Quantifying Gene Expression in Living Cells with Ratiometric Bimolecular Beacons

Abstract

Molecular beacons (MBs), a class of oligonucleotide-based probes, have enabled researchers to study various RNA molecules in their native live-cell contexts. However, it is also increasingly recognized that, when delivered into cells, MBs have the tendency to be sequestered into the nucleus where they may generate false positive signals. In an attempt to overcome this issue, MBs have been synthesized with chemically modified oligonucleotide backbones to confer greater biostability. Alternatively, strategies have been developed to minimize nuclear entry. In the latter approach, we have combined functional elements of MBs with functional elements of siRNAs that facilitate nuclear export to create a new RNA imaging platform called ratiometric bimolecular beacons (RBMBs). We showed that RBMBs exhibited long-term cytoplasmic retention, and hence a marginal level of false positive signals in living cells. Subsequent studies demonstrated that RBMBs could sensitively and accurately quantify mRNA transcripts engineered to contain multiple tandem repeats of an MB target sequence at the single-molecule level. In this chapter, we describe the synthesis of RBMBs and their applications for absolute quantification and tracking of single mRNA transcripts in cells.