Bisulfite-free and Base-resolution Analysis of 5-methylcytidine and 5-hydroxymethylcytidine in RNA with Peroxotungstate

Overview

Journal Chem Commun (Camb)

Publisher Royal Society of Chemistry

Specialty Chemistry

Date 2019 Feb 7

PMID 30723849

Citations 22

Authors

Fang Yuan

Ying Bi

Paulina Siejka-Zielinska

Ying-Lin Zhou

Xin-Xiang Zhang

Chun-Xiao Song

Affiliations

Soon will be listed here.

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.

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