Enzymatic Polymerization of Phosphonate Nucleosides

✍ Scribed by Marleen Renders; Roel Lievrouw; Marcela Krecmerová; Antonin Holý; Piet Herdewijn

• Publisher: John Wiley and Sons
• Year: 2008
• Tongue: English
• Weight: 304 KB
• Volume: 9
• Category: Article
• ISSN: 1439-4227
• DOI: 10.1002/cbic.200800494

✦ Synopsis

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 K~M~ 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.