Bisulfite-free and base-resolution analysis of 5-methylcytidine and 5-hydroxymethylcytidine in RNA with peroxotungstate

Abstract

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), two of the best-studied DNA modifications, play crucial roles in normal development and disease in mammals. Although 5-methylcytidine (m⁵C) and 5-hydroxymethylcytidine (hm⁵C) have also been identified in RNA, their distribution and biological function in RNA remain largely unexplored, due to the lack of suitable sequencing methods. Here, we report a base-resolution sequencing method for hm⁵C in RNA. We applied the selective oxidation of hm⁵C to trihydroxylated-thymine (^thT) mediated by peroxotungstate. ^thT was subsequently converted to T during cDNA synthesis using a thermostable group II intron reverse transcriptase (TGIRT). Base-resolution analysis of the hm⁵C sites in RNA was performed using Sanger sequencing. Furthermore, in combination with the TET enzyme oxidation of m⁵C to hm⁵C in RNA, we expand the use of peroxotungstate oxidation to detect m⁵C in RNA at base-resolution. By using this method, we confirmed three known m⁵C sites in human tRNA, demonstrating the applicability of our method in analyzing real RNA samples.