Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Confined space facilitates G-quadruplex formation

Nature Nanotechnology volume 12, pages 582–588 (2017)Cite this article

Subjects

Abstract

Molecular simulations suggest that the stability of a folded macromolecule increases in a confined space due to entropic effects. However, due to the interactions between the confined molecular structure and the walls of the container, clear-cut experimental evidence for this prediction is lacking. Here, using DNA origami nanocages, we show the pure effect of confined space on the property of individual human telomeric DNA G-quadruplexes. We induce targeted mechanical unfolding of the G-quadruplex while leaving the nanocage unperturbed. We find that the mechanical and thermodynamic stabilities of the G-quadruplex inside the nanocage increase with decreasing cage size. Compared to the case of diluted or molecularly crowded buffer solutions, the G-quadruplex inside the nanocage is significantly more stable, showing a 100 times faster folding rate. Our findings suggest the possibility of co-replicational or co-transcriptional folding of G-quadruplex inside the polymerase machinery in cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Experimental strategy to unfold human telomere G-quadruplex in a DNA nanocage.
Figure 2: Synthesis and characterization of individual DNA origami constructs.
Figure 3: Mechanical unfolding of human telomeric G-quadruplexes at room temperature.
Figure 4: Refolding kinetics of G-quadruplexes in DNA nanocages.
Figure 5: Transition kinetics and free energy diagrams of telomeric G-quadruplex in nanocages.

Similar content being viewed by others

References

  1. Tang, Y.-C. et al. Structural features of the GroEL-GroES nano-cage required for rapid folding of encapsulated protein. Cell 125, 903–914 (2006).

    Article  CAS  Google Scholar 

  2. Brinker, A. et al. Dual function of protein confinement in chaperonin-assisted protein folding. Cell 107, 223–233 (2001).

    Article  CAS  Google Scholar 

  3. Baumketner, A., Jewett, A. & Shea, J. E. Effects of confinement in chaperonin assisted protein folding: rate enhancement by decreasing the roughness of the folding energy landscape. J. Mol. Biol. 332, 701–713 (2003).

    Article  CAS  Google Scholar 

  4. Jewett, A. I., Baumketner, A. & Shea, J.-E. Accelerated folding in the weak hydrophobic environment of a chaperonin cavity: creation of an alternate fast folding pathway. Proc. Natl Acad. Sci. USA 101, 13192–13197 (2004).

    Article  CAS  Google Scholar 

  5. Takagi, F., Koga, N. & Takada, S. How protein thermodynamics and folding mechanisms are altered by the chaperonin cage: molecular simulations. Proc. Natl Acad. Sci. USA 100, 11367–11372 (2003).

    Article  CAS  Google Scholar 

  6. Zhou, H.-X. Protein folding and binding in confined spaces and in crowded solutions. J. Mol. Recognit. 17, 368–375 (2004).

    Article  CAS  Google Scholar 

  7. Zhou, H.-X. & Dill, K. A. Stabilization of proteins in confined spaces†. Biochemistry 40, 11289–11293 (2001).

    Article  CAS  Google Scholar 

  8. Eggers, D. K. & Valentine, J. S. Molecular confinement influences protein structure and enhances thermal protein stability. Protein Sci. 10, 250–261 (2001).

    Article  CAS  Google Scholar 

  9. Senske, M., Smith, A. E. & Pielak, G. J. Protein stability in reverse micelles. Angew. Chem. Int. Ed. 55, 3586–3589 (2016).

    Article  CAS  Google Scholar 

  10. Zhou, H. X., Rivas, G. & Minton, A. P. Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Annu. Rev. Biophys. 37, 375–397 (2008).

    Article  CAS  Google Scholar 

  11. Mitchell, M., Gillis, A., Futahashi, M., Fujiwara, H. & Skordalakes, E. Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA. Nat. Struct. Mol. Biol. 17, 513–518 (2010).

    Article  CAS  Google Scholar 

  12. Jansson, L. I. et al. Structural basis of template-boundary definition in Tetrahymena telomerase. Nat. Struct. Mol. Biol. 22, 883–888 (2015).

    Article  CAS  Google Scholar 

  13. Koirala, D. et al. Intramolecular folding in three tandem guanine repeats of human telomeric DNA. Chem. Commun. 48, 2006–2008 (2012).

    Article  CAS  Google Scholar 

  14. Li, W., Hou, X.-M., Wang, P.-Y., Xi, X.-G. & Ming Li, M. Direct measurement of sequential folding pathway and energy landscape of human telomeric G-quadruplex structures. J. Am. Chem. Soc. 135, 6423–6426 (2013).

    Article  CAS  Google Scholar 

  15. Han, H. & Hurley, L. H. G-quadruplex DNA: a potential target for anti-cancer drug design. Trends Pharmacol. Sci. 21, 136–142 (2000).

    Article  CAS  Google Scholar 

  16. Balasubramanian, S. & Neidle, S. G-quadruplex nucleic acids as therapeutic targets. Curr. Opin. Chem. Biol. 13, 345–353 (2009).

    Article  CAS  Google Scholar 

  17. Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).

    Article  CAS  Google Scholar 

  18. Moffitt, J. R., Chemla, Y. R., Smith, S. B. & Bustamante, C. Recent advances in optical tweezers. Annu. Rev. Biochem. 77, 205–228 (2008).

    Article  CAS  Google Scholar 

  19. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

    Article  CAS  Google Scholar 

  20. Koirala, D. et al. A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligands. Nat. Chem. 3, 782–787 (2011).

    Article  CAS  Google Scholar 

  21. Venczel, E. A. & Sen, D. Parallel and antiparallel G-DNA structures from a complex telomeric sequence. Biochemistry 32, 6220–6228 (1993).

