HYPERMUTAGENESIS OF RNA USING HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE AND BIASED DNTP CONCENTRATIONS

The finding of G --> A hypermutated retroviral genomes in which up to 40% of guanines may be substituted by adenines was proposed to result from the depletion of the intracellular dCTP concentration and suggested a means to hypermutagenize nucleic acids, Using a RNA/reverse transcriptase ratio of approximate to 1:30, comparable to that within the retroviral replication complex, G --> A hypermutants were produced in a simple in vitro reaction using highly biased dNTP concentrations-i.e., a low ratio of [dCTP]/[dTTP]. Up to 38% of G residues could be substituted, the proportion being inversely proportional to the concentration of dCTP, As G --> A hypermutation resulted from elongation beyond multiple rG.dT mismatches, U --> C hypermutants resulting from multiple rU.dG mismatches were sought, and found, during cDNA synthesis using low [dATP] and high [dGTP]. Mixed G --> A and U --> C hypermutants could also be produced under conditions of low [dCTP] plus low [dATP] and high [dTTP] plus high [dGTP]. Hypermutagenesis should allow jumping through, and subsequent exploration of, sequence space to a greater degree than heretofore and, in conjunction with genetic screening, might be of use in the search of proteins or ribozymes with novel or enhanced properties.