C, 40-65°C, 40-60°C, 40-55°C, 40-50°C, 45-60°C, 45-55°C, 45-50°C, 50-95°C, 50-80°C, 50-70°C, 50-65°C, 50-60°C, 50-55°C, 60-95°C, 60-80°C). React at temperatures of 60-70℃, 60-65℃, 65-70℃, 65-80℃, 65-95℃, 70-75℃, 75-80℃, 70-95℃, 80-85℃, 85-90℃, 80-95℃, 90-95℃, for example, approximately 37℃, approximately 42℃, approximately 50℃, approximately 55℃, approximately 60℃, approximately 65℃, approximately 70℃, approximately 75℃, approximately 80℃, approximately 95℃) for 3-15 minutes (e.g., 3-12 minutes, 3-10 minutes, 5-15 minutes, 5-12 minutes, 5-10 minutes, 8-15 minutes, 8-12 minutes, 8-10 minutes, for example, approximately 5 minutes, approximately 10 minutes, approximately 15 minutes).

[0054] In some implementations, in step (2), the nucleic acid molecules to be tested are reacted with nitrite at 45-95°C (e.g., 45-60°C, 45-55°C, 45-50°C, 50-95°C, 50-80°C, 50-70°C, 50-65°C, 50-60°C, 50-55°C, 60-95°C, 60-80°C, 60-70°C, 60-65°C, 65-70°C, 65-80°C, 65-95°C, 70-75°C, 75-80°C). React for 3-12 minutes (e.g., 3-12 minutes, 3-10 minutes, 5-12 minutes, 5-10 minutes, 8-12 minutes, 8-10 minutes, 8-12 minutes, 8-10 minutes, 8-12 minutes, 8-10 minutes, e.g., about 5 minutes, about 10 minutes) at temperatures of 0℃, 70-95℃, 80-85℃, 85-90℃, 80-95℃, 80-95℃, or approximately 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 95℃, or approximately 95℃) for 3-12 minutes (e.g., 3-12 minutes, 3-10 minutes, 5-12 minutes, 5-10 minutes, 8-12 minutes, 8-10 minutes, or approximately 5 minutes, about 10 minutes).

[0055] In some embodiments, the method further includes a step of preprocessing the nucleic acid molecules to be tested prior to step (2).

[0056] In some implementations, the pretreatment includes purifying, fragmenting, denaturing, or any combination thereof, the nucleic acid molecules to be tested.

[0057] The method of this application utilizes nitrite to deaminate adenine in the nucleic acid molecule to hypoxanthine, while N6-methyladenine cannot be deaminated due to its stable chemical properties. Therefore, by analyzing the signals of adenine or hypoxanthine before and after the conversion, m 6 A modifies the level or m 6 Detection of site A.

[0058] Therefore, it can be understood that in some implementations, the method can achieve m by directly detecting the signal of adenine or hypoxanthine in the product of the deamination catalytic reaction of the nucleic acid to be tested. 6 A. Detection of modification level. In some implementations, the signal of adenine or hypoxanthine in the deamination-catalyzed product of the nucleic acid to be tested can be detected by methods such as mass spectrometry, enzyme fragmentation, and / or chromatography to achieve m 6 A. Detection of modification level.

[0059] Because hypoxanthine pairs complementaryly with cytosine during reverse transcription or DNA amplification, it is read as guanine; N6-methyladenine retains its complementary pairing with thymine or uracil and is still read as adenine. Therefore, in some embodiments, the method can achieve m [the desired result] by detecting the sequence information of the amplified product or reverse transcription product of the nucleic acid after deamination catalysis. 6 A modifier level and m 6Detection of site A. In some embodiments, the method can achieve m by detecting the sequence information of the amplified product or reverse transcription product of the product after deamination catalysis of the test nucleic acid, for example, through sequencing or hybridization. 6 A modifier level and m 6 Detection of site A.

[0060] In some embodiments, the method further includes the steps of purifying, reverse transcribing and / or amplifying the nucleic acid molecules obtained in the previous step before step (2) and after step (3).

[0061] In some embodiments, in step (3), the detection includes nucleotide composition analysis of nucleic acid molecules by sequencing or hybridization, mass spectrometry (e.g., triple tandem quadrupole mass spectrometry), enzyme fragmentation, and / or chromatography.

[0062] In some implementations, the detection includes nucleotide composition analysis of nucleic acid molecules via sequencing.

[0063] In some embodiments, the method further includes: performing nucleotide composition analysis on the nucleic acid molecule to be tested that has not been treated in step (2) (e.g., not reacted with nitrite).

[0064] In some embodiments, the method further includes comparing the nucleotide composition of the product nucleic acid molecule described in step (3) and the test nucleic acid molecule that has not reacted with nitrite to determine the content and / or location information of N6-methyladenine in the test nucleic acid molecule.

[0065] In some implementations, the method is used for the detection of N6-methyladenine at the whole genome or transcriptome level.

[0066] In some implementations, the method is used for the detection of N6-methyladenine in a target nucleic acid molecule.

[0067] It is readily understood by those skilled in the art that, in order to facilitate the detection of N6-methyladenine in the target nucleic acid molecule, increasing the copy number and proportion of the target nucleic acid molecule in the sample through specific amplification is significantly advantageous.

[0068] Therefore, in some embodiments, the method further includes a step of amplifying the product nucleic acid molecule obtained in the previous step before step (2) and after step (3). In some embodiments, the amplification includes using primers specific to the target nucleic acid molecule.

[0069] In some embodiments where the nucleic acid molecule to be tested is DNA, the amplification is DNA amplification, which preferably includes the use of primers specific to the target nucleic acid molecule.

[0070] In some embodiments where the nucleic acid molecule to be tested is RNA, the amplification includes reverse transcription and DNA amplification, preferably including the use of target nucleic acid molecule-specific reverse transcription primers or target nucleic acid molecule-specific cDNA amplification primers.

[0071] In some implementations, step (3) includes nucleotide composition analysis of the amplification product.

[0072] In some embodiments, the nucleic acid molecule to be tested can be derived from any living organism (e.g., eukaryotic cells, prokaryotic cells, viruses, and viroids) or a non-living organism (e.g., a nucleic acid molecule library). The nucleic acid molecule to be tested can exist in single-stranded or double-stranded form.

[0073] In some implementations, the nucleic acid molecule to be tested is RNA, DNA, a DNA / RNA hybrid, or any combination thereof.

[0074] In some implementations, the nucleic acid molecule to be tested is RNA (e.g., mRNA).

[0075] In some embodiments, the method includes: (i) adding reverse transcription-silent carrier RNA to the test sample in step (1), (ii) adding reverse transcription-silent carrier RNA to the reaction system in step (2), and / or, (ii) adding reverse transcription-silent carrier RNA to the reaction product in step (2);

[0076] The reverse transcription-silencing vector RNA can block or inhibit the synthesis of cDNA using itself as a template.

[0077] In some embodiments, the method includes adding the reverse transcription-silencing vector RNA to the test sample in step (1).

[0078] In some embodiments, the method includes: a step of purifying the nucleic acid molecule to be tested before step (2); and / or, a step of reverse transcription, amplification and purification of the product nucleic acid molecule obtained in the previous step before step (3) and after step (2).

[0079] In some embodiments, the reverse transcription-silencing vector RNA contains one or more blocking groups capable of blocking or inhibiting the synthesis of cDNA using itself as a template.

[0080] In some embodiments, the reverse transcription-silencing vector RNA contains one or more N1-cyanoethylinosine (ce1I).

[0081] In some embodiments, the reverse transcription-silencing vector RNA comprises at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% of N1-cyanoethyl inosine (ce1I); wherein the percentage is the ratio of the amount of N1-cyanoethyl inosine (ce1I) to the total number of bases in the reverse transcription-silencing vector RNA.

[0082] In some implementations, the nucleic acid molecule to be tested is RNA (e.g., mRNA).

[0083] In some implementations, the total RNA in the sample to be tested is greater than or equal to 10 nanograms, greater than or equal to 20 nanograms, greater than or equal to 50 nanograms, greater than or equal to 100 nanograms, greater than or equal to 1000 nanograms, or greater than or equal to 10000 nanograms.

[0084] In some implementations, the total RNA in the sample to be tested is greater than or equal to 100 nanograms, greater than or equal to 1000 nanograms, or greater than or equal to 10000 nanograms.

[0085] In some embodiments, the nucleic acid molecule to be tested is mRNA, and the total amount of mRNA in the sample to be tested is greater than or equal to 0.1 nanograms, greater than or equal to 0.2 nanograms, greater than or equal to 0.5 nanograms, greater than or equal to 1.0 nanograms, greater than or equal to 1.5 nanograms, greater than or equal to 2.0 nanograms, greater than or equal to 5.0 nanograms, greater than or equal to 10.0 nanograms, or greater than or equal to 100 nanograms.

