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.

Nanomechanical recognition measurements of individual DNA molecules reveal epigenetic methylation patterns

Abstract

Atomic force microscopy1 (AFM) is a powerful tool for analysing the shapes of individual molecules and the forces acting on them. AFM-based force spectroscopy provides insights into the structural and energetic dynamics2,3,4 of biomolecules by probing the interactions within individual molecules5,6, or between a surface-bound molecule and a cantilever that carries a complementary binding partner7,8,9. Here, we show that an AFM cantilever with an antibody tether can measure the distances between 5-methylcytidine bases in individual DNA strands with a resolution of 4 Å, thereby revealing the DNA methylation pattern, which has an important role in the epigenetic control of gene expression. The antibody is able to bind two 5-methylcytidine bases of a surface-immobilized DNA strand, and retracting the cantilever results in a unique rupture signature reflecting the spacing between two tagged bases. This nanomechanical approach might also allow related chemical patterns to be retrieved from biopolymers at the single-molecule level.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: A single-molecule force spectroscopy experiment reveals the molecular distance between two 5-methylcytosine bases in a DNA strand.
Figure 2: A two-step unbinding event between antibody and methylcytosine-containing ssDNA.
Figure 3: Two-step unbinding from ssDNA with nine 5-methylcytidines separated by six nucleotides.
Figure 4: Single nucleotide resolution enables 5-methylcytidine sequencing and the detection of single epigenetic changes.

References

  1. Binnig, G., Quate, C. F. & Gerber, C. Atomic force microscope. Phys. Rev. Lett. 56, 930–933 (1986).

    Article  CAS  Google Scholar 

  2. Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E. Energy landscapes of receptor–ligand bonds explored with dynamic force spectroscopy. Nature 397, 50–53 (1999).

    Article  CAS  Google Scholar 

  3. Baumgartner, W. et al. Cadherin interaction probed by atomic force microscopy. Proc. Natl Acad. Sci. USA 97, 4005–4010 (2000).

    Article  CAS  Google Scholar 

  4. Oesterhelt, F. et al. Unfolding pathways of individual bacteriorhodopsins. Science 288, 143–146 (2000).

    Article  CAS  Google Scholar 

  5. Rief, M., Oesterhelt, F., Heymann, B. & Gaub, H. E. Single molecule force spectroscopy on polysaccharides by atomic force microscopy. Science 275, 1295–1297 (1997).

    Article  CAS  Google Scholar 

  6. Oberhauser, A. F., Marszalek, P. E., Erickson, H. P. & Fernandez, J. M. The molecular elasticity of the extracellular matrix protein tenascin. Nature 393, 181–185 (1998).

    Article  CAS  Google Scholar 

  7. Lee, G. U., Chrisey, L. A. & Colton, R. J. Direct measurement of the forces between complementary strands of DNA. Science 266, 771–773 (1994).

    Article  CAS  Google Scholar 

  8. Moy, V. T., Florin, E. L. & Gaub, H. E. Intermolecular forces and energies between ligands and receptors. Science 266, 257–259 (1994).

    Article  CAS  Google Scholar 

  9. Hinterdorfer, P., Baumgartner, W., Gruber, H. J., Schilcher, K. & Schindler, H. Detection and localization of individual antibody–antigen recognition events by atomic force microscopy. Proc. Natl Acad. Sci. USA 93, 3477–3481 (1996).

    Article  CAS  Google Scholar 

  10. Braslavsky, I., Hebert, B., Kartalov, E. & Quake, S. R. Sequence information can be obtained from single DNA molecules. Proc. Natl Acad. Sci. USA 100, 3960–3964 (2003).

    Article  CAS  Google Scholar 

  11. Greenleaf, W. J. & Block, S. M. Single-molecule, motion-based DNA sequencing using RNA polymerase. Science 313, 801 (2006).

    Article  CAS  Google Scholar 

  12. Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).

    Article  CAS  Google Scholar 

  13. Branton, D. et al. The potential and challenges of nanopore sequencing. Nature Biotechnol. 26, 1146–1153 (2008).

    Article  CAS  Google Scholar 

  14. Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nature Nanotech. 4, 265–270 (2009).

    Article  CAS  Google Scholar 

  15. Chang, S. et al. Tunnelling readout of hydrogen-bonding-based recognition. Nature Nanotech. 4, 297–301 (2009)

    Article  CAS  Google Scholar 

  16. Smith, S. B., Cui, Y. & Bustamante, C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271, 795–799 (1996).

    Article  CAS  Google Scholar 

  17. Essevaz-Roulet, B., Bockelmann, U. & Heslot, F. Mechanical separation of the complementary strands of DNA. Proc. Natl Acad. Sci. USA 94, 11935–11940 (1997).

    Article  CAS  Google Scholar 

  18. Rief, M., Clausen-Schaumann, H. & Gaub, H. E. Sequence-dependent mechanics of single DNA molecules. Nat. Struct. Biol. 6, 346–349 (1999).

