CuAAC, strain-promoted azide–alkyne cycloaddition (SPAAC) and
azide–phosphine Staudinger ligation reactions. Although the ver-
satility of azide group has been well exploited in labeling and
imaging glycans, proteins, and lipids, incorporation of clickable
azide group into nucleic acids has remained a challenge [17–21].
This is largely due to the (1) instability of azide substrates under
solid-phase oligonucleotide synthesis conditions and (2) lack of
effective methods to incorporate metabolically stable azide group
into cellular RNA. We have recently developed a tool box of azide-
modified UTP analogs, which can be conveniently introduced into
RNA oligonucleotides by transcription reaction [22–24]. The
azide-labeled RNAs have been subsequently functionalized post-
transcriptionally with several biophysical probes by using click reac-
tions. Notably, endogenous RNA polymerases specifically
incorporate one of the nucleotide analogs, 5-azidomethyl UTP
(AMUTP, Fig.1), into cellular RNA transcripts [24]. This labeling
process further facilitated the visualization of newly synthesized
RNA in fixed and live cells by using CuAAC and SPAAC reactions
with a suitable fluorescent alkyne substrate.
Here, we provide a detailed stepwise protocol for in situ label-
ing of cellular RNA with azide group and subsequently image newly
transcribed RNA by using click reactions. In the first part, experi-
mental procedure to image cellular RNA in fixed cells by using
CuAAC reaction is described. In the second part, we enumerate
the steps to visualize azide-labeled cellular RNA in live cells by
using SPAAC reaction with a fluorescent cyclooctyne probe.2 Materials
Prepare all solutions using autoclaved water and analytical grade
reagents, and store at room temperature (unless indicated other-
wise). In this work, we have used fluorescent dyes Alexa 594 alkyne
and Cy3 DBCO (Fig.1). Store these probes in a deep freezer ( 20
or 40 C). In this work, we have used the transfecting agent
DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammo-
nium methylsulfate), which is procured from Roche. Follow our
reported stepwise procedure to prepare AMUTP from 5-
azidomethyluridine (AMU), which is now available with Jena Bio-
science GmbH [23, 24]. Store media and reagents required for cell
culture experiments at 4C (unless indicated otherwise).2.1 Buffer Solutions
for Cell Culture and
Click Reactions
- HEPES buffered saline (HBS): 20 mM HEPES, 150 mM
NaCl, pH 7.4. Weigh 2.6 g of HEPES and 4.38 g of NaCl in
a 500 mL graduated cylinder. Allow HEPES and NaCl to
dissolve completely by adding 400 mL of autoclaved water.
Adjust the pH of the buffer to 7.4 by dropwise addition of
1 M NaOH and finally adjust the volume to 500 mL by adding
360 Anupam A. Sawant et al.