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Single-molecule dynamic structural biology with vertically arranged DNA on a fluorescence microscope

Nature Methods volume 22, pages 135–144 (2025)Cite this article

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Abstract

The intricate interplay between DNA and proteins is key for biological functions such as DNA replication, transcription and repair. Dynamic nanoscale observations of DNA structural features are necessary for understanding these interactions. Here we introduce graphene energy transfer with vertical nucleic acids (GETvNA), a method to investigate DNA–protein interactions that exploits the vertical orientation adopted by double-stranded DNA on graphene. This approach enables the dynamic study of DNA conformational changes via energy transfer from a probe dye to graphene, achieving spatial resolution down to the Ångström scale at subsecond temporal resolution. We measured DNA bending induced by adenine tracts, bulges, abasic sites and the binding of endonuclease IV. In addition, we observed the translocation of the O6-alkylguanine DNA alkyltransferase on DNA, reaching single base-pair resolution and detecting preferential binding to adenine tracts. This method promises widespread use for dynamical studies of nucleic acids and nucleic acid–protein interactions with resolution so far reserved for traditional structural biology techniques.

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Fig. 1: GETvNA principle.
Fig. 2: DNA bending originating from bulges and A-tracts.
Fig. 3: DNA bending induced by Endo IV.
Fig. 4: AGT diffusion on dsDNA.

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Data availability

Raw and processed data supporting the findings of this work are available on Zenodo at https://doi.org/10.5281/zenodo.13794574 (ref. 89).

Code availability

A home-made Python script used to process the raw data is available as Supplementary Software alongside example data. The Python scripts used to generate the plots presented in the figures of this work, based on processed data, are available on Zenodo at https://doi.org/10.5281/zenodo.13794574 (ref. 89).

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Acknowledgements

We thank the members of the Tinnefeld group for discussions and feedback. L.R. acknowledges S. Krause who suggested preliminary experiments leading to the discovery of GETvNA. Furthermore, we thank P. Schüler, T. Schröder and J. Zähringer for fruitful discussions. P.T. and I.K. thank, for financial support by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) under grant numbers TI 329/14-1 and KA 5449/2-1, the excellence cluster e-conversion under Germany’s Excellence Strategy – EXC 2089/1 – 390776260, and by the Center for NanoScience (CeNS). P.T thanks funding by the Federal Ministry of Education and Research (BMBF, 13N16929) and the Free State of Bavaria under the Excellence Strategy of the Federal Government and the Länder through the ONE MUNICH Project Munich Multiscale Biofabrication. L.R. acknowledges support by the Studienstiftung des deutschen Volkes. A.M.S. is thankful for the support by the Alexander von Humboldt foundation under reference Ref 3.2-ARG-1220722-GF-P. I.K. acknowledges support by the National Science Center of Poland (Sonata 2019/35/D/ST5/00958). K.C. and A.A. were supported by the US National Science Foundation (DMR-1827346) and the Human Frontier Science Program (RGP0047/2020). The supercomputer time was provided through ACESSS allocation grant MCA05S028 (A.A.) and the Leadership Resource Allocation MCB20012 on Frontera of the Texas Advanced Computing Centre (A.A). I.T. acknowledges financial support by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation), under grant number TE671/7-1. A.M.V. acknowledges financial support by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) under project number 522200875.

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Author notes

  1. Alan M. Szalai

    Present address: Centro de Investigaciones en Bionanociencias, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina

  2. These authors contributed equally: Alan M. Szalai, Giovanni Ferrari, Lars Richter.

Authors and Affiliations

  1. Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Munich, Germany

    Alan M. Szalai, Giovanni Ferrari, Lars Richter, Jakob Hartmann, Merve-Zeynep Kesici, Bosong Ji, Martin R. J. Dagleish, Annika Jaeger, Izabela Kamińska, Andrés M. Vera & Philip Tinnefeld

  2. Department of Physics, Center for Biophysics and Quantitative Biology, and Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Kush Coshic & Aleksei Aksimentiev

  3. Rudolf Virchow Center, University of Würzburg, Würzburg, Germany

    Ingrid Tessmer

  4. Institute of Physical Chemistry of the Polish Academy of Sciences, Warsaw, Poland

    Izabela Kamińska

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Contributions

P.T., A.M.S. and L.R. conceived the concept and experiments. A.M.S., G.F. and L.R. designed the experiments and the analysis pipeline, and curated data. A.M.S., G.F., L.R., J.H., M.-Z.K., B.J., A.J., A.M.V. and I.K. conducted experiments. A.M.S., G.F. and M.R.J.D. developed the analysis software. A.M.S., G.F., L.R., J.H., M.-Z.K. and B.J. analyzed data. K.C. and A.A. contributed the MD simulations. A.M.V. contributed to the design of studies involving Endo IV and their interpretation. I.T. contributed to the design and interpretation of experiments involving AGT and contributed AGT samples. B.J., M.-Z.K. and A.M.V. prepared Endo IV samples. I.K. prepared graphene-on-glass samples, optimized their preparation protocol and interpreted data. P.T. supervised the study. A.M.S. supervised data acquisition, analysis and visualization. A.M.S., G.F., L.R. and P.T. interpreted data and wrote the paper. All authors reviewed and approved the final paper.

