We have used R-loop formation and direct hybridization techniques to analyze the kinetics by which RNA is displaced from a heteroduplex by DNA of identical sequence. Using random walk simulations we were able to calculate the step times for a single displacement reaction. For RNA with a GC content of 57-60% the data indicate an RNA exchange probability of 50.06%, which is indicative of a modest destabilization of the heteroduplex compared with a DNA duplex in the presence of magnesium. The average step time for the reversible exchange of a single nucleotide is 345.0 (± 1.3) ms/step. An acceleration of the displacement reaction was observed in the absence of magnesium. A comparison with step times for elongation shows that RNA displacement would not be rate limiting to transcription elongation under two conditions: (i) if magnesium is eliminated from the newly synthesized heteroduplex; (ii) if displacement is kept in a forward only exchange mode through binding of the emerging RNA. Distamycin, a minor groove binding drug, is very effective as a 'catalyst' of RNA displacement. This effect is likely to be due to preferential binding of distamycin to the minor groove of the DNA duplex as opposed to the heteroduplex. This kinetic assay could therefore serve as a convenient assay for the determination of binding preferences of nucleic acid ligands.
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