Site-specific RNA modification via initiation of in vitro transcription reactions with m6A and isomorphic emissive adenosine analogs

• 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
• Subjects: Chemistry
• ISSN: 2633-0679; 2633-0679
• DOI: 10.1039/d4cb00045e

The templated enzymatic incorporation of adenosine and its analogs, including m6A, thA and tzA 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.The templated enzymatic incorporation of adenosine and its analogs, including m6A, thA and tzA 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.