Enzymatic Polymerization of Phosphonate Nucleosides
- 14 November 2008
- journal article
- research article
- Published by Wiley in ChemBioChem
- Vol. 9 (17), 2883-2888
- https://doi.org/10.1002/cbic.200800494
Abstract
5′-O-Phosphonomethyl-2′-deoxyadenosine (PMdA) proved to be a good substrate of the Therminator polymerase. In this article, we investigated whether the A, C, T and U analogues of this phosphonate nucleoside (PMdN) series can function as substrates of natural DNA polymerases. PMdT and PMdU could only be polymerized enzymatically to a limited extent. Nevertheless, PMdA and PMdC could be incorporated into a DNA duplex with complete chain elongation by all the DNA polymerases tested. A mixed sequence of four nucleotides containing modified C, T and A residues could be obtained with the Vent(exo−) and Therminator polymerases. The kinetic values for the incorporation of PMdA by Vent(exo−) polymerase were determined; a reduced KM value was found for the incorporation of PMdA compared to the natural substrate. Future polymerase directed evolution studies will allow us to select an enzyme with a heightened capacity to process these modified DNA building blocks into modified strands.Keywords
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