Fluorescent PNA Probes as Hybridization Labels for Biological RNA

Abstract

Fluorescent labeling of biological RNA is complicated by the narrow range of nucleoside triphosphates that can be used for biological synthesis (i.e., transcription) as well as the inability to site-specifically incorporate them into long RNA transcripts. Noncovalent strategies for labeling RNA rely on attaching fluorescent dyes to hybridization probes which deliver the dye to a specific region of the RNA through Watson-Crick base pairing. This report demonstrates the use of high-affinity peptide nucleic acid (PNA) probes in labeling mRNA transcripts with thiazole orange donor and Alexa-594 acceptor fluorophores. The PNA probes were targeted to sequences flanking splice sites in a pre-mRNA such that before splicing the PNAs were separated by \u3e300 nucleotides (nts) whereas after splicing the separation decreased t