Polymerase-Dependent DNA Synthesis from Phosphoramidate-Activated Nucleotides

Abstract

Nucleoside triphosphate mimetics, which are substrates for polymerases, can be used in the enzymatic synthesis of nucleic acids. Alternatively, they might also become reversible or irreversible enzyme inhibitors. In order to analyze the effects of 5′-phosphoramidate modification of deoxynucleotide in DNA synthesis, 3-phosphono-L-Ala-dNMP (N = A, T, or G) were evaluated as substrates of HIV-1 RT, Vent (exo−), and Therminator polymerase, respectively. The DNA-dependent DNA polymerase activity is significantly higher for Vent exo− polymerase than for HIV-1 RT, which is reflected by the capacity of Vent exo− polymerase to efficiently synthesize DNA without stalling effects. In addition, Vent (exo−) polymerase proved to be more accurate than Therminator polymerase, based on Watson–Crick base-pairing. The optimal yield (88%–97%) of full-length elongation can be obtained in 60 minutes by Vent (exo−) polymerase at 0.025 U/μL, with the phosphoramidate analogues as substrates. These data led us to conclude that the optimal pyrophosphate mimetic for the enzyme-catalyzed synthesis of DNA is polymerase dependent.