[0086] In some embodiments, the nucleic acid molecule to be tested is mRNA, and the total amount of mRNA in the sample to be tested is greater than or equal to 2.0 nanograms, greater than or equal to 5.0 nanograms, greater than or equal to 10.0 nanograms, or greater than or equal to 100 nanograms.

[0087] Based on the disclosure of this application, those skilled in the art will readily understand that the use of the reverse transcription-silencing vector RNA can significantly reduce RNA loss during RNA purification, and therefore can be advantageously applied to the detection of N6-methyladenine in test samples with extremely low RNA input.

[0088] Therefore, in some embodiments using reverse transcription-silencing vector RNA, the total amount of RNA in the test sample is greater than or equal to 2 nanograms, greater than or equal to 5 nanograms, greater than or equal to 10 nanograms, greater than or equal to 20 nanograms, greater than or equal to 50 nanograms, greater than or equal to 100 nanograms, greater than or equal to 1000 nanograms, or greater than or equal to 10000 nanograms.

[0089] In some embodiments using reverse transcription-silencing vector RNA, the total amount of RNA in the test sample is greater than or equal to 10 nanograms, greater than or equal to 20 nanograms, greater than or equal to 50 nanograms, greater than or equal to 100 nanograms, greater than or equal to 1000 nanograms, or greater than or equal to 10000 nanograms.

[0090] In some embodiments where reverse transcription-silencing vector RNA is used and the nucleic acid molecule to be tested is mRNA, the total amount of mRNA in the sample to be tested is greater than or equal to 0.01 nanograms, greater than or equal to 0.02 nanograms, greater than or equal to 0.05 nanograms, greater than or equal to 0.1 nanograms, greater than or equal to 0.2 nanograms, greater than or equal to 0.5 nanograms, greater than or equal to 1.0 nanograms, greater than or equal to 2.0 nanograms, greater than or equal to 5.0 nanograms, or greater than or equal to 10.0 nanograms.

[0091] In some embodiments where reverse transcription-silencing vector RNA is used and the nucleic acid molecule to be tested is mRNA, the total amount of mRNA in the sample to be tested is greater than or equal to 0.1 ng, greater than or equal to 0.2 ng, greater than or equal to 0.5 ng, greater than or equal to 1.0 ng, greater than or equal to 2.0 ng, greater than or equal to 5.0 ng, or greater than or equal to 10.0 ng.

[0092] In some embodiments, the amount of the reverse transcription-silencing vector RNA added is 10-100 times (e.g., 10-20 times, 20-50 times, 50-100 times) the amount of RNA in the sample to be tested.

[0093] Reagent test kit

[0094] On the other hand, this application provides a kit comprising a carbonyl compound and a nitrite, as well as a transcription-silencing vector RNA; the reverse transcription-silencing vector RNA is capable of blocking or inhibiting cDNA synthesis using itself as a template.

[0095] In some embodiments, the carbonyl compound is selected from compounds of formula I and any combination thereof.

[0096] Where R1 is an aldehyde group, C 1-3 Alkyl (e.g., methyl, ethyl, or n-propyl) or halogen-substituted C 1-3 Alkyl groups (e.g., trichloromethyl, trifluoromethyl), where R2 is H or C. 1-3 Alkyl (e.g., methyl, ethyl, or n-propyl), phenyl, or HC(=O)-CH(Br)-; or,

[0097] R1 and R2 form cyclic structures with the carbonyl groups to which they are attached, such as ninhydrin.

[0098] In some embodiments, the carbonyl compound is selected from: glyoxal, 2,3-butanedione, ninhydrin, 2-bromomalondialdehyde, acetone aldehyde, trichloroacetaldehyde, phenylglyoxal, and any combination thereof.

[0099] In some embodiments, the carbonyl compound is selected from glyoxal, 2-bromomalondialdehyde, 2,3-butanedione, phenylglyoxal, trichloroacetaldehyde, ninhydrin, and any combination thereof.

[0100] In some embodiments, the carbonyl compound is glyoxal.

[0101] In some embodiments, the reverse transcription-silencing vector RNA contains one or more blocking groups capable of blocking or inhibiting the synthesis of cDNA using itself as a template. In some embodiments, the reverse transcription-silencing vector RNA contains one or more N1-cyanoethylinosine (ce1I).

[0102] In some embodiments, the reverse transcription-silencing vector RNA comprises at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% of N1-cyanoethyl inosine (ce1I); wherein the percentage is the ratio of the amount of N1-cyanoethyl inosine (ce1I) to the total number of bases in the reverse transcription-silencing vector RNA.

[0103] In some embodiments, the nitrite is selected from sodium nitrite, potassium nitrite, and combinations thereof.

[0104] In some embodiments, the kit further comprises one or more selected from the following:

[0105] (i) dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), or combinations thereof;

[0106] (ii) at least one of p-toluenesulfonic acid, phosphoric acid, 2-(N-morpholine)ethanesulfonic acid (MES), sodium acetate, 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-1,4-diethanesulfonic acid (PIPES), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPPS), and tris(hydroxymethyl)aminomethane (TRIS);

[0107] (iv) Substances for preparing 2-(N-morpholine) ethanesulfonic acid (MES) buffer, substances for preparing sodium acetate buffer, substances for preparing 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, substances for preparing piperazine-1,4-diethanesulfonic acid (PIPES) buffer, substances for preparing 4-hydroxyethylpiperazine ethanesulfonic acid (HEPPS) buffer, substances for preparing tris(hydroxymethyl)aminomethane (TRIS) buffer, and any combination thereof;

[0108] (v) Substances for preparing phosphate buffer, substances for preparing triethylamine acetate (TEAA) buffer, and combinations thereof;

[0109] (vi) Phosphate buffer, triethylamine acetate (TEAA) buffer, or a combination thereof;

[0110] (vii) Hydrochloric acid, boric acid, acetic acid, and any combination thereof;

[0111] In some embodiments, the triethylamine acetate (TEAA) buffer contains 45–50 v / v% formamide and has a pH of 8.0–9.0.

[0112] In some embodiments, the phosphate buffer contains 40–60 v / v% dimethyl sulfoxide (DMSO) at a pH of 7.1–8.0.

[0113] The substances used to prepare the buffer solution described in this application can exist in either dry powder or solution form. The substances used to prepare the buffer solution are well-known in the art and can be selected according to experimental needs and conventional methods in the art.

[0114] For example, the materials used to prepare the 2-(N-morpholine)ethanesulfonic acid (MES) buffer include 2-morpholine ethanesulfonic acid and optionally sodium hydroxide. The materials used to prepare the sodium acetate buffer include sodium acetate and optionally acetic acid. The materials used to prepare the 3-(N-morpholino)propanesulfonic acid (MOPS) buffer include 3-(N-morpholino)propanesulfonic acid and optionally sodium hydroxide. The materials used to prepare the piperazine-1,4-diethanesulfonic acid (PIPES) buffer include piperazine-1,4-diethanesulfonic acid and optionally sodium hydroxide. The materials used to prepare the 4-hydroxyethylpiperazine ethanesulfonic acid (HEPPS) buffer include 4-hydroxyethylpiperazine ethanesulfonic acid and optionally sodium hydroxide. The materials used to prepare the tris(hydroxymethyl)aminomethane (TRIS) buffer include tris(hydroxymethyl)aminomethane and optionally hydrochloric acid.

[0115] In some implementations, the kit also includes reagents for mass spectrometry detection and / or nucleic acid sequencing.

[0116] In some embodiments, the kit also includes instructions. In some embodiments, the instructions describe the method as described above.

[0117] On the other hand, this application provides the use of the kit described above in the detection of N6-methyladenine in nucleic acid molecules.

[0118] On the other hand, this application provides an RNA purification method, comprising:

[0119] (1) Provide a purified sample containing RNA, and a reverse transcription-silencing vector RNA;

[0120] (2) Add the reverse transcription-silencing vector RNA to the sample to be purified;

[0121] (3) The RNA of the sample containing the reverse transcription-silencing vector RNA was purified;

[0122] The reverse transcription-silencing vector RNA contains one or more blocking groups that can block or inhibit the synthesis of cDNA using itself as a template.

[0123] In some embodiments, the reverse transcription-silencing vector RNA molecule contains one or more N1-cyanoethyl inosine (ce1I).

[0124] In some embodiments, the reverse transcription-silencing vector RNA comprises at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% of N1-cyanoethyl inosine (ce1I); wherein the percentage is the ratio of the amount of N1-cyanoethyl inosine (ce1I) to the total number of bases in the reverse transcription-silencing vector RNA.

[0125] In some embodiments, the purified product of the method is used for nucleic acid processing involving reverse transcription. In some embodiments, the purified product of the method is used for the detection of N6-methyladenine in RNA.

[0126] In some implementations, the total RNA content in the sample to be tested is greater than or equal to 2 nanograms, greater than or equal to 5 nanograms, greater than or equal to 10 nanograms, greater than or equal to 20 nanograms, greater than or equal to 50 nanograms, greater than or equal to 100 nanograms, greater than or equal to 1000 nanograms, or greater than or equal to 10000 nanograms.

