Delivery of molecular beacons for live-cell imaging and analysis of RNA

Over the past decade, a variety of oligonucleotide-based probes have been developed that allow for direct visualization of RNA molecules in living cells. Of these, molecular beacons have garnered a particularly high degree of interest due to their simple yet exquisite unimolecular stem-loop design that allows for the efficient conversion of target recognition into a specific fluorescent signal. As a result of their favorable fluorescent enhancement and their high specificity, molecular beacons have been used for a wide range of applications, including the monitoring of RNA expression and localization in living cells, cancer cell detection, and the study of viral infections. In this chapter we describe a general methodology that can be followed for the imaging and analysis of RNA in living cells using molecular beacons. Several commonly employed methods for delivering molecular beacons into the cytosol are discussed including toxin-based cell membrane permeabilization, microinjection, and microporation. Strategies for acquiring ratiometric measurements are also described.