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2-Formyl-dATP as Substrate for Polymerase Synthesis of Reactive DNA Bearing an Aldehyde Group in the Minor Groove.
- Source :
-
ChemPlusChem [Chempluschem] 2020 Jun; Vol. 85 (6), pp. 1164-1170. - Publication Year :
- 2020
-
Abstract
- 2-Formyl-2'-deoxyadenosine triphosphate (d <superscript>CHO</superscript> 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 <superscript>CHO</superscript> 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 <superscript>CHO</superscript> 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.<br /> (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
Details
- Language :
- English
- ISSN :
- 2192-6506
- Volume :
- 85
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- ChemPlusChem
- Publication Type :
- Academic Journal
- Accession number :
- 32496002
- Full Text :
- https://doi.org/10.1002/cplu.202000287