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2‐Formyl‐dATP as Substrate for Polymerase Synthesis of Reactive DNA Bearing an Aldehyde Group in the Minor Groove
Author(s) -
Krömer Matouš,
Brunderová Mária,
Ivancová Ivana,
Poštová Slavětínská Lenka,
Hocek Michal
Publication year - 2020
Publication title -
chempluschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.801
H-Index - 61
ISSN - 2192-6506
DOI - 10.1002/cplu.202000287
Subject(s) - chemistry , aldehyde , hydroxylamine , stereochemistry , deoxyadenosine , nucleotide , polymerase , dna , protecting group , dna polymerase , biochemistry , organic chemistry , gene , alkyl , catalysis
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 of d CHO ATP was based on the preparation of protected 2‐dihydroxyethyl‐2′‐deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The d CHO 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.