2-Formyl-dATP as Substrate for Polymerase Synthesis of Reactive DNA Bearing an Aldehyde Group in the Minor Groove
Abstrakt
2-Formyl-2'-deoxyadenosine triphosphate (d(CHO)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 ofd(CHO)ATPwas based on the preparation of protected 2-dihydroxyethyl-2'-deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage.
Thed(CHO)ATPtriphosphate 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.