Reverse transcriptase incorporation of 1,5-anhydrohexitol nucleotides

Several reverse transcriptases were studied for their ability to accept anh ydrohexitol triphosphates, having a conformationally restricted six-membere d ring, as substrate for template-directed synthesis of HNA. It was found t hat AMV, M-MLV, M-MLV (H-), RAV2 and HIV-1 reverse transcriptases were able to recognise the anhydrohexitol triphosphate as substrate and to efficient ly catalyse the incorporation of one non-natural anhydrohexitol nucleotide opposite a natural complementary nucleotide. However, only the dimeric enzy mes, the RAV2 and HIV-1 reverse transcriptases, seemed to be able to furthe r extend the primer with another anhydrohexitol building block. Subsequentl y, several HIV-1 mutants (4xAZT, 4xAZT/L100I, L74V, M184V and K65A) were li kewise analysed, resulting in selection of K65A and, in particular, M184V a s the most succesful mutant HIV-1 reverse transcriptases capable of elongat ing a DNA primer with several 1,5-anhydrohexitol adenines in an efficient w ay. Results of kinetic experiments in the presence of this enzyme revealed that incorporation of one anhydrohexitol nucleotide of adenine or thymine g ave an increased (for 1,5-anhydrohexitol-ATP) and a slightly decreased (for 1,5-anhydrohexitol-TTP) K-m value in comparison to that of their natural c ounterparts. However, no more than four analogues could be inserted under t he experimental conditions required for selective incorporation. Investigat ion of incorporation of the altritol anhydrohexitol nucleotide of adenine i n the presence of M184V and Vent (exo(-)) DNA polymerase proved that an adj acent hydroxyl group on C3 of 1,5-anhydrohexitol-ATP has a detrimental effe ct on the substrate activity of the six-ring analogue. These results could be rationalised based on the X-ray structure of HIV-1 reverse transcriptase .