A quantitative study of the flexibility contributed to RNA structures by nicks and single-stranded gaps

Disulfide crosslinking via thiol-disulfide interchange was applied to quantitate the relative flexibility contributed by nicks and single-stranded gaps in an RNA structure. An RNA duplex comprised of three strands was constructed containing the disulfide crosslink precursors 1 and 2 at opposite ends of the duplex on opposite strands. The third strand was of varying length to yield a nick or single-stranded gaps of 1, 2, or 3 nt, Crosslinking rates indicated relative flexibilities of the resulting two-helix junctions. Crosslinking in the nicked duplex occurred two orders of magnitude slower than in a duplex containing a 3-nt gap. Rates of crosslinking in duplexes with 3-and 2-nt gaps showed only modest dependence on the gap sequence, Many natural RNAs, including ribozymes, contain two-helix junctions related to the model system described here, The data suggest that two-helix junctions containing a nick in one strand will retain substantial rigidity, whereas one or more single-stranded nucleotides at a two-helix junction allow significant flexibility.