Phosphodiester substrates for incorporation of nucleotides in DNA using HIV-1 reverse transcriptase.

In previous research we demonstrated that some amino acid derivatives of deoxyadenosine 5'-O-monophosphate act as substrates for incorporation into DNA by HIV-1 reverse transcriptase while retaining the canonical base-pair selectivity for all natural bases. Thus, some amino acids mimic the pyrophosphate group in the polymerization process with this enzyme. Herein we extended this study to the evaluation of a range of potential new leaving groups with aromatic and aliphatic structures carrying one or two carboxylic acid functions. Out of this series, the isophthalic acid derivative of deoxyadenosine 5'-O-monophosphate gave single-nucleotide incorporation results similar to those obtained with the L-aspartic acid phosphoramidate of deoxyadenosine monophosphate. The glycolic acid analogue is a better substrate than the glycine congener, supporting the good leaving group properties of a phosphodiester linkage. These investigations provide new insight into the structural requirements for leaving groups that can mimic the pyrophosphate moiety of nucleoside triphosphates.