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

Abstract

Binding of small oligonucleotides to the periphery of folded RNA can provide insight into the secondary structure of complex RNA in solution. To discriminate between bound and unbound fluorescein-labeled 2′-O-methyl RNA probes, we use ionically coated gold nanoparticles to selectively adsorb unbound probes and quench their fluorescence. The target is the 3′ untranslated region of Bombyx mori R2 RNA. Fluorescence indicates that R2 sequences complementary to some of the probes are accessible for binding in the three-dimensional structure. Hybridization occurs under homogeneous conditions in the absence of the gold nanoparticles so that steric issues associated with chip-based assays are avoided. The assay is compatible with well plate formats, takes less than 5 min, and requires only 2 pmol or less of unlabeled target RNA per probe sequence tested.

Keywords

• RNA secondary structure
• gold nanoparticles
• fluorescence quenching
• 2′-O-methyl RNA