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The finding of G--&gt;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 approximately 1:30, comparable to that within the retroviral replication complex, G--&gt;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--&gt;A hypermutation resulted from elongation beyond multiple rG.dT mismatches, U--&gt;C hypermutants resulting from multiple rU.dG mismatches were sought, and found, during cDNA synthesis using low [dATP] and high [dGTP]. Mixed G--&gt;A and U--&gt;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.

Hypermutagenesis of RNA using human immunodeficiency virus type 1 reverse transcriptase and biased dNTP concentrations.