[0127] In some embodiments using reverse transcription-silencing vector RNA, the total amount of RNA in the test sample is greater than or equal to 10 nanograms, greater than or equal to 20 nanograms, greater than or equal to 50 nanograms, greater than or equal to 100 nanograms, greater than or equal to 1000 nanograms, or greater than or equal to 10000 nanograms.

[0128] In some embodiments where the RNA to be purified is mRNA, the total amount of mRNA in the sample to be purified is greater than or equal to 0.01 nanograms, greater than or equal to 0.02 nanograms, greater than or equal to 0.05 nanograms, greater than or equal to 0.1 nanograms, greater than or equal to 0.2 nanograms, greater than or equal to 0.5 nanograms, greater than or equal to 1.0 nanograms, greater than or equal to 2 nanograms, greater than or equal to 5 nanograms, greater than or equal to 100 nanograms, greater than or equal to 1000 nanograms, or greater than or equal to 10000 nanograms.

[0129] In some embodiments where the RNA to be purified is mRNA, the total amount of mRNA in the sample to be purified is greater than or equal to 0.1 nanograms, greater than or equal to 0.2 nanograms, greater than or equal to 0.5 nanograms, greater than or equal to 1.0 nanograms, greater than or equal to 2 nanograms, greater than or equal to 5 nanograms, greater than or equal to 10 nanograms, greater than or equal to 20 nanograms, greater than or equal to 50 nanograms, greater than or equal to 100 nanograms, greater than or equal to 1000 nanograms, or greater than or equal to 10000 nanograms.

[0130] In some embodiments, the amount of reverse transcription-silencing vector RNA added is 10-100 times (e.g., 10-20 times, 20-50 times, 50-100 times) the amount of RNA in the sample to be purified.

[0131] On the other hand, this application provides the use of reverse transcription-silencing vector RNA in RNA purification;

[0132] The reverse transcription-silencing vector RNA contains one or more blocking groups that can block or inhibit the synthesis of cDNA using itself as a template.

[0133] In some embodiments, the reverse transcription-silencing vector RNA contains one or more N1-cyanoethylinosine (ce1I).

[0134] In some embodiments, the reverse transcription-silencing vector RNA comprises at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% of N1-cyanoethyl inosine (ce1I); wherein the percentage is the ratio of the amount of N1-cyanoethyl inosine (ce1I) to the total number of bases in the reverse transcription-silencing vector RNA.

[0135] Terminology Definition

[0136] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0137] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0138] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0139] As used herein, unless the context clearly indicates otherwise, the term "adenine" or "A" as used herein covers adenine itself, as well as nucleotides or nucleosides containing adenine (e.g., adenine ribonucleotide, adenine deoxyribonucleotide, adenine ribonucleotide residue, adenine deoxyribonucleotide residue, adenosine, deoxyadenosine); the terms "N6-methyladenine," "6mA," "m6A," or "m 6 The term "A" encompasses N6-methyladenine itself, as well as nucleotides or nucleosides containing N6-methyladenine (e.g., ribonucleotides, deoxyribonucleotides, ribonucleotide residues, deoxyribonucleotide residues, nucleosides, deoxynucleosides); the term "hypoxanthine" or "I" encompasses hypoxanthine itself, as well as nucleotides or nucleosides containing hypoxanthine (e.g., ribonucleotides, deoxyribonucleotides, ribonucleotide residues, deoxyribonucleotide residues, nucleosides, deoxynucleosides); the term "xanthine" or "X" encompasses xanthine itself, as well as nucleotides or nucleosides containing xanthine (e.g., ribonucleotides, deoxyribonucleotides, ribonucleotide residues, deoxyribonucleotide residues, nucleosides, deoxynucleosides); the term "guanine" or "G" encompasses guanine itself, as well as nucleotides or nucleosides containing guanine (e.g., nucleosides, deoxyribonucleotides, ribonucleotide residues, deoxyribonucleotide residues, nucleosides, deoxynucleosides); The terms “glyconucleotide,” “deoxyribonucleotide,” “ribonucleotide residue,” “deoxyribonucleotide residue,” “nucleoside,” and “deoxynucleoside” are used to refer to nucleotides or nucleosides containing thymine. The term “cytosine” or “C” covers cytosine itself and nucleotides or nucleosides containing cytosine. The term “uracil” or “U” covers uracil itself and nucleotides or nucleosides containing uracil.

[0140] As used herein, unless the context clearly indicates otherwise, the terms “m6A,” “6mA,” and “m” will be used interchangeably. 6The three terms “A” all encompass N6-methyladenine itself in DNA, RNA, or DNA / RNA hybrids, as well as nucleotides or nucleosides containing N6-methyladenine (e.g., ribonucleotides, deoxyribonucleotides, ribonucleotide residues, deoxyribonucleotide residues, nucleosides, deoxynucleosides), and are used interchangeably.

[0141] As used herein, unless the context clearly indicates otherwise, the compounds referred to herein have the meaning commonly understood by those skilled in the art, such as the following compounds and their corresponding CAS numbers: 2-(N-morpholine)ethanesulfonic acid (MES, CAS: 4432-31-9), sodium acetate (CAS: 127-09-3), 3-(N-morpholine)propanesulfonic acid (MOPS, CAS: 1132-61-2), piperazine-1,4-diethanesulfonic acid (PIPES, CAS: 5625-37-6), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPPS, CAS: 7365-45-9), or tris(hydroxymethyl)aminomethane (TRIS, CAS: 77-86-1). Glyoxal (CAS: 107-22-2), acetone aldehyde (CAS: 78-98-8), 2,3-butanedione (CAS: 431-03-8), ninhydrin (CAS: 485-47-2), 2-bromomalondialdehyde (CAS: 2065-75-0), trichloroacetaldehyde (CAS: 75-87-6), phenylglyoxal (CAS: 1075-06-5), formamide (CAS: 75-12-7), p-toluenesulfonic acid (CAS: 104-15-4), phosphoric acid (CAS: 7664-38-2), sodium hydroxide (CAS: 1310-73-2), hydrochloric acid (CAS: 7647-01-0), acetic acid (CAS: 64-19-7).

[0142] As used herein, the various buffer solutions used herein have the meanings commonly understood by those skilled in the art and can be prepared according to conventional methods in the art as needed for experiments. For example, the 2-(N-morpholine)ethanesulfonic acid (MES) buffer described herein can be prepared from 2-(N-morpholine)ethanesulfonic acid (MES), optionally with the pH adjusted as needed using sodium hydroxide solution. For example, the sodium acetate buffer used herein can be prepared from sodium acetate, optionally with the pH adjusted as needed using acetic acid. For example, the 3-(N-morpholino)propanesulfonic acid (MOPS) buffer used herein can be prepared from 3-(N-morpholino)propanesulfonic acid, optionally with the pH adjusted as needed using sodium hydroxide. For example, the piperazine-1,4-diethanesulfonic acid (PIPES) buffer used herein can be prepared from piperazine-1,4-diethanesulfonic acid, optionally with the pH adjusted as needed using sodium hydroxide. For example, the 4-hydroxyethylpiperazine ethanesulfonic acid (HEPPS) buffer used in this paper can be prepared from 4-hydroxyethylpiperazine ethanesulfonic acid, and the pH can be adjusted with sodium hydroxide as needed. Similarly, the tris(hydroxymethyl)aminomethane (TRIS) buffer used in this paper can be prepared from tris(hydroxymethyl)aminomethane, and the pH can be adjusted with hydrochloric acid as needed.

[0143] Beneficial effects of the invention

[0144] Compared to the currently more powerful m6A detection technology in the field, GLORI (referred to as GLORI 1.0 in this paper), the novel m6A detection method provided in this application (GLORI 2.0 and its optimized technology GLORI 3.0) has one or more of the following beneficial effects:

[0145] (1) Significantly reduced RNA degradation;

[0146] (2) Significantly higher detection sensitivity (e.g., GLORI 1.0 can measure m6A across the entire transcriptome with mRNA input of more than 50 nanograms; GLORI 2.0 can measure m6A across the entire transcriptome with mRNA input as low as 2 nanograms; GLORI 3.0 can measure m6A across the entire transcriptome with total RNA input as low as 10 nanograms), thus making it more suitable for the quantitative detection of m6A in samples with ultra-low nucleic acid (e.g., RNA) content (e.g., subcellular components of complex tissues, rare cell types sorted by FACS) or low abundance RNA;

[0147] (3) It can better maintain the integrity of RNA, thus making it more suitable for quantitative detection of specific m6A sites (e.g., low abundance sites).

