Nanomechanical recognition measurements of individual DNA molecules reveal epigenetic methylation patterns

Journal name:
Nature Nanotechnology
Volume:
5,
Pages:
788–791
Year published:
DOI:
doi:10.1038/nnano.2010.212
Received
Accepted
Published online

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.

At a glance

Figures

  1. A single-molecule force spectroscopy experiment reveals the molecular distance between two 5-methylcytosine bases in a DNA strand.
    Figure 1: A single-molecule force spectroscopy experiment reveals the molecular distance between two 5-methylcytosine bases in a DNA strand.

    a, ssDNA is coupled to an aldehyde-bearing glass surface through an amine group at its 3′-end, and the antibody is tethered via a lysine residue or its natural oligosaccharide and a flexible PEG crosslinker to the cantilever tip. The two Fab-arms and the Fc-arm of the antibody are indicated. Panels 1, 2, 3 and 4 correspond to different states that occur upon retracting the cantilever from the surface, including the elongation of the PEG and DNA strands and the sequential breaking of the two methylcytosine antibody bonds. b, These molecular changes are reflected in a force–distance curve, with the rupture distance in the curve (points 2 to 3) corresponding to the spacing between two 5-methylcytidines in a DNA strand.

  2. A two-step unbinding event between antibody and methylcytosine-containing ssDNA.
    Figure 2: A two-step unbinding event between antibody and methylcytosine-containing ssDNA.

    a, Examples of antibody–DNA ruptures with two unbinding events (indicated by red arrows, 1 and 2). The sketches show the possible binding positions of the antibody on the DNA, with 4, 8, 12 and 16 nucleotides (nt) separation. b, Distribution of the distances between two unbinding events. The thick red line represents the experimental probability density function of distances constructed from distance values of 224 force curves. The thin lines are the result of a multiple Gaussian fit. Inset: possible pairs of dual antibody binding.

  3. Two-step unbinding from ssDNA with nine 5-methylcytidines separated by six nucleotides.
    Figure 3: Two-step unbinding from ssDNA with nine 5-methylcytidines separated by six nucleotides.

    a, Examples of force–distance curves with sketches showing the possible binding positions of the antibody on the DNA. b, Distribution of the rupture distance between two unbinding events. The thick red line represents the experimental probability density function of distances constructed from 150 force curves. The thin lines are the result of a multiple Gaussian fit.

  4. Single nucleotide resolution enables 5-methylcytidine sequencing and the detection of single epigenetic changes.
    Figure 4: Single nucleotide resolution enables 5-methylcytidine sequencing and the detection of single epigenetic changes.

    a, Distance distribution from a DNA containing six 5-methylcytidines separated by 3, 8, 1, 8 and 3 nucleotides. The thick red line represents the experimental probability density function of distances constructed from distance values of 298 force curves. The thin lines are the result of a multiple Gaussian fit. b, Three possible sequences can be reconstructed (see text for explanation) from the peaks corresponding to 3, 8, 9, 11, 12, 17, 20 and 23 nucleotides in the distance distribution in a. c, Removal of a single methyl group from the DNA. The distance distribution shows similar peaks when compared to those in a, except for the absence of the peak corresponding to 17 nucleotides. The thick red line represents the experimental probability density function of distances constructed from distance values of 216 force curves. The thin lines are the result of a multiple Gaussian fit.

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Affiliations

  1. Institute for Biophysics, Johannes Kepler University of Linz, A-4040 Linz, Austria

    • Rong Zhu,
    • Ferry Kienberger,
    • Johannes Preiner,
    • Jan Hesse,
    • Andreas Ebner,
    • Vassili Ph. Pastushenko,
    • Hermann J. Gruber &
    • Peter Hinterdorfer
  2. Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Johannes Kepler University of Linz, A-4040 Linz, Austria

    • Rong Zhu &
    • Peter Hinterdorfer
  3. Center for Advanced Bioanalysis GmbH, 4020 Linz, Austria

    • Stefan Howorka,
    • Jan Hesse &
    • Peter Hinterdorfer
  4. University College London, Department of Chemistry, London WC1H 0AJ, UK

    • Stefan Howorka
  5. Department of Internal Medicine I, Elisabethinen Hospital, A-4010 Linz, Austria

    • Johannes Pröll
  6. Present address: Red Cross Transfusion Service of Upper Austria, A-4020 Linz, Austria

    • Johannes Pröll

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.

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