RNA Detection

Chapter 16

Optimizing Molecular Beacons for Intracellular

Analysis of RNA

Mingming Chen, Yantao Yang, Christopher J. Krueger,

and Antony K. Chen

Abstract

Conventional molecular beacons (MBs) have been used extensively for imaging specific endogenous RNAs
in living cells, but their tendency to generate false-positive signals as a result of nuclease degradation and/or
nonspecific binding limits sensitive and accurate imaging of intracellular RNAs. In an attempt to overcome
this limitation, MBs have been synthesized with various chemically modified oligonucleotide backbones to
confer greater biostability. We have recently developed a new MB architecture composed of 2^0 -O-methyl
RNA (2Me), a fully phosphorothioate (PS) modified loop domain and a phosphodiester stem (2Me/
PSLOOPMB). We showed that this new MB exhibits a marginal level of false-positive signals and enables
accurate single-molecule imaging of target RNA in living cells. In this chapter, we describe detailed
methods that led us to conclude that, among various PS-modified configurations, the 2Me/PSLOOPMB
is an optimal design for intracellular RNA analysis.

Key wordsMolecular beacons, 2^0 -O-methyl RNA, Phosphorothioate, Ratiometric imaging, Live-cell

RNA detection, False-positive signals

1 Introduction

Over the past decade, it has become evident that cell fate and

disease evolution are significantly influenced by the expression

level, trafficking, and localization of specific RNA molecules. To

better understand the molecular mechanisms underlying these

processes, a variety of oligonucleotide-based probes have been

developed to enable direct visualization of RNA molecules in living

cells. One widely employed technology is the molecular beacon

(MB), a stem-loop-forming oligonucleotide probe labeled with a

fluorescent reporter and a quencher at the two termini [1]. In the

nonhybridized state, the fluorophore is held in close proximity to

the quencher as a result of self-annealing of the short arm sequences

that form the stem domain. Hybridization of the loop domain to

target RNA transcripts opens the stem, restoring MB fluorescence

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_16,©Springer Science+Business Media LLC 2018

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