Skip to main content
SpringerLink
Log in

FRET Assay for Ligands Targeting the Bacterial A-Site RNA

  • Protocol
  • First Online:
Non-Natural Nucleic Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1973))

Abstract

A robust, fluorescence-based analysis and discovery platform is described for bacterial A-site binders. The assay relies on an incorporated isomorphic fluorescent uridine analog, which substitutes the A-site’s U1406 and serves as a FRET donor to an A-site bound coumarin-labeled aminoglycoside that serves as the FRET acceptor. Binding efficiency of unlabeled A-site ligands can be determined by competition experiments, where the acceptor-labeled aminoglycoside is displaced. The replacement efficiency is gauged by the concentration-dependent loss of the sensitized FRET acceptor’s signal with concomitant restoration of the donor’s emission. Plotting the relative emission intensity of both the donor and acceptor as a function of ligand concentration followed by fitting of the data points to a dose-response curve yields IC50 values, one possible measure of the antibiotic potency of new A-site binders.

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 119.00
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. McCoy LS, Xie Y, Tor Y (2011) Antibiotics that target protein synthesis. WIREs RNA 2:209–232

    Article  CAS  PubMed  Google Scholar 

  2. Knowles DJC, Foloppe N, Matassova NB, Murchie AIH (2002) The bacterial ribosome, a promising focus for structure-based drug design. Curr Opin Pharmacol 2:501–506

    Article  CAS  PubMed  Google Scholar 

  3. Hermann T (2005) Drugs targeting the ribosome. Curr Opin Struct Biol 15:355–366

    Article  CAS  PubMed  Google Scholar 

  4. Auerbach T, Bashan A, Harms J, Schluenzen F, Zarivach R, Bartels H, Agmon I, Kessler M, Pioletti M, Franceschi F, Yonath A (2002) Antibiotics targeting ribosomes: crystallographic studies. Curr Drug Targets Infect Disord 2:169–186

    Article  CAS  PubMed  Google Scholar 

  5. Carter AP, Clemons WM, Brodersen DE, Morgan-Warren RJ, Wimberly BT, Ramakrishnan V (2000) Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407:340–348

    Article  CAS  PubMed  Google Scholar 

  6. Gale EF, Cundliffe E, Renolds PE, Richmond MH, Waring MJ (1981) The molecular basis of antibiotic action. Wiley, London

    Google Scholar 

  7. Moazed D, Noller HF (1987) Interaction of antibiotics with functional sites in 16S ribosomal-RNA. Nature 327:389–394

    Article  CAS  PubMed  Google Scholar 

  8. Brodersen DE, Clemons WM Jr, Carter AP, Morgan-Warren RJ, Wimberly BT, Ramakrishnan V (2000) The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell 103:1143–1154

    Article  CAS  PubMed  Google Scholar 

  9. Harms JM, Bartels H, Schlünzen F, Yonath A (2003) Antibiotics acting on the translational machinery. J Cell Sci 116:1391–1393

    Article  CAS  PubMed  Google Scholar 

  10. Wirmer J, Westhof E, Minoru F (2006) Molecular contacts between antibiotics and the 30S ribosomal particle. Methods Enzymol 415:180–202

    Article  CAS  PubMed  Google Scholar 

  11. Schlünzen F, Zarivach R, Harms J, Bashan A, Tocilj A, Albrecht R, Yonath A, Franceschi F (2001) Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria. Nature 413:814–821

    Article  PubMed  Google Scholar 

  12. Vicens Q, Westhof E (2003) RNA as a drug target: the case of aminoglycosides. Chembiochem 4:1018–1023

    Article  CAS  PubMed  Google Scholar 

  13. Francois B, Russell RJM, Murray JB, Aboul-ela F, Masquida B t, Vicens Q, Westhof E (2005) Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: role of the number of rings and positive charges in the specific binding leading to miscoding. Nucleic Acids Res 33:5677–5690

    Article  CAS  PubMed  Google Scholar 

  14. Purohit P, Stern S (1994) Interactions of a small RNA with antibiotic and RNA ligands of the 30S subunit. Nature 370:659–662

    Article  CAS  PubMed  Google Scholar 

  15. Fourmy D, Recht MI, Blanchard SC, Puglisi JD (1996) Structure of the A site of escherichia coli 16S ribosomal RNA complexed with an aminoglycoside antibiotic. Science 274:1367–1371

