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Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension

Nature Methods volume 11, pages 456–462 (2014)Cite this article

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Abstract

Single-molecule measurements of DNA twist and extension have been used to reveal physical properties of the double helix and to characterize structural dynamics and mechanochemistry in nucleoprotein complexes. However, the spatiotemporal resolution of twist measurements has been limited by the use of angular probes with high rotational drag, which prevents detection of short-lived intermediates or small angular steps. We introduce gold rotor bead tracking (AuRBT), which yields >100× improvement in time resolution over previous techniques. AuRBT employs gold nanoparticles as bright low-drag rotational and extensional probes, which are monitored by instrumentation that combines magnetic tweezers with objective-side evanescent darkfield microscopy. Our analysis of high-speed structural dynamics of DNA gyrase using AuRBT revealed an unanticipated transient intermediate. AuRBT also enables direct measurements of DNA torque with >50× shorter integration times than previous techniques; we demonstrated high-resolution torque spectroscopy by mapping the conformational landscape of a Z-forming DNA sequence.

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Figure 1: AuRBT design and data collection.
Figure 2: Angular resolution of AuRBT.
Figure 3: High-resolution measurement of twist-stretch coupling.
Figure 4: High-resolution analysis of gyrase dynamics at 1 mM ATP.
Figure 5: AuRBT torque spectroscopy.

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Acknowledgements

We thank J.M. Berger and members of the Bryant group for many useful discussions and comments on the manuscript, and A. Bekshaev, P. Ruijgrok and M. Dijk for providing Matlab code used for computing Mie scattering parameters and optical forces. This work was supported by a Stanford Interdisciplinary Graduate Fellowship and the Natural Sciences and Engineering Research Council of Canada (award NSERC PGS-D3) to P.L., by a Stanford Bio-X graduate fellowship to A.B., by a Pew Scholars Award and US National Institutes of Health grants OD004690 and GM106159 to Z.B., and by a Swiss National Science Foundation Fellowship to F.C.O.

Author information

Author notes

  1. Aakash Basu & Elsa M Tretter

    Present address: Present addresses: Howard Hughes Medical Institute and Laboratory of Sensory Neuroscience, The Rockefeller University, New York, New York, USA (A.B.), and Nurix Inc., San Francisco, California, USA (E.M.T.).,

Authors and Affiliations

  1. Department of Applied Physics, Stanford University, Stanford, California, USA

    Paul Lebel & Aakash Basu

  2. Department of Bioengineering, Stanford University, Stanford, California, USA

    Paul Lebel, Aakash Basu, Florian C Oberstrass & Zev Bryant

  3. Department of Molecular and Cell Biology, University of California, Berkeley, California, USA

    Elsa M Tretter

  4. Department of Structural Biology, Stanford University Medical Center, Stanford, California, USA

    Zev Bryant

Authors
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Contributions

P.L. designed and built instrumentation, wrote data acquisition code, performed experiments and analyzed data. A.B. aided in developing and interpreting DNA gyrase experiments, collaborated on data acquisition code and performed calibration experiments to establish evanescent nanometry procedures. F.C.O. synthesized molecules for torque spectroscopy and aided in static torque assay development. E.M.T. purified and characterized E. coli GyrA and GyrB subunits for gyrase experiments. Z.B. conceived and supervised the project. P.L. and Z.B. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Zev Bryant.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1–3 and Supplementary Notes 1–3 (PDF 9057 kb)

High-speed darkfield imaging and angle tracking.

An 80-nm gold rotor bead was attached above a 420 bp torsionally constrained DNA segment (Supplementary Table 3 “bottom constrained” tether), held under 26 pN of tension and imaged at 6.3 kHz. A total of ∼150 ms of raw images (pixel size, 104.5 nm) are shown alongside cumulative plots of the fitted x-y positions and the corresponding calculated angle. The elapsed time in milliseconds is displayed above the image panel. x-y markers are color-coded in time to distinguish current positions from accumulated data. (MOV 2863 kb)

Comparison of predicted and observed torque-measurement distributions.

The binned data in Figure 5d are compared with the model prediction in Figure 5e by displaying torque measurement distributions –log(P(τ)) for successive values of imposed twist θ. Predicted distributions were calculated (equations S9–S12) using parameters obtained from fitting the mean torque 〈τ〉(θ) alone. The distribution is unimodal at low twist values that favor B-DNA. At intermediate twist values, the distribution becomes bimodal as formation of a Z-DNA domain becomes thermodynamically accessible; the B-Z coexistence regime is characterized by a broad torque distribution, which narrows asymmetrically as the B-Z transformation approaches completion. (MOV 5140 kb)

Supplementary Software

AuRBT data acquisition in Matlab. These files may be used to implement the data acquisition and PSF fitting procedure described in Online Methods. “Initialize.m” initializes hardware, and “Acquire.m” performs acquisition and x-y feedback. “gsolve2d.c” should be compiled as a Matlab executable, which is called by “Acquire.m” for Gaussian fitting. (ZIP 11 kb)

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Lebel, P., Basu, A., Oberstrass, F. et al. Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension. Nat Methods 11, 456–462 (2014). https://doi.org/10.1038/nmeth.2854

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