THE RNA SUBSTRATE OF THE TOBACCO RINGSPOT VIRUS DERIVED HAIRPIN RIBOZYME AND ITS DNA ANALOG ADOPT DISTINCT ORDERED STRUCTURES IN SOLUTION -AN INTRINSIC PHOTOAFFINITY STUDY

The conformational states adopted in solution by the RNA and DNA subst rates of the ribozyme derived from the negative strand of the satellit e RNA of the tobacco ringspot virus (-sTRSV) were covalently trapped u sing 4-thio(deoxy)uridine intrinsic photolabeling. The nature of the c rosslinks and the behavior of the unsubstituted oligomers on native ge l electrophoresis demonstrate that, in the cleavage buffer, the RNA su bstrate self-associates as an imperfect duplex. The corresponding diss ociation constant is close to 10(-6)M at 10 degrees C and increases wi th temperature. The DNA substrate, on the other hand, folds as a hairp in which remains stable at temperatures up to 37 degrees C. Unexpected ly, molecular mechanics predicts the existence of both the DNA and RNA hairpins and it is likely that such a folding of the RNA substrate is prevented by interactions of solvent molecules and counter-ions with the RNA backbone. The existence of a stable hairpin form of the DNA an alog under cleavage conditions is in agreement with its poor binding a ffinity to the (-)sTRSV ribozyme (Chowrira et al., Biochemistry, 1991, 30, 8518-8522 and Dos Santos et al., Nucleic Acids Res., 1993, 21, 20 1-207). The data reported here demonstrate the unique advantage of the intrinsic photolabeling methodology which allows simultaneous charact erization of at least two ordered forms of the oligomers at micromolar and submicromolar strand concentration and provides rough estimation of their apparent dissociation constants.