RNA Detection

Chapter 15

Quantifying Gene Expression in Living Cells

with Ratiometric Bimolecular Beacons

Yantao Yang, Mingming Chen, Christopher J. Krueger,

Andrew Tsourkas, and Antony K. Chen

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.

Key wordsMolecular beacons, Ratiometric bimolecular beacons, RNA imaging, Single-molecule

detection, Single-molecule fluorescence in situ hybridization, Quantifying gene expression

1 Introduction

Subcellular trafficking and localization of specific RNA molecules

are crucial activities underlying many cellular processes, including

embryonic development, stem cell differentiation and neuronal

growth. Knowledge of RNA activities at the single-molecule level

is expected to facilitate understanding of the roles that RNAs play in

the regulation of these processes. One tool that has been used to

study RNA in living cells is the molecular beacon [1], a class of

antisense stem-loop-forming oligonucleotide (oligo) probe with

the ends labeled with a fluorophore and a quencher. In the unhy-

bridized state, self-complementation of the stem sequences at the

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_15,©Springer Science+Business Media LLC 2018

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