QUANTITATIVE HYBRIDIZATION KINETICS OF DNA PROBES TO RNA IN SOLUTION FOLLOWED BY DIFFUSIONAL FLUORESCENCE CORRELATION-ANALYSIS

Binding kinetics in solution of six N,N,N'N'-tetramethyl-5-carboxyrhod amine-labeled oligodeoxyribonucleotide probes to a 101mer target RNA c omprising the primer binding site for HIV-1 reverse transcriptase were characterized using fluorescence correlation spectroscopy (FCS). FCS allows a sensitive, non-radioactive real time observation of hybridiza tion of probes to the RNA target in the buffer of choice without separ ation of free and bound probe. The binding process could directly be m onitored by the change in translational diffusion time of the 17mer to 37mer DNA probe upon specific hybridization with the larger RNA targe t. The characteristic diffusion time through a laser-illuminated open volume element with 0.5 mu m in diameter increased from 0.13-0.2 ms (f ree) to 0.37-0.50 ms (bound), depending an the probe. Hybridization wa s approximated by biphasic irreversible second-order reaction kinetics , yielding first-phase association rate constants between 3 x 10(4) an d 1.5 x 10(6) M(-1) s(-1) for the different probes. These varying init ial rates reflected the secondary structures of probes and target site s, being consistent with a hypothetical binding pathway starting from loop-loop interactions in a kissing complex, and completion of hybridi zation requiring an additional interaction involving single-stranded r egions of both probe and target. FCS thus permits rapid screening for suitable antisense nucleic acids directed against an important target like HIV-1 RNA with low consumption of probes and target.