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
Open Access articles citing this article.
Biophysical Reviews Open Access 10 July 2021
Scientific Reports Open Access 18 October 2013
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Binnig, G., Quate, C. F. & Gerber, C. Atomic force microscope. Phys. Rev. Lett. 56, 930–933 (1986).
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).
Baumgartner, W. et al. Cadherin interaction probed by atomic force microscopy. Proc. Natl Acad. Sci. USA 97, 4005–4010 (2000).
Oesterhelt, F. et al. Unfolding pathways of individual bacteriorhodopsins. Science 288, 143–146 (2000).
Rief, M., Oesterhelt, F., Heymann, B. & Gaub, H. E. Single molecule force spectroscopy on polysaccharides by atomic force microscopy. Science 275, 1295–1297 (1997).
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).
Lee, G. U., Chrisey, L. A. & Colton, R. J. Direct measurement of the forces between complementary strands of DNA. Science 266, 771–773 (1994).
Moy, V. T., Florin, E. L. & Gaub, H. E. Intermolecular forces and energies between ligands and receptors. Science 266, 257–259 (1994).
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).
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).
Greenleaf, W. J. & Block, S. M. Single-molecule, motion-based DNA sequencing using RNA polymerase. Science 313, 801 (2006).
Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).
Branton, D. et al. The potential and challenges of nanopore sequencing. Nature Biotechnol. 26, 1146–1153 (2008).
Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nature Nanotech. 4, 265–270 (2009).
Chang, S. et al. Tunnelling readout of hydrogen-bonding-based recognition. Nature Nanotech. 4, 297–301 (2009)
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).
Essevaz-Roulet, B., Bockelmann, U. & Heslot, F. Mechanical separation of the complementary strands of DNA. Proc. Natl Acad. Sci. USA 94, 11935–11940 (1997).
Rief, M., Clausen-Schaumann, H. & Gaub, H. E. Sequence-dependent mechanics of single DNA molecules. Nat. Struct. Biol. 6, 346–349 (1999).
Krautbauer, R., Rief, M. & Gaub, H. E. Unzipping DNA oligomers. Nano Lett. 3, 493–496 (2003).
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).
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).
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).
Rein, T., DePamphilis, M. L. & Zorbas, H. Identifying 5-methylcytosine and related modifications in DNA genomes. Nucleic Acids Res. 26, 2255–2264 (1998).
Huang, Y. et al. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS One 5, e8888 (2010)
Ebner, A. et al. A new, simple method for linking of antibodies to atomic force microscopy tips. Bioconjug. Chem. 18, 1176–1184 (2007).
Schlapak, R. et al. Glass surfaces grafted with high-density poly(ethylene glycol) as substrates for DNA oligonucleotide microarrays. Langmuir 22, 277–285 (2006).
Kienberger, F., Mueller, H., Pastushenko, V. & Hinterdorfer, P. Following single antibody binding to purple membranes in real time. EMBO Rep. 5, 579–583 (2004).
Saphire, E. O. et al. Contrasting IgG structures reveal extreme asymmetry and flexibility. J. Mol. Biol. 319, 9–18 (2002).
Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448 (1993).
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).
Schneider, G. & Fechner, U. Computer-based de novo design of drug-like molecules. Nat. Rev. Drug. Discov. 4, 649–663 (2005).
Pröll, J. et al. Ultra-sensitive immunodetection of 5′-methyl cytosine for DNA methylation analysis on oligonucleotide microarrays. DNA Res. 13, 37–42 (2006).
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).
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.).
The authors declare no competing financial interests.
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
This article is cited by
Biophysical Reviews (2021)
Cytosolic targeting factor AKR2A captures chloroplast outer membrane-localized client proteins at the ribosome during translation
Nature Communications (2015)
Nature Communications (2014)
Scientific Reports (2013)
Nature Nanotechnology (2012)