Nucleic Acids Research Advance Access published online on July 13, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp587
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Methods Online |
Improvement of RNA secondary structure prediction using RNase H cleavage and randomized oligonucleotides
Department of Chemistry and Biochemistry, Canisius College, 2001 Main St., Buffalo, NY 14208, USA
*To whom correspondence should be addressed. Tel: +1 716 888 2342; Fax: +1 716 888 3112; Email: childsdj{at}canisius.edu
Received April 12, 2009. Revised June 24, 2009. Accepted June 25, 2009.
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.