Chapter 27
Posttranscriptional Suzuki-Miyaura Cross-Coupling Yields
Labeled RNA for Conformational Analysis and Imaging
Manisha B. Walunj and Seergazhi G. Srivatsan
Abstract
Chemical labeling of RNA by using chemoselective reactions that work under biologically benign conditions is increasingly becoming valuable in the in vitro and in vivo analysis of RNA. Here, we describe a
modular RNA labeling method based on a posttranscriptional Suzuki-Miyaura coupling reaction, which
works under mild conditions and enables the direct installation of various biophysical reporters and tags.
This two-part procedure involves the incorporation of a halogen-modified UTP analog (5-iodouridine5
0 -triphosphate) by a transcription reaction. Subsequent posttranscriptional coupling with boronic acid/
ester substrates in the presence of a palladium catalyst provides access to RNA labeled with (a) fluorogenic
environment-sensitive nucleosides for probing nucleic acid structure and recognition, (b) fluorescent
probes for microscopy, and (3) affinity tags for pull-down and immunoassays. It is expected that this
method could also become useful for imaging nascent RNA transcripts in cells if the nucleotide analog
can be metabolically incorporated and coupled with reporters by metal-assisted cross-coupling reactions.
Key words Nucleotide analog, Suzuki-Miyaura reaction, Bio-orthogonal reaction, Posttranscriptional chemical modification, RNA labeling, RNA imaging, Environment-sensitive probe
1 Introduction
Several chemoselective transformations that can be carried out
under biologically benign conditions serve as valuable tools to
label and study biomacromolecular structure and function in both
cell-free and cellular environments [1–4]. Some of the prominent
examples of chemical labeling approaches, which have been categorized as bio-orthogonal reactions, include azide-alkyne cycloaddition [5], Staudinger ligation [6], and inverse electron demand
Diels-Alder reactions [7]. While these reactions have been conveniently implemented in labeling proteins, sugars, and DNA, their
use in labeling RNA has not been very straightforward due to
inherent low stability of RNA [8]. In this regard, we and other
groups have designed posttranscriptional and chemo-enzymatic
labeling approaches employing above-said bio-orthogonal
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_27, © Springer Science+Business Media, LLC, part of Springer Nature 2020
473
Posttranscriptional Suzuki-Miyaura Cross-Coupling Yields
Labeled RNA for Conformational Analysis and Imaging
Manisha B. Walunj and Seergazhi G. Srivatsan
Abstract
Chemical labeling of RNA by using chemoselective reactions that work under biologically benign conditions is increasingly becoming valuable in the in vitro and in vivo analysis of RNA. Here, we describe a
modular RNA labeling method based on a posttranscriptional Suzuki-Miyaura coupling reaction, which
works under mild conditions and enables the direct installation of various biophysical reporters and tags.
This two-part procedure involves the incorporation of a halogen-modified UTP analog (5-iodouridine5
0 -triphosphate) by a transcription reaction. Subsequent posttranscriptional coupling with boronic acid/
ester substrates in the presence of a palladium catalyst provides access to RNA labeled with (a) fluorogenic
environment-sensitive nucleosides for probing nucleic acid structure and recognition, (b) fluorescent
probes for microscopy, and (3) affinity tags for pull-down and immunoassays. It is expected that this
method could also become useful for imaging nascent RNA transcripts in cells if the nucleotide analog
can be metabolically incorporated and coupled with reporters by metal-assisted cross-coupling reactions.
Key words Nucleotide analog, Suzuki-Miyaura reaction, Bio-orthogonal reaction, Posttranscriptional chemical modification, RNA labeling, RNA imaging, Environment-sensitive probe
1 Introduction
Several chemoselective transformations that can be carried out
under biologically benign conditions serve as valuable tools to
label and study biomacromolecular structure and function in both
cell-free and cellular environments [1–4]. Some of the prominent
examples of chemical labeling approaches, which have been categorized as bio-orthogonal reactions, include azide-alkyne cycloaddition [5], Staudinger ligation [6], and inverse electron demand
Diels-Alder reactions [7]. While these reactions have been conveniently implemented in labeling proteins, sugars, and DNA, their
use in labeling RNA has not been very straightforward due to
inherent low stability of RNA [8]. In this regard, we and other
groups have designed posttranscriptional and chemo-enzymatic
labeling approaches employing above-said bio-orthogonal
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_27, © Springer Science+Business Media, LLC, part of Springer Nature 2020
473