Skip to main content
SpringerLink
Log in

Posttranscriptional Suzuki-Miyaura Cross-Coupling Yields Labeled RNA for Conformational Analysis and Imaging

  • Protocol
  • First Online:
RNA Tagging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2166))

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-iodouridine-5′-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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Prescher JA, Bertozzi CR (2005) Chemistry in living systems. Nat Chem Biol 1:13–21

    Article  CAS  Google Scholar 

  2. Weisbrod SH, Marx A (2011) Novel strategies for the site-specific covalent labelling of nucleic acids. Chem Commun 47:7018

    Article  Google Scholar 

  3. Spicer CD, Davis BG (2014) Selective chemical protein modification. Nat Commun 5:4740

    Article  CAS  Google Scholar 

  4. Holstein JM, Rentmeister A (2016) Current covalent modification methods for detecting RNA in fixed and living cells. Methods 98:18–25

    Article  CAS  Google Scholar 

  5. McKay CS, Finn MG (2014) Click chemistry in complex mixtures: bioorthogonal bioconjugation. Chem Biol 21:1075–1101

    Article  CAS  Google Scholar 

  6. van Berkel SS, van Eldijk MB, van Hest JCM (2011) Staudinger ligation as a method for bioconjugation. Angew Chem Int Ed 50:8806–8827

    Article  Google Scholar 

  7. Wu H, Devaraj NK (2016) Inverse electron-demand Diels-Alder bioorthogonal reactions. Top Curr Chem 374:3

    Article  Google Scholar 

  8. George JT, Srivatsan SG (2017) Posttranscriptional chemical labeling of RNA by using bioorthogonal chemistry. Methods 120:28–38

    Article  CAS  Google Scholar 

  9. El-Sagheer AH, Brown T (2010) New strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead ribozymes. Proc Natl Acad Sci U S A 107:15329–15334

    Article  CAS  Google Scholar 

  10. Rao H, Tanpure AA, Sawant AA, Srivatsan SG (2012) Enzymatic incorporation of an azide-modified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization. Nat Protoc 7:1097–1112

    Article  CAS  Google Scholar 

  11. Samanta A, Krause A, Jäschke A (2014) A modified dinucleotide for site-specific RNA-labelling by transcription priming and click chemistry. Chem Commun 50:1313–1316

    Article  CAS  Google Scholar 

  12. Someya T, Ando A, Kimoto M, Hirao I (2015) Site-specific labeling of RNA by combining genetic alphabet expansion transcription and copper-free click chemistry. Nucleic Acids Res 43:6665–6676

    Article  CAS  Google Scholar 

  13. Holstein JM, Stummer D, Rentmeister A (2016) Enzymatic modification of 5’-capped RNA and subsequent labeling by click chemistry. Methods Mol Biol 428:45–60

    Article  Google Scholar 

  14. Jao CY, Salic A (2008) Exploring RNA transcription and turnover in vivo by using click chemistry. Proc Natl Acad Sci U S A 105:15779–15784

    Article  CAS  Google Scholar 

  15. Grammel M, Hang H, Conrad NK (2012) Chemical reporters for monitoring RNA synthesis and poly (A) tail dynamics. Chembiochem 13:1112–1115

    Article  CAS  Google Scholar 

  16. Sawant AA, Tanpure AA, Mukherjee PP, Athavale S, Kelkar A, Galande S, Srivatsan SG (2016) A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA. Nucleic Acids Res 44:e16

    Article  Google Scholar 

  17. Nguyen K, Fazio M, Kubota M, Nainar S, Feng C, Li X, Atwood SX, Bredy TW, Spitale RC (2017) Cell-selective bioorthogonal metabolic labeling of RNA. J Am Chem Soc 139:2148–2151

    Article  CAS  Google Scholar 

  18. Sawant AA, Galande S, Srivatsan SG (2018) Imaging newly transcribed RNA in cells by using a clickable azide-modified UTP analog. Methods Mol Biol 1649:359–371

    Article  CAS  Google Scholar 

  19. Zhang Y, Kleiner RE (2019) A metabolic engineering approach to incorporate modified pyrimidine nucleosides into cellular RNA. J Am Chem Soc 141:3347–3351. https://doi.org/10.1021/jacs.8b11449

  20. Defrancq E, Messaoudi S (2017) Palladium-mediated labeling of nucleic acids. Chembiochem 18:426–443

    Article  CAS  Google Scholar 

  21. Jbara M, Maity SK, Brik A (2017) Palladium in the chemical synthesis and modification of proteins. Angew Chem Int Ed 56:10644–10655

