Conversion of stable RNA hairpin to a metastable dimer in frozen solution

Abstract

Previous studies employing a 79-nucleotide (nt) RNA indicated that this RNA could form two bands in a native polyacrylamide gel while one band was observed in a denaturing gel. This report describes an investigation on the nature of the two corresponding structures and the segment responsible for forming the slower mobility band. Sedimentation equilibrium of the 79-nt RNA was consistent with the two gel bands corresponding to monomer and dimer forms. The portion of the RNA required for dimer formation was explored using a secondary structure prediction algorithm of two 79-nt RNAs linked in a head-to-tail fashion. The predicted structure suggested that the first 21-nt at the 5′ end of each RNA formed a self-complementary duplex. A ribonuclease H assay carried out with RNA prepared as monomer (M), or a mixture of monomer and dimer (M/D), gave results consistent with the predicted M and D structures. Gel mobility experiments on 5′ and 3′ segments of the 79-nt RNA also indicated that dimer formation was due to the 21-nt 5′ end. Studies on the 21-nt RNA molecule and sequence variants showed that this sequence can form a hairpin and a dimer complex. Unexpectedly, the hairpin to dimer conversion was shown to occur at high efficiency in frozen solution, although little or no conversion was observed above 0°C. The results indicate that a freezing environment can promote formation of intermolecular RNA complexes from stable RNA hairpins, supporting the notion that this environment could have played a role in the evolution of RNA complexity.