[0148] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0149] Figure 1: Optimized deamination system. (a) Glyoxal and nitrite-mediated deamination protocol in GLORI 1.0. The process consists of three consecutive steps: G protection, A deamination, and G deprotection. (b) RNA fragment analysis showing the degradation of total RNA in each step of GLORI 1.0. The positions of 18S rRNA and 28S rRNA are indicated by blue arrows. (c, d) Speculated mechanism of glyoxal-mediated G protection and A activation. In the nitrosation reaction, G* cannot be deamination to X or O, while A* is rapidly deamination to I. (e) Showing the key reagents and reaction times required for the latest ultrafast, one-pot deamination process (GLORI 2.0). (f) AI conversion rates of GLORI 1.0 and the updated chemical reaction (GLORI 2.0). (g) G to X and G to O conversion rates of GLORI 1.0 and the updated chemical reaction (GLORI 2.0). (h) RNA fragment analysis shows the total RNA degradation under GLORI 1.0 treatment and the update chemical reaction (GLORI 2.0).

[0150] Figure 2: Glyoxal and nitrite-catalyzed deamination. (a) Agarose gel showing the degradation of total RNA in GLORI 1.0. (b) Nitrite-mediated G to X and G to O conversions.

[0151] Figure 3: Optimization of GLORI chemical reactions. (a) A-1 and GX conversions under the A-deamination step conditions of GLORI 1.0 (reaction mixture containing 750 mM NaNO2, 1.41 M glyoxal, H3BO3, DMSO, 40 mM MES, and water; incubation at 16°C for 8 hours). (b) AI conversion without glyoxal in the reaction mixture. (c) AI conversion under different concentrations of glyoxal and NaNO2 (incubation at 16°C for 8 hours). (d) AI conversion at different times and temperatures (reaction mixture containing 1 M NaNO2, 2.29 M glyoxal, H3BO3, DMSO, 40 mM MES, and water). (e) AI conversion at reaction temperatures above 50°C. (f) AI conversion under different acid conditions (reaction mixture containing 1 M NaNO2, 2.29 M glyoxal, acid, DMSO, 40 mM MES, and water). (g) RNA fragment analysis showed that total RNA was degraded at reaction temperatures above 50°C. (n = 2 independent samples).

[0152] Figure 4: GLORI 2.0 can quantify the m6A methylome. (a) Exemplary GLORI 2.0 protocol: mRNA is first split and then treated with the latest chemical reactions to achieve efficient deamination of unmethylated As. The treated RNA sample undergoes a sequencing library construction process. The sequencing results are aligned with a ternary genome reference, and the "A rate" of each site is calculated as the methylation level of m6A modification. (b) Exemplary single-tube library construction protocol. (c) Motivational analysis of m6A sites identified by GLORI 2.0. (d) Methylation levels of m6A sites with different patterns. (e) A map showing the distribution of m6A sites detected by GLORI 2.0.

[0153] Figure 5: Identification and quantification of m6A across the entire genome by GLORI 2.0. (a) Venn diagram showing the overlap of m6A sites identified by GLORI 2.0 in two biological replicates. (b) Methylation levels of m6A detected by GLORI 2.0 in the two replicates. Color gradients represent the density of overlapping modification sites. (c) Correlation between the expected (x-axis) and detected (y-axis) m6A ratios of five synthetic RNA pattern sequences and five defined methylation levels. Pearson correlation coefficients and p-values ​​based on two-sided t-tests are shown. (d) Venn diagram showing the overlap of m6A sites identified by GLORI 1.0 and GLORI 2.0. (e) Methylation levels of m6A detected by GLORI 1.0 and GLORI 2.0. Color gradients represent the density of overlapping modification sites. (f) Representative view of four m6A sites (red lines) and methylation levels detected by GLORI 1.0 and GLORI 2.0. The methylation levels detected by GLORI 1.0 and GLORI 2.0 were almost identical. (g) m6A methylation levels detected by GLORI 2.0 in 2 ng and 50 ng mRNA. The color gradient represents the density of overlapping modification sites.

[0154] Figure 6: Validation of the accuracy of GLORI 2.0 in identifying and quantifying m6A. (a) m6A levels of all individual modification sites detected by GLORI 2.0 between STM2457-treated and untreated HEK293T cells. The color gradient represents the density of overlapping modification sites. (b) m6A levels of HEK 293T mRNA samples treated with or untreated with STM2457 were measured using GLORI 2.0. (c) m6A content of HEK 293T mRNA in STM2457-treated or untreated samples was determined by quantitative LC-MS / MS, n = 2 independent biological samples. (d) Venn diagram showing the overlap of m6A sites in HEK293T mRNA with single-base resolution sites detected in IVT RNA. (e) Distribution map of m6A sites in HEK293T mRNA and sites detected in IVT RNAs. (f) Analysis of sites detected in IVT RNA.

[0155] Figure 7: Comparison of m6A methylation sites detected by GLORI 2.0 and GLORI 1.0. (a) The box plot shows the readout coverage of m6A sites shared in the GLORI 2.0 dataset (sites identified in both GLORI 1.0 and GLORI 2.0) and those identified only by GLORI 2.0. (b) m6A methylation levels of 50 ng mRNA detected by GLORI 1.0 and GLORI 2.0. The color gradient represents the density of overlapping modification sites. (c) Distribution of m6A sites detected by GLORI 1.0 and GLORI 2.0 in different mRNA inputs. (d) The graph shows the m6A methylation levels identified by GLORI 1.0 and GLORI 2.0 in different mRNA inputs.

[0156] Figure 8: Identification and quantification of m6A at specific sites. (a) Site-specific m6A quantification protocol supported by GLORI 2.0. Full-length mRNA was treated with a mild ultrafast deamination reaction to achieve efficient deamination of unmethylated A. The treated RNA was reverse transcribed and amplified into amplicons by PCR. Sanger sequencing and NGS were used to identify and quantify m6A. (b) Quantification of methylation level at m6A4220 site in 28S rRNA. (c) Quantification of methylation level of SLC7A5_3483 by fragment-Sanger sequencing, fragment-NGS, and transcriptome GLORI. (d) Quantification of methylation level of 19 m6A sites in HEK293T mRNA by amplicon-Sanger sequencing, amplicon-NGS, and transcriptome GLORI, respectively. The color gradient represents the methylation ratio of these m6A sites. The horizontal bars on the right side of the heatmap show the expression level of genes containing the above m6A sites.

[0157] Figure 9: Identification and quantification of m6A by GLORI 2.0-Sanger sequencing. (af) Quantification of methylation levels at different m6A sites in HEK293T mRNA by amplicon-Sanger sequencing.

[0158] Figure 10: Characterization of m6A detected by GLORI 2.0 from low-volume RNA. (a, b) Characterization analysis of m6A detected by GLORI 2.0 from 20 nanograms of total RNA.

[0159] Figure 11: A novel strategy for preparing vector RNA. (a) Ideal properties of vector RNA. Cellular RNA can be reverse transcribed normally to obtain qualified cDNA; however, vector RNA is artificially modified, which hinders the reverse transcription process. (b) Vector RNA preparation protocol. Total RNA from MEF cell lines undergoes deamination and cyanoethylation to obtain vector RNA containing a high-density reverse transcription termination signal, N1-cyanoethylinosine (ce1I). (c) A can be deainated to I via a mild and ultrafast deamination reaction; I can undergo cyanoethylation with acrylonitrile to generate ce1I. (d) HPLC-MS / MS analysis of ribonucleosides shows that the conversion efficiency of this two-step chemical reaction exceeds 99%. Chromatograms of C (blue), U (green), and I (orange) are scaled to the left Y-axis, while chromatograms of A (black) and m6A (purple) are scaled to the right Y-axis. (e) RNA fragment distribution analysis of AI-transformed RNA and cyanoethylated RNA. The positions of 18S rRNA and 28S rRNA are indicated by blue arrows. (f) DNA fragment analysis of sequencing libraries. The blue dashed box represents the primer dimer fragment; the red dashed box represents the DNA library derived from cellular mRNA.

[0160] Figure 12: Ce1I disrupts base pairing, thereby inhibiting the elongation of first-strand cDNA synthesis. (a) Cyanoethylation of inosine. I reacts with acrylonitrile to generate ce1I. (c) During library construction, vector RNA cannot form sequencing libraries, while cellular RNA can undergo library construction processing to obtain qualified sequencing libraries.

[0161] Figure 13: GLORI 3.0 can perform m6A analysis with ultra-low input RNA. (a) Exemplary protocol of GLORI 3.0: mRNA extraction; splitting of mRNA and vector RNA, followed by treatment with glyoxal and nitrite to achieve efficient deamination of unmethylated As; the treated RNA sample enters the sequencing library construction process. During library construction, cellular mRNA can generate qualified sequencing libraries, while vector RNA cannot. (b) Comparison of library quality between GLORI 2.0 and GLORI 3.0 in terms of valid reads and identified m6A sites using 20 ng total RNA input. “Clean reads” refers to the remaining reads after quality control, aptamer trimming, and deduplication. (c) m6A methylation levels detected by GLORI 2.0 and GLORI 3.0 with different amounts of input RNA. Color gradients represent the density of overlapping modification sites. (d) Venn diagram showing the overlap of m6A sites identified by GLORI 3.0 from three groups of low-volume RNA inputs. (e) The graph shows the m6A methylation levels measured by GLORI 2.0 from 50 ng mRNA and by GLORI 3.0 from 10 ng total RNA. (f) The graph shows the distribution of m6A sites detected by GLORI 3.0 in three groups of low-quantity RNA.

