RNA Detection

Chapter 18

In Vivo Visualization and Function Probing of Transport

mRNPs Using Injected FIT Probes

Jasmine Chamiolo, Imre Gaspar, Anne Ephrussi, and Oliver Seitz

Abstract

Fluorogenic hybridization methods, such as the use of FIT probes, enable the in vivo detection of specific
mRNAs transcribed from their endogenous, genetically nonmodified loci. Here, we describe the design,
synthesis and injection of nuclease resistant FIT probes into developingDrosophilaoocytes to detect
endogenous localizing mRNAs as wells as to probe function of structural RNA elements.

Key wordsmRNA transport, Injection, Live cell imaging, Locked nucleic acid, Forced intercalation

of thiazole orange

1 Introduction

To understand the steps of the complex and highly dynamic bio-

genesis of mRNPs, such as transcription, splicing, nuclear export,

cytoplasmic localization, translation, storage, and decay, the

mRNAs of interest have to be studied in vivo in their subcellular

context and in relation to their continuously changing binding

partners that compose the RNPs.

Hybridization techniques using conventional chromogenic or

fluorescent labels require a differentiation step—mostly washes—to

discriminate between target and background. Since such steps can-

not be performed in vivo, probes with different types of fluorogenic

labeling were developed over the past 2 decades: molecular beacons

[1], hybridizing RNA aptamers [2], exciton-controlled

hybridization-sensitive fluorescent oligonucleotide (ECHO [3,

4 ]) and forced intercalation of thiazole orange (FIT) probes [5].

Through different mechanisms these probe molecules substantially

increase their fluorescence upon hybridization to target (respon-

siveness) and when delivered to target cells—e.g., by microinjec-

tion—they enable the detection of endogenous transcript

molecules with great specificity. FIT probes rely on the fluorescence

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_18,©Springer Science+Business Media LLC 2018

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