    Article  CAS  Google Scholar 

  22. Yu, Z. et al. Click chemistry assisted single-molecule fingerprinting reveals a 3D biomolecular folding funnel. J. Am. Chem. Soc. 134, 12338–12341 (2012).

    Article  CAS  Google Scholar 

  23. An, N., Fleming, A. M., Middleton, E. G. & Burrows, C. J. Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity. Proc. Natl Acad. Sci. USA 111, 14325–14331 (2014).

    Article  CAS  Google Scholar 

  24. Zhou, J. et al. Formation and stabilization of G-quadruplex in nanosized water pools. Chem. Commun. 46, 1700–1702 (2010).

    Article  CAS  Google Scholar 

  25. Gore, J., Ritort, F. & Bustamante, C. Bias and error in estimates of equilibrium free-energy differences from nonequilibrium measurements. Proc. Natl Acad. Sci. USA 100, 12564–12569 (2003).

    Article  CAS  Google Scholar 

  26. Bennett, C. H. Efficient estimation of free energy differences from Monte Carlo data. J. Comput. Phys. 22, 245–268 (1976).

    Article  Google Scholar 

  27. Crooks, G. E. Path-ensemble averages in systems driven far from equilibrium. Phys. Rev. E 61, 2361–2366 (2000).

    Article  CAS  Google Scholar 

  28. Collin, D. et al. Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies. Nature 437, 231–234 (2005).

    Article  CAS  Google Scholar 

  29. Dhakal, S. et al. Structural and mechanical properties of individual human telomeric G-quadruplexes in molecularly crowded solutions. Nucleic Acids Res. 41, 3915–3923 (2013).

    Article  CAS  Google Scholar 

  30. Dudko, O. K., Hummer, G. & Szabo, A. Theory, analysis, and interpretation of single-molecule force spectroscopy experiments. Proc. Natl Acad. Sci. USA 105, 15755–15760 (2008).

    Article  CAS  Google Scholar 

  31. Greenleaf, W. J., Frieda, K. L., Foster, D. A., Woodside, M. T. & Block, S. M. Direct observation of hierarchical folding in single riboswitch aptamers. Science 319, 630–633 (2008).

    Article  CAS  Google Scholar 

  32. Xie, M., Podlevsky, J. D., Qi, X., Bley, C. J. & Chen, J. J.-L. A novel motif in telomerase reverse transcriptase regulates telomere repeat addition rate and processivity. Nucleic Acids Res. 38, 1982–1996 (2010).

    Article  CAS  Google Scholar 

  33. Paeschke, K., Simonsson, T., Postberg, J., Rhodes, D. & Lipps, H. J. Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. Nat. Struct. Mol. Biol. 12, 847–854 (2005).

    Article  CAS  Google Scholar 

  34. Miyoshi, D., Karimata, H. & Sugimoto, N. Hydration regulates thermodynamics of G-quadruplex formation under molecular crowding conditions. J. Am. Chem. Soc. 128, 7957–7963 (2006).

    Article  CAS  Google Scholar 

  35. Dai, J. et al. Structure of the intramolecular human telomeric G-quadruplex in potassium solution: a novel adenine triple formation. Nucleic Acids Res. 35, 2440–2450 (2007).

    Article  CAS  Google Scholar 

  36. Pelliccioli, A. P. & Wirz, J. Photoremovable protecting groups: reaction mechanisms and applications. Photochem. Photobiol. Sci. 1, 441–458 (2002).

    Article  Google Scholar 

  37. Luchette, P., Abiy, N. & Mao, H. Microanalysis of clouding process at the single droplet level. Sens. Actuat. B 128, 154–160 (2007).

    Article  CAS  Google Scholar 

  38. Mao, H. & Luchette, P. An integrated laser-tweezers instrument for microanalysis of individual protein aggregates. Sens. Actuat. B 129, 764–771 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This project was supported by the Japan Society for the Promotion of Science (JSPS) and the National Science Foundation (NSF) under the JSPS-NSF International Collaborations in Chemistry (ICC) (CHE-1415883, to H.S. and H.M.). H.M. acknowledges support from NSF (CHE-1609504). M.E. acknowledges supports from JSPS KAKENHI (grant nos. 24104002, 15H03837 and 16K14033).

Author information

Authors and Affiliations

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

    Prakash Shrestha, Sagun Jonchhe & Hanbin Mao

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

    Tomoko Emura, Kumi Hidaka & Hiroshi Sugiyama

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

    Masayuki Endo & Hiroshi Sugiyama

Authors
  1. Prakash Shrestha
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sagun Jonchhe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tomoko Emura
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kumi Hidaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Masayuki Endo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hiroshi Sugiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hanbin Mao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.M. and H.S. conceived the project. H.M., H.S., M.E. and P.S. designed experiments. H.M. supervised mechanical unfolding experiments. H.S. and M.E. supervised origami synthesis and characterization. P.S. performed mechanical unfolding experiments and prepared origami constructs. P.S. and H.M. carried out data analyses. S.J. performed mechanical unfolding experiments. T.E. and K.H. prepared and characterized origami constructs. H.M., P.S., M.E. and H.S. co-wrote the manuscript.

Corresponding authors

Correspondence to Masayuki Endo, Hiroshi Sugiyama or Hanbin Mao.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 2231 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shrestha, P., Jonchhe, S., Emura, T. et al. Confined space facilitates G-quadruplex formation. Nature Nanotech 12, 582–588 (2017). https://doi.org/10.1038/nnano.2017.29

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2017.29

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing