Single-nucleotide resolution of N⁶-adenine methylation sites in DNA and RNA by nitrite sequencing

Abstract

A single-nucleotide resolution sequencing method of N6-Adenine methylation sites in DNA and RNA is described. Using sodium nitrite under acidic conditions, chemoselective deamination of unmethylated adenines readily occurs, without competing...

NT-seq: a chemical-based sequencing method for genomic methylome profiling

“…As the methyl groups of 6mA and 4mC are located at the amino groups of adenine and cytosine, 6mA and 4mC can block the deamination of adenine and cytosine under nitrite conditions. Recently, liquid chromatography mass spectrometry (LC-MS) results showed that N 6 -methyladenosine (m 6 A) was converted to N 6 -nitroso-m6A (m 6 A-NO) but not deaminated inosine by nitrite treatment [ 21 , 22 ]. While the nitrite treatment has been adapted to detect DNA 6mA and RNA m 6 A, it has been only tested in oligos or targeted RNA locus as a proof-of-concept for methylation sequencing [ 21 , 22 ].…”

“…Recently, liquid chromatography mass spectrometry (LC-MS) results showed that N 6 -methyladenosine (m 6 A) was converted to N 6 -nitroso-m6A (m 6 A-NO) but not deaminated inosine by nitrite treatment [ 21 , 22 ]. While the nitrite treatment has been adapted to detect DNA 6mA and RNA m 6 A, it has been only tested in oligos or targeted RNA locus as a proof-of-concept for methylation sequencing [ 21 , 22 ]. Moreover, it can also be used to distinguish 5mC from cytosine because the deamination rate of 5mC in nitrite treatment has been reported to be up to 4.5-fold higher than cytosine [ 32 ] (Fig.…”

“…For adenine methylation, although it was reported more than 60 years ago [ 19 , 20 ], such a condition that could be applied for sequencing was not clarified until recently. Deamination induced by nitrous acid has been shown only to deaminate unmethylated adenine but not 6mA, which was utilized to develop nitrite sequencing [ 21 ] and NOseq [ 22 ] for DNA 6mA or RNA m6A detection in oligos or targeted sequencing settings. As far as we know, nitrite treatment has not been applied for genomic methylome profiling because generating the genomic sequencing library in such conditions and following bioinformatic analysis are still challenging [ 23 ].…”

Quantification and mapping of DNA modifications

“…A single-nucleotide resolution mapping analysis of 6mA in DNA and RNA was recently developed using nitrite labeling-mediated sequencing (Figure 4E). 194 In this study, treatment of DNA or RNA by sodium nitrite under acidic conditions led to the chemoselective deamination of unmethylated adenines, but not the 6mA. The deamination of adenines resulted in hypoxanthine bases, which were read as guanine during replication and reverse transcription, while 6mA resisted deamination and was read as adenine.…”

Synthesis of Nucleobase‐Modified Oligonucleotides by Post‐Synthetic Modification in Solution

“…The acid‐mediated nitrate reaction allowed for a single‐nucleotide resolution sequencing method of N 6 ‐adenine methylation sites in DNA and RNA (Scheme 22). [85] In the presence of sodium nitrate in 2.3 % or 5 % acetic acid solution, the adenine base in the oligonucleotide 82 was transformed into inosine base 84 , which was read as a guanine base by polymerases and reverse transcriptases. On the contrary, N 6 ‐methyladenine 83 was subjected to the same conditions to produce N 6 ‐nitroso‐ N 6 ‐methyladenine 85 , which was read as an adenine base by the enzymes.…”