Single-Molecule Analysis of RNA Dynamics in Living Cells Using Molecular Beacons

Over the past decade, emerging evidence has indicated that long intergenic noncoding RNAs (lincRNAs), a class of RNA transcripts greater than 200 nt in length, function as key regulators of gene expression in cellular physiology and pathogenesis. Greater understanding of lincRNA activities, particularly in the context of subcellular localization and dynamic regulation at the single-molecule level, is expected to provide in-depth understanding of molecular mechanisms that regulate cell behavior and disease evolution. We have recently developed a fluorescence-imaging approach to investigate RNA dynamics in living cells at the single-molecule level. This approach entails the use of molecular beacons (MBs), which are a class of stem-loop forming oligonculeotide-based probes that emit detectable fluorescence upon binding to target sequence, and tandem repeats of MB target sequences integrated to the target RNA sequence. Binding of the MBs to the tandem repeats could illuminate the target RNA as a bright spot when imaged by conventional fluorescence microscopy, making the MB-based imaging approach a versatile tool for RNA analysis across laboratories. In this chapter, we describe the development of the MB-based approach and its application for imaging single NEAT1 lincRNA transcripts in living cells.