KINETICS OF SPONTANEOUS DISPLACEMENT OF RNA FROM HETERODUPLEXES BY DNA

We have used R-loop formation and direct hybridization techniques to a nalyze the kinetics by which RNA is displaced from a heteroduplex by D NA of identical sequence. Using random walk simulations we were able t o calculate the step times for a single displacement reaction. For RNA with a GC Content of 57-60% the data indicate an RNA exchange probabi lity 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 nucleot ide is 345.0 (+/-1.3) ms/step. An acceleration of the displacement rea ction was observed in the absence of magnesium. A comparison with step times for elongation shows that RNA displacement would not be rate li miting to transcription elongation under two conditions: (i) if magnes ium is eliminated from the newly synthesized heteroduplex; (ii) if dis placement is kept in a forward only exchange mode through binding of t he emerging RNA. Distamycin, a minor groove binding drug, is very effe ctive 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 th erefore serve as a convenient assay for the determination of binding p references of nucleic acid ligands.