    Article  CAS  Google Scholar 

  19. Krautbauer, R., Rief, M. & Gaub, H. E. Unzipping DNA oligomers. Nano Lett. 3, 493–496 (2003).

    Article  CAS  Google Scholar 

  20. Liphardt, J., Onoa, B., Smith, S. B., Tinoco, I. J. & Bustamante, C. Reversible unfolding of single RNA molecules by mechanical force. Science 292, 733–737 (2001).

    Article  CAS  Google Scholar 

  21. Voulgarakis, N. K., Redondo, A., Bishop, A. R. & Rasmussen, K. O. Sequencing DNA by dynamic force spectroscopy: limitations and prospects. Nano Lett. 6, 1483–1486 (2006).

    Article  CAS  Google Scholar 

  22. Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl Acad. Sci. USA 89, 1827–1831 (1992).

    Article  CAS  Google Scholar 

  23. Rein, T., DePamphilis, M. L. & Zorbas, H. Identifying 5-methylcytosine and related modifications in DNA genomes. Nucleic Acids Res. 26, 2255–2264 (1998).

    Article  CAS  Google Scholar 

  24. Huang, Y. et al. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS One 5, e8888 (2010)

    Article  Google Scholar 

  25. Ebner, A. et al. A new, simple method for linking of antibodies to atomic force microscopy tips. Bioconjug. Chem. 18, 1176–1184 (2007).

    Article  CAS  Google Scholar 

  26. Schlapak, R. et al. Glass surfaces grafted with high-density poly(ethylene glycol) as substrates for DNA oligonucleotide microarrays. Langmuir 22, 277–285 (2006).

    Article  CAS  Google Scholar 

  27. Kienberger, F., Mueller, H., Pastushenko, V. & Hinterdorfer, P. Following single antibody binding to purple membranes in real time. EMBO Rep. 5, 579–583 (2004).

    Article  CAS  Google Scholar 

  28. Saphire, E. O. et al. Contrasting IgG structures reveal extreme asymmetry and flexibility. J. Mol. Biol. 319, 9–18 (2002).

    Article  CAS  Google Scholar 

  29. Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448 (1993).

    Article  CAS  Google Scholar 

  30. Kohl, A. et al. Designed to be stable: Crystal structure of a consensus ankyrin repeat protein. Proc. Natl Acad. Sci. USA 100, 1700–1705 (2003).

    Article  CAS  Google Scholar 

  31. Schneider, G. & Fechner, U. Computer-based de novo design of drug-like molecules. Nat. Rev. Drug. Discov. 4, 649–663 (2005).

    Article  CAS  Google Scholar 

  32. Pröll, J. et al. Ultra-sensitive immunodetection of 5′-methyl cytosine for DNA methylation analysis on oligonucleotide microarrays. DNA Res. 13, 37–42 (2006).

    Article  Google Scholar 

  33. Erlanger, B. F. & Beiser, S. M. Antibodies specific for ribonucleosides and ribonucleotides and their reaction with DNA. Proc. Natl Acad. Sci. USA 52, 68–74 (1964).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank L. Wildling, R. Schlapak, C. Riese, C. Hesch, J. Jacak and C. Rankl for expert technical assistance, and G. Kada, G. Schütz, D. Blaas, A. Frischauf and C. Aberger for enlightening discussions. This work was supported by the Gen-Au project ‘Ultra sensitive Proteomics and Genomics’ from the Austrian federal ministry for education, science and culture (R.Z., S.H., J.Pröll, P.H.), the Austrian science fund project P15295 (H.J.G.), the Austria Nano-Initiative/NABIOS (R.Z., S.H., H.J.G., P.H.), the European Commission grant ‘Single Molecule Workstation (SMW)’ no. NMP4-SE-2008-213717 (R.Z., F.K., P.H.), and the Austrian Science Fund project L422-N20 (J.H.).

Author information

Authors and Affiliations

Authors

Contributions

R.Z. performed the experiments and data evaluation. S.H. developed the surface chemistry and co-wrote the paper. J.Pröll performed the surface chemistry and selected the DNA sequences. F.K., J.Preiner and A.E. discussed the results. J.H. contributed to the surface chemistry. V.Ph.P. programmed the data evaluation. H.J.G. developed the tip chemistry. P.H. led the experimental design and wrote the paper.

Corresponding author

Correspondence to Peter Hinterdorfer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 378 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zhu, R., Howorka, S., Pröll, J. et al. Nanomechanical recognition measurements of individual DNA molecules reveal epigenetic methylation patterns. Nature Nanotech 5, 788–791 (2010). https://doi.org/10.1038/nnano.2010.212

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2010.212

This article is cited by

Search

Quick links

Find nanotechnology articles, nanomaterial data and patents all in one place. Visit Nano by Nature Research