[0162] Figure 14: Characterization of m6A sites detected by GLORI 3.0 from low-volume RNA input. (a) Comparison of effective reads and m6A sites detected by GLORI 2.0 and GLORI 3.0 in 100 ng total RNA. “Clean reads” refers to the remaining reads after quality control, aptamer trimming, and deduplication. (b, c) Characterization of m6A detected by GLORI 3.0 in 100 ng total RNA. (d) m6A levels of all individual modification sites detected by GLORI 3.0 in 100 ng total RNA between STM2457-treated and untreated HEK293T cells. Color gradients indicate the density of overlapping modification sites. (e) m6A levels of all individual modification sites detected by GLORI 3.0 in 20 ng total RNA between STM2457-treated and untreated HEK293T cells. Color gradients indicate the density of overlapping modification sites.

[0163] Figure 15: An example illustrating the quantitative accuracy of GLORI 3.0. This is a representative view of the four m6A sites (red lines) and their methylation levels detected by GLORI 2.0 and GLORI 3.0 from different RNA inputs. The methylation levels detected by GLORI 2.0 and GLORI 3.0 are almost identical. Detailed Implementation

[0164] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not to limit the invention), and are not intended to limit the scope of protection claimed by the invention.

[0165] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0166] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0167] GLORI 1.0

[0168] As mentioned above, based on extensive research, the inventors of this application have developed a new generation of GLORI technology, GLORI 2.0 and GLORI 3.0, on the basis of the previously reported GLORI technology (hereinafter referred to as GLORI 1.0). These technologies solve the RNA degradation problem of GLORI 1.0 and overcome its limitations when applied to extremely low-volume RNA samples.

[0169] For a detailed description of GLORI 1.0, please refer to Liu C, et al. (Liu C, Sun H, Yi Y, Shen W, Li K, Xiao Y, Li F, Li Y, Hou Y, Lu B, Liu W, Meng H, Peng J, Yi C, Wang J. Absolute quantification of single-base m 6 A methylation in the mammalian transcriptome using GLORI.Nat Biotechnol.2023Mar;41(3):355-366.doi:10.1038 / s41587-022-01487-9.) and patent application WO 2022 / 257354 A1; the full text of which is incorporated herein by reference.

[0170] To more clearly describe the present invention, an exemplary experimental scheme for GLORI 1.0 is provided below:

[0171] GLORI 1.0 Chemical Reaction

[0172] (1) RNA protection: Take 200 ng of fragmented RNA, add nuclease-free water to a final volume of 14 μL, add 6 μL of glyoxal solution and 20 μL of DMSO reagent, mix well by pipetting, and incubate at 50℃ for 30 min in a PCR instrument. During incubation, prepare a saturated boric acid solution. Take approximately 100 mg of boric acid into a 1.5 mL EP tube, add 400 μL of nuclease-free water, vortex to mix at room temperature, let stand for 10 min, centrifuge, and collect the supernatant, which is the saturated boric acid solution. After RNA incubation for 30 min, add 10 μL of saturated boric acid solution, mix well by pipetting, and continue incubating at 50℃ in a PCR instrument for 30 min. After incubation, place on ice for later use. During incubation, prepare a 5M sodium nitrite solution.

[0173] (2) RNA deamination reaction: Prepare the deamination reaction solution in advance, containing 15 μL of 5M sodium nitrite, 8 μL of 500mM MES buffer (pH 6), and 10 μL of glyoxal solution, plus 17 μL of nuclease-free water, for a total of 50 μL. Mix the protected RNA from the previous step with the deamination reaction solution by pipetting and aspirating, and centrifuge the liquid on the tube wall to the bottom of the tube. Incubate the reaction solution in a PCR instrument at 16℃ for 8 hours, or incubate it in a PCR instrument at 50℃ for 30 minutes. After the deamination reaction, the product can be purified: transfer 100 μL of RNA reaction solution to a 1.5 mL EP tube, add 4 μL of glycogen and 10 μL of 3M sodium acetate solution, and 250 μL of pre-chilled anhydrous ethanol, mix by inverting, and incubate at -20℃ for at least 1 hour.

[0174] (3) RNA deprotection reaction: First, prepare the 1M TEAA solution: Mix 6.95 mL of triethylamine and 2.86 mL of glacial acetic acid, add water to a final volume of 30 mL, adjust the pH to 8.6 using triethylamine, and then add water to a final volume of 50 mL. Next, prepare the deprotection reagent: Take 10 mL of the 1M TEAA solution, add 9.5 mL of deionized formamide solution and 0.5 mL of nuclease-free water. Mix thoroughly by inverting the container, and store at 4°C in the dark.

[0175] After ethanol precipitation, the RNA was centrifuged and washed with 75% ethanol, then air-dried at room temperature. 50 μL of deprotection reagent was added, and the EP tube was heated in a 95°C mixer for 10 min, followed by rapid cooling on ice. 2 μL of glycogen and 5 μL of 3M sodium acetate solution, along with 145 μL of pre-chilled anhydrous ethanol, were added, and the mixture was inverted and mixed thoroughly. The tube was then incubated at -20°C for at least 1 hour.

[0176] Example 1: G (guanosine) protection leads to RNA degradation

[0177] To examine RNA degradation issues with GLORI 1.0, total RNA was placed in each of the GLORI 1.0 treatment steps, and RNA integrity was analyzed after each treatment (Fig. 1a). Of the three main steps (1. G(guanosine)-protection, 2. A(adenosine)-deamination, 3. G(guanosine)-deprotection), the "guanosine (G)-protection" step was found to cause severe RNA degradation, while the other two steps were relatively mild and did not impair RNA integrity (Fig. 1b and Fig. 2a).

[0178] Example 2: Mild and ultrafast deamination to reduce RNA degradation (GLORI 2.0)

[0179] Furthermore, the inventors of this application designed a novel deamination reaction process that does not include prior G protection: glyoxal, boric acid, and sodium nitrite are added together, and the A base is deaminated in a one-pot reaction (Figure 1e). The experimental steps are as follows:

[0180] Prepare the deamination reaction solution in advance, containing 15 μL of 5M sodium nitrite, 8 μL of 500mM MES buffer (pH 6), 16 μL of glyoxal solution, 20 μL of DMSO reagent, and 10 μL of saturated boric acid solution. Add RNA and bring the volume to 100 μL with nuclease-free water. Mix the reaction solution by pipetting and centrifuging to remove excess liquid from the tube wall. Incubate the reaction solution in a 16°C PCR instrument for 8 hours. Transfer 100 μL of the RNA reaction solution to a 1.5 mL EP tube, add 4 μL of glycogen, 10 μL of 3M sodium acetate solution, and 250 μL of pre-chilled anhydrous ethanol. Mix by inverting and incubate at -20°C for at least 1 hour.

[0181] After ethanol precipitation, the RNA was centrifuged and washed with 75% ethanol, then air-dried at room temperature. 50 μL of deprotection reagent was added, and the EP tube was heated in a 95°C mixer for 10 min, followed by rapid cooling on ice. 2 μL of glycogen and 5 μL of 3M sodium acetate solution, along with 145 μL of pre-chilled anhydrous ethanol, were added, and the mixture was inverted and mixed thoroughly. The tube was then incubated at -20°C for at least 1 hour.

[0182] The results showed that, without prior G protection, approximately 90% of the A to I conversion was achieved, along with a lower G deamination rate (Figure 3a).

[0183] It is currently speculated that the reason why this embodiment can achieve a significantly high A to I conversion efficiency and a low G deamination rate without prior G protection is as follows:

[0184] The initial design of the G protection step in GLORI 1.0 was based on the following rationale: research in the 1960s found that in nitrite-mediated deamination, the deamination of A was much slower than that of G; G undergoes deamination to form xanthine nucleoside (X) or oxopurine (O; Figure 2b); therefore, G was protected with glyoxal before A deamination (forming a stable structure G*). However, reflecting on the A deamination mediated by GLORI 1.0, the inventors of this application hypothesize that its mechanism should be significantly different from that of nitrite-mediated A deamination: it is hypothesized that the rapid A deamination in GLORI 1.0 is promoted by the formation of the key intermediate A*, which is formed in the presence of glyoxal, similar to the formation of G* (Figure 1c). Examining the chemical structure of A*, it is speculated that the weak nucleophile (i.e., the outer 6-amino group of A) has been transformed into a strong nucleophile at the N6 position of A*, thereby significantly enhancing its interaction with nitrite (Figure 1d). Therefore, the deamination of A via A* should be much greater than the deamination of G (and G*), which may form the basis for removing the dedicated G-protected step.

