RNA Probing Methods: New Probes for the Detection and Visualization of Endogenous RNA in Fixed and Live Tissue

Abstract

In recent years, RNA’s structure and function has been increasingly analyzed through the utilization of experimental methods, such as RNA in situ hybridization (ISH) and electrophoretic mobility shift assays. Oligonucleotide probes are used for the detection and visualization of target RNAs for obtaining greater insight into the mechanism of gene expression and RNA function. RNA Fluorescence In-Situ Hybridization (FISH) and molecular beacons are popular tools used to detect and track endogenous RNAs. An additional tool utilized to study RNA function is the triplex forming oligonucleotide (TFO), which is a short probe that can bind to a purine-rich double stranded region within a target RNA to form a triple helix. In our study, a new probe was designed and evaluated for its efficiency in detecting a model RNA using peptide nucleic acid (PNA) TFOs. The PNA TFO probe was tested to determine the extent to which it formed a triple helix with a model RNA hairpin using two methods: 1) spectrofluorometer assays and 2) gel electrophoresis. Triplex formation was confirmed using the gel electrophoresis assay. Moreover, new theoretical probes were designed using the PinMol and TFOFinder software. Moving forward, these probes will be further tested using the experimental methodologies described in this study to evaluate their efficiency in detecting and visualizing endogenous RNA in live and fixed tissue.