RNA Detection

Chapter 20

Method for Imaging Live-Cell RNA Using an RNA Aptamer

and a Fluorescent Probe

Shin-ichi Sato, Kenji Yatsuzuka, Yousuke Katsuda, and Motonari Uesugi

Abstract

Live-cell imaging of mRNA dynamics is increasingly important to understanding spatially restricted gene
expression. We recently developed a convenient and versatile method that uses a gene-specific RNA aptamer
and a fluorescent probe to enable spatiotemporal imaging of endogenous mRNAs in living cells. The
method was validated by live-cell imaging of the endogenous mRNA ofβ-actin. The new RNA-imaging
technology might be useful for live-cell imaging of any RNA molecules.

Key wordsLive-cell imaging, mRNA, RNA aptamer, Small molecule, Chemical biology

1 Introduction

Imaging of RNAs in living cells is a powerful approach to under-

standing the intracellular dynamics of RNA and for measuring

spatiotemporal gene expression. A number of different methods

have been developed for detecting RNA targets [1–15]. The use of

molecular beacons, the fluorescence of which changes upon bind-

ing to native mRNA targets, offers one of the best approaches

[16–19]. Although such molecular beacons could provide valuable

information about RNA dynamics in live cells, this method suffers

from several potential drawbacks: injection of beacons into living

cells might damage the cells, and poor stability of beacons in cells

would produce background fluorescence. New technology for

RNA imaging is needed to overcome these drawbacks. We recently

developed a convenient and versatile method that permits spatio-

temporal imaging of specific native RNAs in living cells [20, 21].

The method employs transfection of a plasmid encoding a gene-

specific RNA aptamer, combined with a cell-permeable synthetic

small molecule, BHQ1-Cy3, the fluorescence of which is restored

only when the RNA aptamer hybridizes with its cognitive mRNA.

Here, we describe the synthesis of BHQ1-Cy3, the selection of the

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_20,©Springer Science+Business Media LLC 2018

305