2-Formyl-dATP as Substrate for Polymerase Synthesis of Reactive DNA Bearing an Aldehyde Group in the Minor Groove.

Abstract

2-Formyl-2'-deoxyadenosine triphosphate (dCHO ATP) was synthesized and tested as a substrate in enzymatic synthesis of DNA modified in the minor groove with a reactive aldehyde group. The multistep synthesis of dCHO ATP was based on the preparation of protected 2-dihydroxyethyl-2'-deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The dCHO ATP triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX). On the other hand, longer sequences (31-mer) with higher number of modifications, or sequences with modifications at adjacent positions did not give full extension. Single-nucleotide extension followed by PEX was used for site-specific incorporation of one aldehyde-linked adenosine into a longer 49-mer sequence. The reactive formyl group was used for cross-linking with peptides and proteins using reductive amination and for fluorescent labelling through oxime formation with an AlexaFluor647-linked hydroxylamine.