Comparisons between Chemical Mapping and Binding to Isoenergetic Oligonucleotide Microarrays Reveal Unexpected Patterns of Binding to the Bacillus subtilis RNase P RNA Specificity Domain

Abstract

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 Mg2+.

Microarrays for identifying binding sites and probing structure of RNAs

“…The analysis of RNA structural motifs binding to microarray probes demonstrates the following features: (i) bulges, internal loops and dangling ends bind strongly to microarray probes; (ii) a terminal stem can also bind strongly, presumably due to invasion of stem by the modified oligonucleotide; (iii) internal stems do not typically bind strongly. Along with further results described below, the data demonstrate that the location of the structural motif in the context of the whole target structure and both secondary and tertiary interactions influence the accessibilities of RNA to probe binding ( 32 , 33 , 62 , 63 ).…”

“… Comparison of calculated free energies of duplexes formed by DNA (purple line), 2′-O-methylRNA (green line), 2′-O-methylRNA including 2′-O-methyl-2,6-diaminopurine riboside (red line), isoenergetic probes (LNA and 2′-O-methylRNA including 2′-O-methyl-2,6-diaminopurine riboside) (blue line) and complementary single-stranded sequence fragments of RNase P RNA from Bacillus subtilis (RNRspBs). This plot has corrections to the plot on Figure S2 of ( 62 ). …”

Mapping Targetable Sites on Human Telomerase RNA Pseudoknot/Template Domain Using 2′-OMe RNA-interacting Polynucleotide (RIPtide) Microarrays

“…In addition, we incorporated 2Ј-OMe substituents on all of the RIPtides displayed on the microarray (Fig. 1, A and B), a modification known to increase oligonucleotide affinity for RNA targets (42,43) as well as stability toward nucleolytic degradation; this modification has previously been used by Turner and co-workers (13)(14)(15)(16)(17)(18) to increase the resolution of RNA structure-mapping by microarray screening.…”

“…Of particular note in the latter regard is the work of Turner and colleagues (15)(16)(17)(18), who employed microarrays of 2Ј-OMe ribonucleotides and locked nucleic acids to perform an independent experimental assessment of the predicted secondary structure for several biologically relevant RNAs. Efforts along these lines to date have involved sparse sampling of ligand sequence space, with at most 320 candidate sequences examined per study, these having been designed on the basis of Watson-Crick complementarity to prospective docking sequences on the RNA target of interest.…”

“…Here, we report the development of microarrays that comprehensively represent all sequences of 2Ј-OMe RNA-interacting polynucleotides (2Ј-OMe RIPtides) having the four natural nucleobases (A, C, G, and U), and ranging in length from 4-to 8-mers. This screening platform includes a total of 87,296 individual oligonucleotide analog probes, vastly exceeding the few hundreds of nucleic acid binders employed in previously reported microarray studies (13)(14)(15)(16)(17)(18).…”