Unit

UNIT 11.11 Site-Specific Fluorescent Labeling of Large RNAs with Pyrene

  1. Mary K. Smalley,
  2. Scott K. Silverman

Published Online: 1 DEC 2004

DOI: 10.1002/0471142700.nc1111s19

Current Protocols in Nucleic Acid Chemistry

Current Protocols in Nucleic Acid Chemistry

How to Cite

Smalley, M. K. and Silverman, S. K. 2004. Site-Specific Fluorescent Labeling of Large RNAs with Pyrene. Current Protocols in Nucleic Acid Chemistry. 19:11.11:11.11.1–11.11.12.

Author Information

  1. University of Illinois at Urbana-Champaign, Urbana, Illinois

Publication History

  1. Published Online: 1 DEC 2004
  2. Published Print: NOV 2004

Abstract

Pyrene is a useful chromophore for monitoring the tertiary structure and folding of large RNAs. This unit describes the general preparation of a large RNA (>80 nucleotides in length) that has been site-specifically modified with pyrene at the 2′-position of an individual internal nucleotide. A protocol is provided for derivatizing a 2′-amino-RNA oligonucleotide with a suitably activated pyrene reagent. This pyrene-labeled oligonucleotide is then assembled with other RNA(s) either by covalent ligation or by noncovalent hybridization to form a full-length structured RNA, which may then be studied by equilibrium and stopped-flow fluorescence spectroscopy.

Keywords:

  • RNA folding;
  • fluorescence;
  • pyrene;
  • ligation;
  • annealing