Efficient fluorescence labeling of a large RNA through oligonucleotide hybridization

Abstract

We present an efficient method of introducing fluorophore labels at selected locations in a large RNA. The method is based on specific and highly efficient hybridization between a fluorophore-containing DNA oligonucleotide and a modular hairpin loop replacing a functionally unimportant hairpin loop in the RNA. We demonstrate its feasibility using a 255-nucleotide RNA derived from the catalytic domain of RNase P from Bacillus subtilis. Hybridization of the DNA oligonucleotide to the modular hairpin loop minimally perturbs the structure and function of this RNA. This labeling scheme should be applicable in studies of RNA conformational dynamics by ensemble and single molecule fluorescence methods.

Keywords

• labeling
• hybridization
• FRET
• single molecule
• C-domain, the catalytic domain of the B. subtilis RNase P RNA consisting of residues 240–409 + 1–85
• FRET, fluorescence resonance energy transfer
• L15-RNA, C-domain containing 10 nucleotides insertion in the L15 loop and a 21-nt extension 3′ to residue #85
• L15m-RNA, L15-RNA with eight nucleotides changes in the enlarged L15 loop
• L15-module, cccAAAUUAAUAACGCUCUUGGUAggg
• L15m-module, cccAAAUAUAUUUCGGACUACGUAggg