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A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligands

Nature Chemistry volume 3, pages 782–787 (2011)Cite this article

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Abstract

Ligands that stabilize the formation of telomeric DNA G-quadruplexes have potential as cancer treatments, because the G-quadruplex structure cannot be extended by telomerase, an enzyme over-expressed in many cancer cells. Understanding the kinetic, thermodynamic and mechanical properties of small-molecule binding to these structures is therefore important, but classical ensemble assays are unable to measure these simultaneously. Here, we have used a laser tweezers method to investigate such interactions. With a force jump approach, we observe that pyridostatin promotes the folding of telomeric G-quadruplexes. The increased mechanical stability of pyridostatin-bound G-quadruplex permits the determination of a dissociation constant Kd of 490 ± 80 nM. The free-energy change of binding obtained from a Hess-like process provides an identical Kd for pyridostatin and a Kd of 42 ± 3 µM for a weaker ligand RR110. We anticipate that this single-molecule platform can provide detailed insights into the mechanical, kinetic and thermodynamic properties of liganded bio-macromolecules, which have biological relevance.

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Figure 1: Schematic of the experimental setup (not to scale).
Figure 2: Mechanical unfolding of Tel-4G with and without PDS.
Figure 3: Folding kinetics of quadruplex structure in the presence or absence of PDS.
Figure 4: Measurement of the dissociation constant between PDS and the telomeric G-quadruplex.
Figure 5: Determination of dissociation constant from a single ligand concentration.

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Acknowledgements

The authors thank the New Faculty Award Program at the Camille and Henry Dreyfus Foundation, Ohio Board of Regents, NSF CHE-1026532, and NIH R15 DK081191-01 for support to H.M., the BBSRC (UK) for a studentship to B.A., and Cancer Research UK for programme funding to S.B. This work was also supported by the Core Research for Evolutional Science and Technology (CREST) of JST and a Grant-in-Aid for Science Research from MEXT (Japan) to H.S.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry and School of Biomedical Sciences, Kent State University, Kent, 44242, Ohio, USA

    Deepak Koirala, Soma Dhakal & Hanbin Mao

  2. Department of Chemistry, The University of Cambridge, Cambridge, CB2 1EW, UK

    Beth Ashbridge, Raphaël Rodriguez & Shankar Balasubramanian

  3. Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8502, Japan

    Yuta Sannohe & Hiroshi Sugiyama

  4. Institute for Integrated Cell Material Sciences (iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto, 606-8501, Japan

    Hiroshi Sugiyama

  5. CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo, 102-0075, Japan

    Hiroshi Sugiyama

  6. Cancer Research UK, Cambridge Research Institute, Li Ka Shing Center, Cambridge, CB2 0RE, UK

    Shankar Balasubramanian

  7. School of Clinical Medicine, The University of Cambridge, Cambridge, CB2 0SP, UK

    Shankar Balasubramanian

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  1. Deepak Koirala
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Contributions

H.M., S.B. and H.S. formulated the concept for the project. H.M. and D.K. designed the experiments. D.K. performed the experiments and analysed the data. S.D. contributed to the kinetics experiment. B.A. and R.R. synthesized the ligands. Y.S. prepared the DNA construct. H.M., S.B. and H.S. wrote the manuscript with D.K.

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Correspondence to Hanbin Mao.

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The authors declare no competing financial interests.

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Koirala, D., Dhakal, S., Ashbridge, B. et al. A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligands. Nature Chem 3, 782–787 (2011). https://doi.org/10.1038/nchem.1126

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