    Article  CAS  PubMed  Google Scholar 

  16. Yoshizawa S, Fourmy D, Puglisi JD (1998) Structural origins of gentamicin antibiotic action. EMBO J 17:6437–6448

    Article  CAS  PubMed  Google Scholar 

  17. Vicens Q, Westhof E (2001) Crystal structure of paromomycin docked into the eubacterial ribosomal decoding A site. Structure 9:647–658

    Article  CAS  PubMed  Google Scholar 

  18. Vicens Q, Westhof E (2002) Crystal structure of a complex between the aminoglycoside tobramycin and an oligonucleotide containing the ribosomal decoding A-site. Chem Biol 9:747–755

    Article  CAS  PubMed  Google Scholar 

  19. Kaul M, Barbieri CM, Pilch DS (2006) Aminoglycoside-induced reduction in nucleotide mobility at the ribosomal RNA A-site as a potentially key determinant of antibacterial activity. J Am Chem Soc 128:1261–1271

    Article  CAS  PubMed  Google Scholar 

  20. Hofstadler SA, Griffey RH (2001) Analysis of noncovalent complexes of DNA and RNA by mass spectrometry. Chem Rev 101:377–390

    Article  CAS  PubMed  Google Scholar 

  21. Haddad J, Kotra LP, Llano-Sotelo B, Kim C, Azucena EF, Liu M, Vakulenko SB, Chow CS, Mobashery S (2002) Design of novel antibiotics that bind to the ribosomal acyltransfer site. J Am Chem Soc 124:3229–3237

    Article  CAS  PubMed  Google Scholar 

  22. Kaul M, Barbieri CM, Pilch DS (2004) Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences. J Am Chem Soc 126:3447–3453

    Article  CAS  PubMed  Google Scholar 

  23. Parsons J, Hermann T (2007) Conformational flexibility of ribosomal decoding-site RNA monitored by fluorescent pteridine base analogues. Tetrahedron 63:3548–3552

    Article  CAS  Google Scholar 

  24. Chao P-W, Chow CS (2007) Monitoring aminoglycoside-induced conformational changes in 16S rRNA through acrylamide quenching. Bioorg Med Chem 15:3825–3831

    Article  CAS  PubMed  Google Scholar 

  25. Wang Y, Hamasaki K, Rando RR (1997) Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region. Biochemistry 36:768–779

    Article  CAS  PubMed  Google Scholar 

  26. Hamasaki K, Rando RR (1997) Specific binding of aminoglycosides to a human rRNA construct based on a DNA polymorphism which causes aminoglycoside-induced deafness. Biochemistry 36:12323–12328

    Article  CAS  PubMed  Google Scholar 

  27. Hamasaki K, Ueno A (2001) Aminoglycoside antibiotics, neamine and its derivatives as potent inhibitors for the RNA-protein interactions derived from HIV-1 activators. Bioorg Med Chem Lett 11:591–594

    Article  CAS  PubMed  Google Scholar 

  28. Xie Y, Dix AV, Tor Y (2009) FRET enabled real time detection of RNA-small molecule binding. J Am Chem Soc 131:17605–17614

    Article  CAS  PubMed  Google Scholar 

  29. Xie Y, Dix AV, Tor Y (2010) Antibiotic selectivity for prokaryotic vs. eukaryotic decoding sites. Chem Commun 46:5542–5544

    Article  CAS  Google Scholar 

Download references

Acknowledgment

We thank the National Institutes of Health (grant number GM 069773) for generous support and Dr. Yun Xie for her insight and assistance.

Author information

Authors and Affiliations

  1. Office of Technology Management, Washington University in St. Louis, St. Louis, MO, USA

    Renatus W. Sinkeldam

  2. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA

    Yitzhak Tor

Authors
  1. Renatus W. Sinkeldam
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yitzhak Tor
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yitzhak Tor .

Editor information

Editors and Affiliations

  1. Department of Chemistry and Biochemistry, Georgia Southern University, Savannah, GA, USA

    Nathaniel Shank

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sinkeldam, R.W., Tor, Y. (2019). FRET Assay for Ligands Targeting the Bacterial A-Site RNA. In: Shank, N. (eds) Non-Natural Nucleic Acids. Methods in Molecular Biology, vol 1973. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9216-4_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9216-4_16

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9215-7

  • Online ISBN: 978-1-4939-9216-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation