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4-Seleno-dTTP: NGS-based mapping of m6A-modified RNA sites

Epigenetic RNA analysis

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)[3]. 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)[4]. 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

  • at single nucleotide resolution
  • without antibodies
  • in an NGS-based approach.

Scheme 1 A: Structure of 4SedTTP

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[4]. 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.

4SedTTP available from Jena Bioscience. Please also note our alternative Selenium labels!

CompoundCat. No.AmountPrice (€)
4SedTTPNU-9791 mg600,00

Selected References:

[1] Jiang et al. (2007) Selenium derivatization of nucleic acids for crystallography. Nucleic Acids Res. 35 (2):477.
[2] Serganov et al. (2005) Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation. Nat. Struct. Mol. Biol. 12 (3):218.
[3] Caton-Williams et al. (2008) Synthesis and DNA-polymerase incorporation of colored 4-selenothymidine triphosphate for polymerase recognition and DNA visualization. Angew. Chem. Int. Ed. Engl. 47 (9):1723.
[4] Hong et al. (2018) Precise Antibody-Independent m6A Identification via 4SedTTP-Involved and FTO-Assisted Strategy at Single-Nucleotide Resolution. J. Am. Chem. Soc. 140 (18):5886.