Interpreting Oligonucleotide Microarray Data To Determine RNA Secondary Structure:  Application to the 3‘ End of Bombyx mori R2 RNA

Abstract

A method to deduce RNA secondary structure on the basis of data from microarrays of 2'-O-methyl RNA 9-mers immobilized in agarose film on glass slides is tested with a 249 nucleotide RNA from the 3' end of the R2 retrotransposon from Bombyx mori. Various algorithms incorporating binding data and free-energy minimization calculations were compared for interpreting the data to provide possible secondary structures. Two different methods give structures with 100 and 87% of the base pairs determined by sequence comparison. In contrast, structures predicted by free-energy minimization alone by Mfold and RNAstructure contain 52 and 72% of the known base pairs, respectively. This combination of high throughput microarray techniques with algorithms using free-energy calculations has potential to allow for fast determination of RNA secondary structure. It should also facilitate the design of antisense and siRNA oligonucleotides.

Selective quenching of fluorescence from unbound oligonucleotides by gold nanoparticles as a probe of RNA structure

“…The threedimensional structure of RNA can often be modeled once the secondary structure is known (Michel and Westhof 1990;Masquida and Westhof 2006), but determination of secondary structure is not trivial. Typical methods for inferring secondary structure include sequence comparison (Pace et al 1999) and chemical or enzymatic mapping (Ziehler and Engelke 2000), where free energy minimization is often combined with either or both of these sets of data (Duan et al 2006). Binding of oligonucleotides by RNA to ascertain exposed sequences can also provide insight into RNA structure (Lewis and Doty 1970;Uhlenbeck et al 1970;Sohail et al 1999;Hopkins and Woodson 2005;Kierzek et al 2006a).…”

Exact calculation of loop formation probability identifies folding motifs in RNA secondary structures

“…The Fold program was used to generate minimum free energy structures. The AllSub program was used to exhaustively enumerate low free energy structures (Wuchty et al 1999;Duan et al 2006). The stochastic program was used to perform stochastic sampling (Ding and Lawrence 2003).…”

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.…”

“…The pioneering work of Southern and co-workers (7)(8)(9)(10)(11)(12) in this area employed microarrays of unmodified oligonucleotides, but synthetically modified nucleic acid analogs (13,14) have also been employed.…”

“…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).…”