[0185] To verify the inventors' above-mentioned mechanistic hypothesis, the inventors omitted glyoxal in the reaction system of GLORI 2.0. The results showed that omitting glyoxal in the new one-step treatment significantly reduced the deamidation of A, further confirming the importance of A activation to A* (Figure 3b). Therefore, it can be concluded that the specific G protection step in GLORI 1.0, which severely impairs RNA integrity, can be removed. It is worth noting that G* can still be formed in the one-stop process; therefore, it is preferable to perform the G deprotection step after the one-stop reaction.

[0186] To further improve A deamination, the one-stop reaction conditions were optimized. First, increasing the glyoxal concentration resulted in an increased A deamination rate (Fig. 3c). Next, increasing the treatment temperature significantly increased A deamination (>99.0%) at 50°C or above, and drastically reduced the treatment time to just a few minutes (Fig. 3d, e). Furthermore, the inventors found that removing boric acid from the GLORI 2.0 one-stop reaction system and replacing it with other acids, such as hydrochloric acid and glacial acetic acid, also resulted in very high deamination efficiency (Fig. 3f), indicating that boric acid provides the acidic environment necessary for nitrosation in the reaction system. Subsequent examination of RNA integrity revealed that although higher temperatures led to some RNA degradation (Fig. 3g), treatment at 50°C for 10 minutes achieved efficient A deamination while maintaining RNA integrity (Fig. 1f-h). Under these conditions, the ratios of G to X, G to O, and cytosine (C) to uracil (U) were also optimal (Fig. 1g and Fig. 3h). Therefore, a mild and ultra-fast deamination process was established, achieving high A to I conversion efficiency without significant RNA degradation.

[0187] Example 3: Precise quantification of m6A methyl group using GLORI 2.0

[0188] Subsequently, GLORI 2.0, combined with the corresponding library construction workflow (Figures 4a and 4b), was applied to detect m6A sites in mRNA isolated from HEK293T cells. At a depth of 80M alignment, 85,057 and 79,574 m6A sites were detected in two biological replicates, respectively, of which 63,777 were shared (approximately 80%, Figure 5a). The m6A proportions were highly consistent between the two experiments (r = 0.9458), demonstrating the high reproducibility of GLORI 2.0 in quantitative m6A analysis (Figure 5b). m6A sites appeared in typical DRACH motifs (D = A, G, or U; R = A or G; H = A, C, or U) and were enriched near the stop codon and in the 3'UTR region of mRNA, which are typical characteristics of m6A (Figures 4c-e).

[0189] To further validate the quantitative capabilities of GLORI 2.0, exogenous RNAs with known m6A proportions (10%, 30%, 60%, 90%, and 100%) were added as internal controls, and a linear correlation was observed between the detected modification levels and the expected levels (R = 0.9914, Fig. 5c). Furthermore, the METTL3 inhibitor STM2457 was used to reduce intracellular m6A levels; this treatment resulted in a significant reduction in m6A levels, with over 98% of m6A sites showing a marked decrease (Fig. 6a). Moreover, the overall m6A levels quantified by GLORI 2.0 were highly consistent with quantitative mass spectrometry methods (Fig. 6b, c). Additionally, GLORI 2.0 was applied to unmodified in vitro transcribed mRNA (IVT-mRNA); unsurprisingly, only a few shared sites were detected between IVT-mRNA and cellular mRNA samples (Fig. 6d). These sites also lacked typical m6A characteristics (Fig. 6e, f).

[0190] Example 4: GLORI 2.0 has higher sensitivity than GLORI 1.0.

[0191] Next, a detailed comparison was made between GLORI 2.0 and 1.0. At the same sequencing depth, GLORI 2.0 captured most (approximately 85%) of the m6A sites identified by GLORI 1.0; simultaneously, GLORI 2.0 identified an additional 27,196 m6A sites, representing approximately 62% more modification sites (Figure 5d). These sites tend to be present in relatively low-abundance mRNA; the superior RNA integrity after GLORI 2.0 treatment helps preserve these modification sites (Figure 7a). It is worth noting that these additional m6A sites are genuine modification sites, as GLORI 1.0 also successfully identified them when sequencing depth was increased. Therefore, GLORI 2.0 is more sensitive than GLORI 1.0 in terms of m6A detection.

[0192] Then, the quantitative performance of GLORI 2.0 and 1.0 was compared. The two versions of GLORI were applied to 50 ng, 10 ng, and 2 ng of mRNA, respectively. For the 50 ng and 10 ng mRNA input amounts, both versions showed consistency in m6A quantification (50 ng mRNA group r = 0.9324; 10 ng mRNA group r = 0.9354, Figures 5e and 7b). Figure 5f shows an example. Taking the four m6A modification sites on the GPAT4 transcript as an example, the modification levels measured by GLORI 1.0 and GLORI 2.0 were almost identical. Further analysis of the proportion of m6A in different motif backgrounds showed no significant difference between GLORI 2.0 and GLORI 1.0 (Figure 4d). However, for the 2 ng mRNA input amount, GLORI 1.0 failed to construct an effective sequencing library, while GLORI 2.0 successfully prepared a sequencing library. GLORI 2.0 not only detected a similar number of m6A sites at input levels of 2 ng or 50 ng mRNA, but the modification ratios and distribution patterns in the two experiments were also highly consistent (Fig. 5g and Fig. 7c, d). Therefore, it can be concluded that GLORI 2.0 is more robust than GLORI 1.0 and exhibits superior performance for low-volume RNA.

[0193] Example 5: Quantification of m6A at specific sites

[0194] While high-throughput m6A quantification across the entire transcriptome is extremely powerful, biological research often focuses on the methylation status of a few mRNA transcripts of interest. Although their throughput is lower, site-specific detection methods simplify data analysis and easily obtain the m6A status of RNA targets.

[0195] After treatment with GLORI, the m6A methylation status at specific sites of the target RNA can be investigated. However, due to severe RNA degradation during GLORI 1.0, targeted amplicons are only occasionally obtained, especially when the target RNA expression level is low. Since GLORI 2.0 can maintain RNA integrity well, its ability to measure m6A at specific sites was further tested. Subsequently, Sanger sequencing or NGS sequencing was performed on the amplicons targeting specific sites to determine the m6A level at those sites (Figure 8a).

[0196] First, the known m6A site (position A4220) in 28S rRNA was tested, where the modification level was near complete. The results showed that GLORI 2.0 could indeed detect a highly modified site, with surrounding adenosine (A) completely converted to guanosine (G) (Fig. 8b). Next, 19 selected m6A modification sites with methylation levels ranging from 30% to 90% were examined (Figs. 8c, d, and 9). The expression levels of these mRNAs ranged from approximately ~0.5 to 270 FPKM, representing very low to high abundance transcripts (Fig. 8d). Subsequently, the target amplicon was submitted for Sanger sequencing and NGS, and the results were compared with the m6A levels obtained from whole-transcriptome GLORI sequencing. As shown in Figs. 8c and d, the m6A levels were very consistent with each other. Notably, for transcripts with FPKM < 3 (generally considered to be of very low expression), whole-transcriptome GLORI sequencing often lacks sufficient sequencing reads to support confident m6A identification, let alone quantification. Conversely, as long as the ternary PCR primers are selected appropriately, applying GLORI 2.0 to the target amplicon method can easily detect and quantify the m6A site with high confidence.

[0197] Furthermore, data from GLORI 2.0 and eTAM-Sanger (see Xiao YL, et al. Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination. Nat Biotechnol. 2023 Jul; 41(7):993-1003. doi:10.1038 / s41587-022-01587-6.) were compared for m6A measurements at specific sites. Eight common mRNA sites were selected for comparison, with expression levels ranging from FPKM 20 to 270. While the two techniques showed highly consistent modification levels for all eight sites, in the eTAM-Sanger data, three of the eight sites (SLC7A5, H2AFX, and OGT transcripts) showed incomplete A deamination. Using the GLORI 2.0 target sequencing method, it was found that the conventional adenosine in these transcripts was completely deaminated (Figure 8c and Figures 9a and b). Therefore, the chemically assisted GLORI method can more efficiently and unbiasedly deaminize transcripts, ensuring the accuracy of targeted m6A measurements.

[0198] Example 6: Construction of a reverse transcription silencing vector RNA for ultra-low starting doses of GLORI

[0199] While GLORI 2.0 significantly improved RNA integrity compared to 1.0 and enabled m6A measurements across the entire transcriptome from mRNAs as low as 2 nanograms, processing extremely small amounts of RNA input remains challenging. For example, when processing animal or clinical samples, the ability to quantify the m6A methyl group from thousands of cells, which may belong to a specific type or even rare subtypes isolated from complex tissues, is particularly desirable. To this end, we extracted RNA from approximately 1000 HEK293T cells, yielding approximately 20 nanograms of total RNA; however, GLORI 2.0 failed to provide a definitive global m6A methylation profile (Figure 10).