Corresponding authors

Correspondence to Alan M. Szalai or Philip Tinnefeld.

Ethics declarations

Competing interests

P.T., A.M.S., L.R., G.F. and I.K. are inventors on a US provisional patent application #18/672,616 related to GETvNA. The other authors declare no competing interests.

Peer review

Peer review information

Nature Methods thanks Pallav Kosuri and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Extended field-of-views of hybrid DNA constructs immobilized on graphene.

Systems containing dsDNA segments of 36 bp, 45 bp, 51 bp and 66 bp length are depicted. For the 36 bp, 51 bp and 66 bp cases the white dotted boxes mark the areas shown in Fig. 1b. The histograms of non-deconvoluted fluorescence lifetime of each detected spot show that the fluorescence lifetimes are homogeneous for large areas. A shift to larger fluorescence lifetimes is observed when the length of the dsDNA segment is increased.

Extended Data Fig. 2 Cramér-Rao lower bound for the localization uncertainty.

σz,CRB denotes the theoretically attained axial precision and z the distance to graphene. The dependency is shown for three different numbers of photons N. An unquenched fluorescence lifetime of 3.51 ns was used. a) \({{SBR}}_{z=\infty }=10\) and b) \({{SBR}}_{z=\infty }=75\).

Extended Data Fig. 3 Contribution of the linker to the axial position of single molecules.

a) Sketch showing the systems used to estimate the contribution of the linker. Left: system containing a dsDNA segment with 66 bp, internally labeled at base #45. Right: system containing a dsDNA segment with 45 bp, labeled at one of its end bases. The negative charges are highlighted, since they are responsible for extending outwards the negatively charged dye (ATTO 542), which is attached through a six-atom carbon linker. b) Distribution of angles with respect to the z-axis obtained for the 40–45 bp segment from the MD simulation trajectory of the 51 bp system. The methodology to calculate this angle was analogous to the one described in the caption of Fig. S7d. c) Height distributions for the two systems described in a). d) Representation of the trigonometric calculations performed to retrieve the linker length assuming a model where the linker is stretched, extending outwards of the dsDNA segment (following the direction of the dsDNA segment for the end-labeled case, and oriented perpendicularly for the internally labeled scenario). The 25° angle used was obtained from the histogram shown in b), and the 1.14 nm height difference was extracted from the histograms shown in c).

Extended Data Fig. 4 Comparison between ranges of bending angles compatible with a given measured energy transfer efficiency for GET and FRET.

a) Sketch showing a simple model for kinked dsDNA, consisting of two rigid cylinders which can rotate around their respective axes (with torsion angles φ and ψ, respectively). They move with respect to each other (\({{\boldsymbol{x}}}_{{\boldsymbol{0}}}{,\,{\boldsymbol{y}}}_{{\boldsymbol{0}}}\) and \({{\boldsymbol{z}}}_{{\boldsymbol{0}}}\) represent the displacements in three dimensions of the bottom of the upper cylinder with respect to the top of the lower one), and bend by an angle \({\boldsymbol{\theta }}\). b) Plot showing the minimum and maximum bending angle \({\boldsymbol{\theta }}\) compatible with a given energy transfer efficiency between 40% and 60%, for GET and FRET. The model shown in a) was considered for the calculations, with 0.34 nm base pair (bp) length, 1 nm dsDNA radius, and 10.5 bp per double helix full turn as physical parameters. Two different labeling strategies were chosen for the two methods: for GET, the kink was positioned at 36 bp distance from graphene, and the dye at 30 bp distance from the kink, in the upper segment; for FRET, the two dyes were both positioned at 8 bp distance from the kink. \({{\boldsymbol{d}}}_{{\boldsymbol{0}}}\) for GET and \({{\boldsymbol{r}}}_{{\boldsymbol{0}}}\) for FRET were set at 17.7 nm and 5 nm respectively. For each value of the energy transfer efficiency \({{\boldsymbol{x}}}_{{\boldsymbol{0}}}{,\,{\boldsymbol{y}}}_{{\boldsymbol{0}}}\) were varied from −1 nm to 1 nm in steps of 0.5 nm, \({{\boldsymbol{z}}}_{{\boldsymbol{0}}}\) was varied between 0 nm and 1 nm (with no steps in between), φ and ψ were varied from −90° to 90° in steps of 1°. For each combination of these parameters, the value of \({\boldsymbol{\theta }}\) leading to the chosen energy transfer efficiency was computed. The plotted minimum and maximum values of \({\boldsymbol{\theta }}\) refer to all the possible combinations of parameters.

