Teaching and old dog new tricks – a heavy atom label for pinpointing m6A-modifications on RNA
Selenium-modified nucleotides have found long-standing use in the structure elucidation of nucleic acids by X-ray crystallography[1,2]. To synthesize Selenium-substituted DNA enzymatically, 4-Seleno-dTTP (4SedTTP) (Scheme 1 A) serves as an excellent substrate analog of dTTP and is readily accepted by DNA-dependent DNA polymerases (Scheme 1 B). When subjecting 4SedTTP to reverse transcription reactions, incorporation by numerous reverse transcriptases is dependent on the methylation pattern of the RNA template (Scheme 1 C). While unmethylated adenosine moieties permit extension of the reverse transcript, elongation is partially stalled at m6A-bearing positions. Combined with FTO-demethylase treatment of the RNA target, 4SedTTP permits the localization of epigenetic m6A- positions
Scheme 1 A Structure of 4SedTTP. B Enzymatic incorporation of 4SedTTP into DNA by DNA-dependent DNA polymerases. Crystallization and X-ray-analysis of the Se-modified Replicon has been proposed for phasing by MAD. C NGS-based FTO demethylase assay for mapping m6A-sites on RNA. The m6A-modified template is treated in presence or absence of FTO demethylase and subjected to reverse transcription using 4SedTTP. Demethylation of the template yields the corresponding full-length cDNA, whereas abortive cDNAs are obtained from the m6A-containing template. After library preparation, NGS permits the localization of m6A-sites.
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