RNA Detection

Chapter 24

Imaging Newly Transcribed RNA in Cells by Using

a Clickable Azide-Modified UTP Analog

Anupam A. Sawant, Sanjeev Galande, and Seergazhi G. Srivatsan

Abstract

Robust RNA labeling and imaging methods that enable the understanding of cellular RNA biogenesis and
function are highly desired. In this context, we describe a practical chemical labeling method based on a
bioorthogonal reaction, namely, azide–alkyne cycloaddition reaction, which facilitates the fluorescence
imaging of newly transcribed RNA in both fixed and live cells. This strategy involves the transfection of
an azide-modified UTP analog (AMUTP) into mammalian cells, which gets specifically incorporated into
RNA transcripts by RNA polymerases present inside the cells. Subsequent posttranscriptional click reaction
between azide-labeled RNA transcripts and a fluorescent alkyne substrate enables the imaging of newly
synthesized RNA in cells by confocal microscopy. Typically, 50μM to 1 mM of AMUTP and a transfection
time of 15–60 min produce significant fluorescence signal from labeled RNA transcripts in cells.

Key wordsNucleotide analog, Click chemistry, Bioorthogonal reaction, Posttranscriptional chemical

modification, Azide–alkyne cycloaddition reaction, RNA labeling, RNA imaging

1 Introduction

Bioconjugation strategy based on chemoselective azide–alkyne

cycloaddition reaction has become a powerful tool to functionalize

RNA both in vitro and in cells [1–6]. Usually alkyne group is

incorporated into RNA by chemical or enzymatic methods and

further functionalization is accomplished by carrying out a

copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction

with the desired probe containing azide group [7–15]. This is

because alkyne-containing phosphoramidite and nucleotide tri-

phosphate (NTP) substrates used in the chemical and enzymatic

RNA labeling methods are highly stable and easily accessible. How-

ever, the use of cytotoxic Cu catalysts in the cycloaddition reaction

between alkyne-labeled nucleic acid and azide counterpart is a

major concern, especially in the study of RNA in live cells [16].

In comparison to alkyne functionality, azide group can undergo

a wider variety of bioorthogonal chemical reactions namely

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_24,©Springer Science+Business Media LLC 2018

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