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 ≈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.