    Article  CAS  Google Scholar 

  22. George JT, Srivatsan SG (2017) Vinyluridine as a versatile chemoselective handle for the posttranscriptional chemical functionalization of RNA. Bioconjug Chem 28:1529–1536

    Article  CAS  Google Scholar 

  23. Walunj MB, Sabale PM, Srivatsan SG (2018) Advances in the application of Pd-mediated transformations in nucleotides and oligonucleotides: palladium-catalyzed modification of nucleosides, nucleotides and oligonucleotides. Elsevier, Amsterdam, pp 269–293

    Book  Google Scholar 

  24. Omumi A, Beach DG, Baker M, Gabryelski W, Manderville RA (2011) Postsynthetic guanine arylation of DNA by Suzuki−Miyaura cross-coupling. J Am Chem Soc 133:42–50

    Article  CAS  Google Scholar 

  25. Cahová H, Jäschke A (2013) Nucleoside-based diarylethene photoswitches and their facile incorporation into photoswitchable DNA. Angew Chem Int Ed 52:3186–3190

    Article  Google Scholar 

  26. Chalker JM, Wood CSC, Davis BG (2009) A convenient catalyst for aqueous and protein Suzuki–Miyaura cross-coupling. J Am Chem Soc 131:16346–16347

    Article  CAS  Google Scholar 

  27. Li N, Lim RKV, Edwardraja S, Lin Q (2011) Copper-free Sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells. J Am Chem Soc 133:15316–15319

    Article  CAS  Google Scholar 

  28. Lercher L, McGouran JF, Kessler BM, Schofield CJ, Davis BG (2013) DNA modification under mild conditions by Suzuki–Miyaura cross-coupling for the generation of functional probes. Angew Chem Int Ed 52:10553–10558

    Article  CAS  Google Scholar 

  29. Walunj MB, Tanpure AA, Srivatsan SG (2018) Posttranscriptional labeling by using Suzuki-Miyaura cross-coupling generates functional RNA probes. Nucleic Acid Res 46:e65

    Article  Google Scholar 

  30. Yusop RM, Unciti-Broceta A, Johansson EMV, Sánchez-Martín RM, Bradley M (2012) Palladium-mediated intracellular chemistry. Nat Chem 3:239–243

    Article  Google Scholar 

  31. Pawar MG, Srivatsan SG (2011) Synthesis, photophysical characterization, and enzymatic incorporation of a microenvironment-sensitive fluorescent uridine analog. Org Lett 13:1114–1117

    Article  CAS  Google Scholar 

  32. Tanpure AA, Srivatsan SG (2011) A microenvironment-sensitive fluorescent pyrimidine ribonucleoside analogue: synthesis, enzymatic incorporation, and fluorescence detection of a DNA abasic site. Chem Eur J 17:12820–12827

    Article  CAS  Google Scholar 

  33. Yamaguchi T, Asanuma M, Nakanishi S, Saito Y, Okazaki M, Dodo K, Sodeoka M (2014) Turn-ON fluorescent affinity labeling using a small bifunctional O-nitrobenzoxadiazole unit. Chem Sci 5:1021–1029

    Article  CAS  Google Scholar 

  34. Boon WR (1952) 6- Dichloro-2-dimethylamino pyrimidine. J Chem Soc 1532

    Google Scholar 

Download references

Acknowledgments

This work was supported by Wellcome Trust-DBT India Alliance (IA/S/16/1/502360) grant to S.G.S. M.B.W. thanks CSIR, India, for a graduate research fellowship. The authors wish to thank Arun Tanpure for discussion and help with work that has led to the optimization of this protocol.

Author information

Authors and Affiliations

  1. Department of Chemistry, Indian Institute of Science Education and Research (IISER), Pune, India

    Manisha B. Walunj & Seergazhi G. Srivatsan

Authors
  1. Manisha B. Walunj
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Seergazhi G. Srivatsan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Seergazhi G. Srivatsan .

Editor information

Editors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Université de Strasbourg, Strasbourg, France

    Manfred Heinlein

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Walunj, M.B., Srivatsan, S.G. (2020). Posttranscriptional Suzuki-Miyaura Cross-Coupling Yields Labeled RNA for Conformational Analysis and Imaging. In: Heinlein, M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0712-1_27

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0712-1_27

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0711-4

  • Online ISBN: 978-1-0716-0712-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation