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.
Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