[0200] The GLORI process involves two purification steps, which presumably may lead to the loss of cellular RNA molecules. This is not a problem when the amount of RNA sample input is unlimited, but it can be detrimental to precious RNA samples. Therefore, to reduce RNA loss during the purification steps, the inventors of this application sought to develop a custom vector RNA for ultra-low starting amounts of GLORI sequencing. An ideal vector RNA would not only mitigate cellular RNA loss but also not interfere with cDNA synthesis. Therefore, the inventors of this application propose a novel vector RNA containing intensive artificial modifications that lead to reverse transcription termination, ultimately preventing the formation of qualified cDNA (Figure 11a).

[0201] To achieve this goal, the inventors utilized the cyanoethylation chemical reaction of inosine: literature from years ago documented that inosine can be efficiently cyanoethylated with acrylonitrile to form N1-cyanoethyl inosine (ce1I), and subsequent studies showed that this completely inhibits the synthesis of first-strand cDNA (Figures 12a and 12b). Therefore, RNA containing I was first generated from total RNA through the aforementioned A deamination treatment; then, it was further highly cyanoethylated (>99%, Figures 11b-d) to generate vector RNA rich in ce1I modification but lacking A or I bases. Due to the mild reaction conditions, the cyanoethylated vector RNA maintained good RNA integrity (Figure 11e). Unsurprisingly, vector RNA containing only ce1I could not successfully construct a library; only in the presence of cellular mRNA could a high-quality sequencing library be obtained (Figures 11f and 12c). Therefore, the vector RNA was reverse-transcribed silencing and automatically removed during the library construction steps in GLORI.

[0202] Example 7: GLORI 3.0 for m6A quantification in ultra-low starting samples

[0203] Next, reverse transcription silent vector RNA was integrated into GLORI 2.0 to develop GLORI 3.0 for determining m6A across the entire transcriptome from ultra-low starting RNA materials. It is easy to understand that the incorporation of reverse transcription silent vector RNA is intended to reduce sample loss during RNA purification; therefore, it can be incorporated simply before the RNA purification step, for example, by incorporating reverse transcription silent vector RNA into the reaction starting RNA material of GLORI 2.0, by incorporating reverse transcription silent vector RNA into the one-to-one reaction (A to I transformation) system of GLORI 2.0, and / or by incorporating reverse transcription silent vector RNA into the one-to-one reaction (A to I transformation) product of GLORI 2.0 (an exemplary reverse transcription silent vector RNA incorporation scheme for GLORI 3.0 is shown in Figure 13a).

[0204] The performance of GLORI 3.0 was tested using two input volumes: 100 ng of total RNA input yielded only a moderate quality m6A methyl group in GLORI 2.0, while 20 ng of total RNA input failed to produce a definitive m6A methyl group in GLORI 2.0 (Fig. 10 and Fig. 14a). With GLORI 3.0, the quality of the sequencing library was significantly improved, and more m6A sites were detected (Fig. 13b and Fig. 14a). The resulting m6A sites exhibited typical DRACH motifs, consistent with those identified with larger mRNA input volumes (Fig. 14b, c). Furthermore, they showed a significant reduction in modification levels after STM2457 treatment (Fig. 14d, e). The quantitative m6A information obtained through GLORI 3.0 was also compared with data from GLORI 2.0 using large RNA inputs (50 ng and 10 ng mRNA), showing a high degree of consistency in the overall m6A modification levels across the transcriptome (Fig. 13c). A typical example is shown in Figure 15.

[0205] GLORI 3.0 was further applied to 10 nanograms of total RNA, equivalent to approximately 500 HEK293T cell RNAs. With only identical alignment readings (approximately 60 M), reliable m6A methyl groups were detected that showed good overlap with data from 20 and 100 nanograms of total RNA (Fig. 13d). Although the number of detected m6A sites increased with increasing RNA input, the methylation percentages and distribution patterns were similar (Fig. 13e, f). Therefore, it can be concluded that GLORI 3.0 is also suitable for global m6A quantification of samples with very low initial RNA volumes.

[0206] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for detecting N6-methyladenine in nucleic acid molecules, comprising the following steps: (1) Provide a sample to be tested, wherein the sample to be tested contains the nucleic acid molecule to be tested; (2) In the presence of carbonyl compounds, the nucleic acid molecules to be tested react with nitrite, causing the adenine in the nucleic acid molecules to be tested to be converted into hypoxanthine; (3) Detect the nucleic acid molecules obtained in the previous step; Prior to step (2) (e.g., before the nucleic acid molecule to be tested reacts with nitrite), the amino group of guanine in the nucleic acid molecule to be tested is not protected.

2. The method of claim 1, wherein, After step (2) and before step (3), the method further includes converting the guanosine adduct in the nucleic acid molecule of the product of step (2) into guanosine.

3. The method of claim 1 or 2, wherein, The carbonyl compound is selected from the compounds shown in Formula I and any combination thereof. Where R1 is an aldehyde group, C 1-3 Alkyl (e.g., methyl, ethyl, or n-propyl) or halogen-substituted C 1-3 Alkyl groups (e.g., trichloromethyl, trifluoromethyl), where R2 is H or C. 1-3 Alkyl (e.g., methyl, ethyl, or n-propyl), phenyl, or HC(=O)-CH(Br)-; or R1 and R2 form cyclic structures with the carbonyl groups they are attached to, such as ninhydrin; Preferably, the carbonyl compound is glyoxal.

4. The method according to any one of claims 1-3, wherein, In step (2), the nucleic acid molecules to be tested are reacted with nitrite in a solution containing the carbonyl compound; Preferably, the solution is water, an aqueous solution of p-toluenesulfonic acid, an aqueous solution of phosphoric acid, 2-(N-morpholine)ethanesulfonic acid (MES) buffer, sodium acetate buffer, 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, piperazine-1,4-diethanesulfonic acid (PIPES) buffer, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPPS) buffer, or tris(hydroxymethyl)aminomethane (TRIS) buffer, or any combination thereof; Preferably, the solution is a 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution; Preferably, the solution is a 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution, wherein the final concentration of 2-(N-morpholine)ethanesulfonic acid (MES) is 10-750 mM (e.g., 10-500 mM, 10-100 mM, 10-60 mM, 10-50 mM, 20-100 mM, 20-60 mM, 20-50 mM, for example, about 40 mM).

5. The method of claim 4, wherein, The pH of the solution is 4.0-6.5 (e.g., 4.0-4.5, 4.0-6.0, 4.5-6.0).

6. The method of claim 4 or 5, wherein, The total molar concentration of the carbonyl compound in the solution is greater than or equal to 50 mM; Preferably, the total molar concentration of the carbonyl compound in the solution is greater than or equal to 1.0 M (e.g., 1.0-2.5 M, 1.0-3.0 M, 1.0-4.0 M, 1.0-5.0 M, 2.0-2.5 M, 2.0-3.0 M, 2.0-4.0 M, 2.0-5.0 M, for example, about 2.3 M); Preferably, the solution contains glyoxal at a concentration greater than or equal to 50 mM, for example greater than or equal to 1.0 M (e.g., 1.0-2.5 M, 1.0-3.0 M, 1.0-4.0 M, 1.0-5.0 M, 2.0-2.5 M, 2.0-3.0 M, 2.0-4.0 M, 2.0-5.0 M, for example, about 2.3 M).

7. The method according to any one of claims 1-6, wherein, The nitrite is selected from sodium nitrite, potassium nitrite, and combinations thereof; Preferably, in step (2), the concentration of the nitrite is 0.5-2.0M, for example 0.5-0.8M, 0.5-1.0M, 0.5-1.2M, 0.5-1.5M, 0.8-1.0M, 0.8-1.2M, 0.8-1.5M, 0.8-2.0M, for example about 0.75M or about 1.0M.

