Site-specific fluorescent labeling of large RNAs with pyrene

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.