Extended Data Fig. 5 Exemplary time traces of systems where the dsDNA segment contained a bulge.

Four example time traces are shown for each bulge (3 A, 5 A, and 7 A). The fluorescence intensity time traces are shown on the left, and the fluorescence decays and corresponding monoexponential fits on the right. For each case, the fitted fluorescence lifetime and the corresponding bending angle are shown on top of the fluorescence decay plots. The rectangles from the intensity time traces highlight the photons used to obtain the fluorescence lifetime decay curves.

Extended Data Fig. 6 Intensity-based smFRET studies of dsDNA containing an AP site in the presence and absence of Endo IV.

The influence of having a PTO modification framed by the 5′-neighboring nucleotide and the AP site is evaluated. a) Example smFRET time traces of the system containing dsDNA with AP site without PTO modification, in the absence (top) and presence (bottom) of Endo IV. On the right, the histograms obtained from the shown traces are depicted. b) FRET efficiency histograms obtained from 75 (dsDNA containing AP, without PTO, in the absence of Endo IV) and 55 traces (dsDNA containing AP, without PTO, in the presence of Endo IV). Here, the FRET efficiencies obtained from every movie frame from all traces are computed together as independent FRET efficiency values. c) and d) Same description as in a) and b), but for systems containing both AP site and PTO modification. 30 traces were analyzed for the population histogram without Endo IV and 66 traces for the case, where Endo IV was added to the solution. Due to the intensity-based measurement protocol, the histograms from b) and d) are weighted by the respective dwell times of each state. This contrasts measurements on graphene, where the fluorescence lifetime of each state is independent of any weighing. As mentioned before, the presented smFRET data are based on the fluorescence intensity and not the fluorescence lifetime as used for GETvNA.

Extended Data Fig. 7 Exemplary time traces of systems where the dsDNA segment contained an AP site and a PTO modification, in the absence of Endo IV.

Seven example time traces are shown. The fluorescence intensity time traces are shown on the left, and the fluorescence decays and corresponding monoexponential fits on the right. For each case, the fitted fluorescence lifetime and the corresponding bending angle are shown next to the fluorescence decays. The rectangles from the intensity time traces highlight the photons used to obtain the fluorescence lifetime decay curve.

Extended Data Fig. 8 Exemplary time traces showing switching between states, corresponding to systems where the dsDNA segment contained an AP site and a PTO modification, in the presence of Endo IV.

Seven exemplary time traces are shown. The fluorescence intensity time traces are depicted on the left, and the fluorescence decays and corresponding monoexponential fits on the right. The color-coded rectangles from the intensity time traces highlight the photons used to obtain each fluorescence lifetime decay curve. The fitted fluorescence lifetime and the corresponding bending angle are shown next to the fluorescence decays.

Extended Data Fig. 9 Distribution of heights obtained from traces not showing switching between states determined by GETvNA in the presence of Endo IV.

The dsDNA contained an AP site and a PTO modification. For every individual trace, a single height was computed, obtained from the fluorescence lifetime fitted using all the photons detected before photobleaching. A three-peak Gaussian distribution was used to fit the experimental distribution. The bending angles corresponding to each subpopulation are also shown.

Extended Data Fig. 10 Exemplary time trace of AGT cluster diffusing on DNA without A-tract.

Left: 60-second-long time window. Right: Zoom-in on the region marked by the gray dotted-rectangle. The fast and slow mode are highlighted by arrows.

Supplementary information

Supplementary Information

Supplementary Discussions 1–3, Figs. 1–28 and Tables 1 and 2.

Reporting Summary

Peer Review File

Supplementary Video 1

MD simulation movie (1,209 ns) for construct containing 36 bp dsDNA.

Supplementary Video 2

MD simulation movie (1,200 ns) for construct containing 51 bp dsDNA.

Supplementary Video 3

MD simulation movie (1,099 ns) for construct containing 66 bp dsDNA.

Supplementary Data Table 1

List of sequences of all the oligonucleotides used in this study.

Supplementary Software

Python script containing routines to analyze the raw data from this study.

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Szalai, A.M., Ferrari, G., Richter, L. et al. Single-molecule dynamic structural biology with vertically arranged DNA on a fluorescence microscope. Nat Methods 22, 135–144 (2025). https://doi.org/10.1038/s41592-024-02498-x

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