Enzymatic polymerization of phosphonate nucleosides.
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.
Renders
Marleen
M
Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
Lievrouw
Roel
R
Krecmerová
Marcela
M
Holý
Antonin
A
Herdewijn
Piet
P
D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009705:Nucleosides / Q000737:chemistry*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D063065:Organophosphonates / Q000737:chemistry*; D013379:Substrate Specificity; D013698:Templates, Genetic
D009705:Nucleosides; D009841:Oligonucleotides; D063065:Organophosphonates; D004259:DNA-Directed DNA Polymerase