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Updated: Jan 29, 2026
Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation
Published on: May 3, 2018
Bisulfite-free and base-resolution analysis of 5-methylcytidine and 5-hydroxymethylcytidine in RNA with
Fang Yuan1, Ying Bi2, Paulina Siejka-Zielinska2
1Ludwig Institute for Cancer Research and Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, OX3 7FZ, UK. chunxiao.song@ludwig.ox.ac.uk and Beijing National Laboratory for Molecular Sciences (BNLMS), MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry, Peking University, Beijing 100871, China. zxx@pku.edu.cn.
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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