Site-specific RNA modification via initiation of in vitro transcription reactions with m 6 A and isomorphic emissive adenosine analogs

The templated enzymatic incorporation of adenosine and its analogs, including m A, A and A into RNA transcripts, has been explored. Enforced transcription initiation with excess free nucleosides and the native triphosphates generates 5'-end modified transcripts, which can be 5'-phosphoryla...

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Bibliographic Details
Published in:RSC chemical biology 2024-05, Vol.5 (5), p.454-458
Main Authors: Cong, Deyuan, Steinbuch, Kfir B, Koyama, Ryosuke, Lam, Tyler V, Lam, Jamie Y, Tor, Yitzhak
Format: Article
Language:English
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Summary:The templated enzymatic incorporation of adenosine and its analogs, including m A, A and A into RNA transcripts, has been explored. Enforced transcription initiation with excess free nucleosides and the native triphosphates generates 5'-end modified transcripts, which can be 5'-phosphorylated and ligated to provide full length, singly modified RNA oligomers. To explore structural integrity, functionality and utility of the resulting non-canonical purine-containing RNA constructs, a MazF RNA hairpin substrate has been synthesized and analyzed for its susceptibility to this endonuclease. Additionally, RNA substrates, containing a singly incorporated isomorphic emissive nucleoside, can be used to monitor the enzymatic reactions in real-time by steady state fluorescence spectroscopy.
ISSN:2633-0679
2633-0679
DOI:10.1039/d4cb00045e