Molecular Beacons: Powerful Tools for Imaging RNA in Living Cells

Abstract

Recent advances in RNA functional studies highlights the pivotal role of these molecules in cell physiology. Diverse methods have been implemented to measure the expression levels of various RNA species, using either purified RNA or fixed cells. Despite the fact that fixed cells offer the possibility to observe the spatial distribution of RNA, assays with capability to real-time monitoring RNA transport into living cells are needed to further understand the role of RNA dynamics in cellular functions. Molecular beacons (MBs) are stem-loop hairpin-structured oligonucleotides equipped with a fluorescence quencher at one end and a fluorescent dye (also called reporter or fluorophore) at the opposite end. This structure permits that MB in the absence of their target complementary sequence do not fluoresce. Upon binding to targets, MBs emit fluorescence, due to the spatial separation of the quencher and the reporter. Molecular beacons are promising probes for the development of RNA imaging techniques; nevertheless much work remains to be done in order to obtain a robust technology for imaging various RNA molecules together in real time and in living cells. The present work concentrates on the different requirements needed to use successfully MB for cellular studies, summarizing recent advances in this area.

RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression

“…This contrasts with the multistep procedure and handling required to synthesize and label molecular fluorescent probes (47) that also are well known to have a low signal-to-background ratio when used in vivo (42). Again when considering fluorescent molecular beacons as a positive miRNA monitoring system (17,18), careful design of the probes needs to be performed first in cellulo before being validated in vitro , and this procedure has to be repeated for each miRNA of interest (48). However, the use of an expression plasmid to monitor the expression of miRNA also has some limitations.…”

Nucleic Acid–Based Nanodevices in Biological Imaging

“…In the presence of a target DNA or RNA molecule, hybridization between the target and the loop sequence of the beacon occurs, and the stronger intermolecular hybridization breaks the weaker stem helix. The spatial separation of the fluorophore and quencher results in the restoration of fluorescence (76, 77). Molecular beacons are generally microinjected into cells to study the transport and localization of endogenous RNAs.…”

Point-of-care Diagnostic Tools to Detect Circulating MicroRNAS as Biomarkers of Disease

“…The use of molecular beacons (MB) as detector of the nucleic acids prevents the need for labeling the sample with fluorescent indicators [43]. This implementation reduces sample preparation times and complexity, while allowing the identification of positive signal in real time [42].…”