RNA Detection

Chapter 19

Visualizing RNA in Live Bacterial Cells Using

Fluorophore- and Quencher-Binding Aptamers

Murat Sunbul, Ankita Arora, and Andres J€aschke

Abstract

To elucidate the roles, dynamics, and regulation of RNAs, it is vital to be able to visualize the RNA of
interest (ROI) in living cells noninvasively. Here, we describe a novel live-cell RNA imaging method using
fluorophore- and quencher-binding aptamers, which can be genetically fused to the ROI. In this method,
new membrane permeable and nonfluorescent fluorophore–quencher conjugates were utilized, and we
showed that their fluorescence increases dramatically upon binding to fluorophore- or quencher-binding
aptamers. This phenomenon allowed for labeling the ROI with many different colored fluorophores and
also dual-color imaging of two distinct RNAs in live bacteria. Our approach uses small RNA tags and small
molecule fluorophores for labeling, thereby minimal perturbation on the function and dynamics of the
RNA of interest is expected.

Key wordsAptamer, Fluorophore, RNA imaging, RNA localization, Contact quenching, Live cell

imaging, Fluorescence microscopy, RNA trafficking, SRB-2 aptamer, DNB aptamer

1 Introduction

RNAs play a diverse role in living cells ranging from translation of

proteins, regulation and silencing of genes to catalysis of chemical

reactions and epigenetics. A mainstay of regulatory processes has

been spatial and temporal localization of various RNAs to specific

sites, which can be dictated by local or external stimuli [1]. Addi-

tionally, it has been previously shown that even bacterial cells

spatially organize their RNAs to promote functional compartmen-

talization in the absence of organelles [2, 3]. Furthermore, locali-

zation of bacterial RNAs can regulate their interactions with

nucleases and control their stability [4]. However, methods to

visualize RNAs and study their dynamics in live bacterial cells are

still in their infancy [5]. Therefore, in order to completely under-

stand the function of RNAs in living cells, new approaches for

imaging of intracellular RNA are of vital importance.

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_19,©Springer Science+Business Media LLC 2018

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