Rapidly photocleavable triazene-functionalized nucleotides for DNA sequencing

Abstract

A method for the preparation of triazene-functionalized nucleotides and their rapidly photocleavable application in DNA sequencing is described. Amine-modified nucleotides can couple with aryl diazonium salts in mild aqueous media, yielding triazene-modified nucleotides, which are then fluorescently labeled to afford the corresponding triazene-functionalized nucleotides. Since 7-deaza-dGTP tends to form azo compounds with aryl diazonium salts, its functionalization is achieved via a triazene-compatible Sonogashira reaction. These triazene-functionalized nucleotides exhibit excellent enzymatic incorporation efficiency, and the triazene linker on DNA can be rapidly photocleaved within 5 s when promoted by a suitable reducing agent. The radical pathway may be the key to accelerating the photocleavage rate, which significantly reduces the time cost for DNA sequencing by synthesis. The application potential of four-color fluorescently labeled reversible terminator nucleotides in single-molecule DNA sequencing by synthesis is also verified.