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Issue 16, 2019
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Bisulfite-free and base-resolution analysis of 5-methylcytidine and 5-hydroxymethylcytidine in RNA with peroxotungstate

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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.

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

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Supplementary files

Article information


Submitted
11 Jan 2019
Accepted
30 Jan 2019
First published
30 Jan 2019

This article is Open Access

Chem. Commun., 2019,55, 2328-2331
Article type
Communication

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

F. Yuan, Y. Bi, P. Siejka-Zielinska, Y. Zhou, X. Zhang and C. Song, Chem. Commun., 2019, 55, 2328
DOI: 10.1039/C9CC00274J

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