Isoenergetic penta- and hexanucleotide microarray probing and chemical mapping provide a secondary structure model for an RNA element orchestrating R2 retrotransposon protein function

Abstract

LNA (locked nucleic acids, i.e. oligonucleotides with a methyl bridge between the 2′ oxygen and 4′ carbon of ribose) and 2,6-diaminopurine were incorporated into 2′-O-methyl RNA pentamer and hexamer probes to make a microarray that binds unpaired RNA approximately isoenergetically. That is, binding is roughly independent of target sequence if target is unfolded. The isoenergetic binding and short probe length simplify interpretation of binding to a structured RNA to provide insight into target RNA secondary structure. Microarray binding and chemical mapping were used to probe the secondary structure of a 323 nt segment of the 5′ coding region of the R2 retrotransposon from Bombyx mori (R2Bm 5′ RNA). This R2Bm 5′ RNA orchestrates functioning of the R2 protein responsible for cleaving the second strand of DNA during insertion of the R2 sequence into the genome. The experimental results were used as constraints in a free energy minimization algorithm to provide an initial model for the secondary structure of the R2Bm 5′ RNA.

NMR structure of a 4 × 4 nucleotide RNA internal loop from an R2 retrotransposon: Identification of a three purine–purine sheared pair motif and comparison to MC-SYM predictions

“…The internal loop studied here (Fig. 1) is found in the R2 retrotransposon RNA from Bombyx mori (Kierzek et al 2008(Kierzek et al , 2009) and occurs in a 320-nucleotide fragment that tightly binds one copy of R2 protein (Christensen et al 2006). The loop is near the leading edge of a 41-nucleotide RNA hairpin that has a conserved secondary structure and codes for a region of partially conserved amino acids in the R2 protein (Kierzek et al 2009).…”

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).…”

The R2 retrotransposon RNA families

“…10 A secondary structure for the B. mori 5' region was first proposed based on single sequence free energy minimization, guided by constraints from chemical mapping and oligonucleotide hybridization experiments. 13 A fragment of 74 nt is unusually insensitive to structural probing. 5 A pseudoknot proposed for this fragment is supported by NMR spectra.…”