A QUANTITATIVE STUDY OF THE FLEXIBILITY CONTRIBUTED TO RNA STRUCTURESBY NICKS AND SINGLE-STRANDED GAPS

Disulfide crosslinking via thiol-disulfide interchange was applied to quantitate the relative flexibility contributed by nicks and single-st randed gaps in an RNA structure. An RNA duplex comprised of three stra nds was constructed containing the disulfide crosslink precursors 1 an d 2 at opposite ends of the duplex on opposite strands. The third stra nd was of varying length to yield a nick or single-stranded gaps of 1, 2, or 3 nt, Crosslinking rates indicated relative flexibilities of th e resulting two-helix junctions. Crosslinking in the nicked duplex occ urred two orders of magnitude slower than in a duplex containing a 3-n t gap. Rates of crosslinking in duplexes with 3-and 2-nt gaps showed o nly modest dependence on the gap sequence, Many natural RNAs, includin g ribozymes, contain two-helix junctions related to the model system d escribed here, The data suggest that two-helix junctions containing a nick in one strand will retain substantial rigidity, whereas one or mo re single-stranded nucleotides at a two-helix junction allow significa nt flexibility.