Comparisons between chemical mapping and binding to isoenergetic oligonucleotide microarrays reveal unexpected patterns of binding to the Bacillus subtilis RNase P RNA specificity domain

Microarrays with isoenergetic pentamer and hexamer 2'-O-methyl oligonucleotide probes with LNA (locked nucleic acid) and 2,6-diaminopurine substitutions were used to probe the binding sites on the RNase P RNA specificity domain of Bacillus subtilis. Unexpected binding patterns were revealed. Because of their enhanced binding free energies, isoenergetic probes can break short duplexes, merge adjacent loops, and/or induce refolding. This suggests new approaches to the rational design of short oligonucleotide therapeutics but limits the utility of microarrays for providing constraints for RNA structure determination. The microarray results are compared to results from chemical mapping experiments, which do provide constraints. Results from both types of experiments indicate that the RNase P RNA folds similarly in 1 M Na(+) and 10 mM Mg(2+).