Improvement of RNA secondary structure prediction using RNase H cleavage and randomized oligonucleotides

Abstract

RNA secondary structure prediction using free energy minimization is one method to gain an approximation of structure. Constraints generated by enzymatic mapping or chemical modification can improve the accuracy of secondary structure prediction. We report a facile method that identifies single-stranded regions in RNA using short, randomized DNA oligonucleotides and RNase H cleavage. These regions are then used as constraints in secondary structure prediction. This method was used to improve the secondary structure prediction of Escherichia coli 5S rRNA. The lowest free energy structure without constraints has only 27% of the base pairs present in the phylogenetic structure. The addition of constraints from RNase H cleavage improves the prediction to 100% of base pairs. The same method was used to generate secondary structure constraints for yeast tRNAPhe, which is accurately predicted in the absence of constraints (95%). Although RNase H mapping does not improve secondary structure prediction, it does eliminate all other suboptimal structures predicted within 10% of the lowest free energy structure. The method is advantageous over other single-stranded nucleases since RNase H is functional in physiological conditions. Moreover, it can be used for any RNA to identify accessible binding sites for oligonucleotides or small molecules.

Molecular paleontology: a biochemical model of the ancestral ribosome

“…RNase H cleavage is broadly used to characterize RNA secondary structure, allowing discrimination between single-stranded and double-stranded RNA (43,44). RNase H recognizes and cleaves RNA/DNA heteroduplexes; double-stranded RNA inhibits cleavage by blocking heteroduplex formation.…”

Allele-Specific Suppression of Mutant Huntingtin Using Antisense Oligonucleotides: Providing a Therapeutic Option for All Huntington Disease Patients

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Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA

“…To directly test the potential ability of DISC to compete with target secondary structure, the cleavage activity of DISC was compared with that of E. coli RNase H, an endoribonuclease that is incapable of cleaving dsRNA ( 30 ), but is instead targeted to ∼8-nt long single-stranded RNA using a complementary short DNA probe ( 31 ). Thus, detection of RNA cleavage by RNase H provides evidence that the target site is solvent-exposed and largely single-stranded.…”