Formation of a metastable intramolecular RNA kissing complex by nanomanipulation

Pan T. X. Li
The stability of an RNA tertiary interaction can be affected by the region linking the two interacting domains. To examine this effect, we designed a simple RNA structure consisting of a pair of hairpins linked by a single-stranded region of 13 to 30 nucleotides. The two hairpins, each with a GACG tetraloop, can form a kissing complex of two GC base pairs. Formation of an intramolecular kissing complex extends the single-stranded linker generating an internal tension that destabilizes the tertiary interaction. Using optical tweezers, we nanomanipulated single RNA molecules into a metastable intramolecular kissing complex by offsetting the internal tension with applied force. When the kissing complex formed, the linker was stretched to nearly the full length of the kissing and hairpin helices. At such an extension, so much tension was exerted that with linkers shorter than 15 nucleotides the hairpins became unstable, which in turn prevented a kissing interaction. The RNA with a 15-nucleotide linker was able to form a transient kissing complex, an unnatural conformation, only when the applied force stretched the linker to >60% of its contour length. A 30-nucleotide linker allowed formation of a thermodynamically stable kissing complex. Experimentally observed effects of the linker length and its tension on the kissing interaction can be reasonably explained by the worm-like-chain polymer model. Our results showed the importance of internal tension and spatial separation of interacting domains in RNA tertiary folding.
DOI