Efficient fluorescence labeling of a large RNA through oligonucleotide hybridization.
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.
Smith
Glenna J
GJ
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.
Sosnick
Tobin R
TR
Scherer
Norbert F
NF
Pan
Tao
T
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D043262:Ribonuclease P / Q000737:chemistry / Q000235:genetics
D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D012329:RNA, Bacterial; D012313:RNA; D043262:Ribonuclease P