Efficient fluorescence labeling of a large RNA through oligonucleotide hybridization

RNA. 2005 Feb;11(2):234-9. doi: 10.1261/rna.7180305. Epub 2004 Dec 21.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacillus subtilis / enzymology
  • Bacillus subtilis / genetics
  • Base Sequence
  • Fluorescent Dyes
  • Molecular Sequence Data
  • Nucleic Acid Hybridization
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / genetics
  • RNA / chemistry*
  • RNA / genetics
  • RNA, Bacterial / chemistry
  • RNA, Bacterial / genetics
  • Ribonuclease P / chemistry
  • Ribonuclease P / genetics

Substances

  • Fluorescent Dyes
  • Oligodeoxyribonucleotides
  • RNA, Bacterial
  • RNA
  • Ribonuclease P