8. The method of any one of claims 1-7, wherein in step (2), the nucleic acid molecule to be tested is reacted with nitrite at 16-95°C (e.g., 16-30°C, 16-50°C, 16-65°C, 16-70°C, 16-80°C, 30-55°C, 30-50°C, 30-65°C, 30-70°C, 30-80°C, 30-95°C, 40-95°C, 40-80°C, 40-70°C, 40-65°C, 40-60°C, 40-55°C, 40-50°C, 45-60°C, 45-55°C, 45-50°C, 50-95°C). React at temperatures of 5℃, 50-80℃, 50-70℃, 50-65℃, 50-60℃, 50-55℃, 60-95℃, 60-80℃, 60-70℃, 60-65℃, 65-70℃, 65-80℃, 65-95℃, 70-75℃, 75-80℃, 70-95℃, 80-85℃, 85-90℃, 80-95℃, 90-95℃ (for example, approximately 37℃, approximately 42℃, approximately 50℃, approximately 55℃, approximately 60℃, approximately 65℃, approximately 70℃, approximately 75℃, approximately 80℃, approximately 95℃). Preferably, in step (2), the nucleic acid molecules to be tested are reacted with nitrite at 16-95℃ (e.g., 16-30℃, 16-50℃, 16-65℃, 16-70℃, 16-80℃, 30-55℃, 30-50℃, 30-65℃, 30-70℃, 30-80℃, 30-95℃, 40-95℃, 40-80℃, 40-70℃, 40-65℃, 40-60℃, 40-55℃, 40-50℃, 45-60℃, 45-55℃, 45-50℃, 50-95℃, 50-80℃, 50-70℃, 50-65℃, 50-60℃, 50-55℃, 60-95℃, 60-80℃, 60...). Reaction time is 3-15 minutes (e.g., 3-12 minutes, 3-10 minutes, 5-15 minutes, 5-12 minutes, 5-10 minutes, 8-15 minutes, 8-12 minutes, 8-10 minutes, for example, approximately 5 minutes, approximately 10 minutes, approximately 15 minutes) at temperatures of -70℃, 60-65℃, 65-70℃, 65-80℃, 65-95℃, 70-75℃, 75-80℃, 70-95℃, 80-95℃, 80-95℃, for example, approximately 37℃, approximately 42℃, approximately 50℃, approximately 55℃, approximately 60℃, approximately 65℃, approximately 70℃, approximately 75℃, approximately 80℃, approximately 95℃) for 3-15 minutes (e.g., 3-12 minutes, 3-10 minutes, 5-15 minutes, 5-12 minutes, 5-10 minutes, 8-15 minutes, 8-12 minutes, 8-10 minutes, for example, approximately 5 minutes, approximately 10 minutes, approximately 15 minutes) for 3-15 minutes at temperatures of -70℃, 60-65℃, 65-70℃, 65-80℃, 65-95℃, 70-75℃, 75-80℃, 70-95℃, 80-85℃, 85-90℃, 80-95 ... Preferably, in step (2), the nucleic acid molecules to be tested are reacted with nitrite at 45-95℃ (e.g., 45-60℃, 45-55℃, 45-50℃, 50-95℃, 50-80℃, 50-70℃, 50-65℃, 50-60℃, 50-55℃, 60-95℃, 60-80℃, 60-70℃, 60-65℃, 65-70℃, 65-80℃, 65-95℃, 70-75℃, 75-80℃). React for 3-12 minutes (e.g., 3-12 minutes, 3-10 minutes, 5-12 minutes, 5-10 minutes, 8-12 minutes, 8-10 minutes, 8-12 minutes, 8-10 minutes, for example, about 5 minutes, about 10 minutes) at temperatures of 70-95℃, 80-85℃, 85-90℃, 80-95℃, 90-95℃, or approximately 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, and 95℃) for 3-12 minutes (e.g., 3-12 minutes, 3-10 minutes, 5-12 minutes, 5-10 minutes, 8-12 minutes, 8-10 minutes, or approximately 5 minutes and 10 minutes).

9. The method of any one of claims 1-8, wherein the method further comprises: A pretreatment step is performed on the nucleic acid molecules to be tested before step (2); Preferably, the pretreatment includes purifying, fragmenting, denaturing, or any combination thereof, the nucleic acid molecules to be tested.

10. The method of any one of claims 1-9, further comprising: Before step (3) and after step (2), the product nucleic acid molecules obtained in the previous step are purified, reverse transcribed and / or amplified.

11. The method according to any one of claims 1-10, wherein, In step (3), the detection includes nucleotide composition analysis of nucleic acid molecules by sequencing or hybridization, mass spectrometry (e.g., triple tandem quadrupole mass spectrometry), enzyme fragmentation and / or chromatography; Preferably, the detection includes nucleotide composition analysis of nucleic acid molecules by sequencing.

12. The method of claim 11, wherein, The method further includes: performing nucleotide composition analysis on the nucleic acid molecules to be tested that have not been treated in step (2) (e.g., not reacted with nitrite); Preferably, the method further includes comparing the nucleotide composition of the product nucleic acid molecule in step (3) and the test nucleic acid molecule that has not reacted with nitrite to determine the content and / or location information of N6-methyladenine in the test nucleic acid molecule.

13. The method of any one of claims 1-12, used for the detection of N6-methyladenine at the whole genome or transcriptome level.

14. The method of any one of claims 1-12, used for the detection of N6-methyladenine in a target nucleic acid molecule; Preferably, the method further includes a step of amplifying the nucleic acid molecule obtained in the previous step before step (2) and after step (3); preferably, the amplification includes using primers specific to the target nucleic acid molecule. Preferably, in step (3), the detection includes nucleotide composition analysis of the amplification product.

15. The method of any one of claims 1-14, wherein, The nucleic acid molecule to be tested is RNA, DNA, DNA / RNA hybrid, or any combination thereof.

16. The method of any one of claims 1-15, wherein, The nucleic acid molecule to be tested is RNA (e.g., mRNA); Preferably, the method includes: (i) adding reverse transcription-silent carrier RNA to the sample to be tested in step (1), (ii) adding reverse transcription-silent carrier RNA to the reaction system in step (2), and / or, (ii) adding reverse transcription-silent carrier RNA to the reaction product in step (2); The reverse transcription-silencing vector RNA can block or inhibit the synthesis of cDNA using itself as a template; Preferably, the method includes: adding the reverse transcription-silencing vector RNA to the sample to be tested in step (1); Preferably, the method includes: a step of purifying the nucleic acid molecule to be tested before step (2); and / or, a step of reverse transcription, amplification and purification of the product nucleic acid molecule obtained in the previous step before step (3) and after step (2).

17. The method of claim 16, wherein, The reverse transcription-silencing vector RNA contains one or more blocking groups capable of blocking or inhibiting the synthesis of cDNA using itself as a template; Preferably, the reverse transcription-silencing vector RNA contains one or more N1-cyanoethyl inosine (ce1I).

18. A kit comprising a carbonyl compound and a nitrite, and a reverse transcription-silencing vector RNA; wherein the reverse transcription-silencing vector RNA is capable of blocking or inhibiting the synthesis of cDNA using itself as a template; Preferably, the carbonyl compound is selected from compounds of formula I and any combination thereof. Where R1 is an aldehyde group, C 1-3 Alkyl (e.g., methyl, ethyl, or n-propyl) or halogen-substituted C 1-3 Alkyl groups (e.g., trichloromethyl, trifluoromethyl), where R2 is H or C. 1-3 Alkyl (e.g., methyl, ethyl, or n-propyl), phenyl, or HC(=O)-CH(Br)-; or, R1 and R2 form cyclic structures with the carbonyl groups they are attached to, such as ninhydrin; Preferably, the carbonyl compound is glyoxal; Preferably, the reverse transcription-silencing vector RNA contains one or more blocking groups capable of blocking or inhibiting the synthesis of cDNA using itself as a template; preferably, the reverse transcription-silencing vector RNA contains one or more N1-cyanoethyl inosine (ce1I); Preferably, the nitrite is selected from sodium nitrite, potassium nitrite, and combinations thereof.

19. The kit of claim 18, further comprising one or more of the following: (i) at least one of p-toluenesulfonic acid, phosphoric acid, 2-(N-morpholine)ethanesulfonic acid (MES), sodium acetate, 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-1,4-diethanesulfonic acid (PIPES), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPPS), and tris(hydroxymethyl)aminomethane (TRIS); (ii) Substances for preparing 2-(N-morpholine) ethanesulfonic acid (MES) buffer, substances for preparing sodium acetate buffer, substances for preparing 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, substances for preparing piperazine-1,4-diethanesulfonic acid (PIPES) buffer, substances for preparing 4-hydroxyethylpiperazine ethanesulfonic acid (HEPPS) buffer, substances for preparing tris(hydroxymethyl)aminomethane (TRIS) buffer, and any combination thereof; (iii) Hydrochloric acid, boric acid, acetic acid, and any combination thereof; Preferably, the kit further comprises reagents for mass spectrometry detection and / or nucleic acid sequencing; Preferably, the kit further includes an instruction manual; preferably, the instruction manual describes the method according to any one of claims 1-17.

20. Use of the kit according to claim 18 or 19 in the detection of N6-methyladenine in nucleic acid molecules.

21. An RNA purification method, comprising: (1) Provide a purified sample containing RNA, and a reverse transcription-silencing vector RNA; (2) Add the reverse transcription-silencing vector RNA to the sample to be purified; (3) The RNA of the sample containing the reverse transcription-silencing vector RNA was purified; The reverse transcription-silencing vector RNA contains one or more blocking groups capable of blocking or inhibiting the synthesis of cDNA using itself as a template; preferably, the reverse transcription-silencing vector RNA contains one or more N1-cyanoethyl inosine (ce1I); Preferably, the purified product of the method is used for nucleic acid processing involving reverse transcription.

22. Application of reverse transcription-silencing vector RNA in RNA purification; in, The reverse transcription-silencing vector RNA contains one or more blocking groups capable of blocking or inhibiting the synthesis of cDNA using itself as a template; preferably, the reverse transcription-silencing vector RNA contains one or more N1-cyanoethyl inosine (ce1I).

Images