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

AUTOR(ES)

Martinez, M A

RESUMO

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 approximately 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.

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