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Ultrahigh-resolution optical trap with single-fluorophore sensitivity

Nature Methods volume 8, pages 335–340 (2011)Cite this article

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Abstract

We present a single-molecule instrument that combines a time-shared ultrahigh-resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, we observed individual single fluorophore–labeled DNA oligonucleotides to bind and unbind complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, we clearly observed coincident angstrom-scale changes in tether extension. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (for example, single-base-pair stepping of DNA translocases) along with the detection of properties of fluorescently labeled protein (for example, internal configuration).

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Figure 1: Combined ultrahigh-resolution optical trap and single-molecule fluorescence microscope setup.
Figure 2: Interlacing and timesharing of optical trap and fluorescence excitation lasers.
Figure 3: Single fluorophore–labeled oligonucleotide hybridization experiment.
Figure 4: Combined measurement of fluorescence and DNA tether extension.
Figure 5: Proposed reaction diagram illustrating the complete process of probe-strand annealing to and melting from complementary tethered DNA.

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Acknowledgements

We thank all members of the Chemla, Ha and DeMarco laboratories for advice. The work was supported by the US National Science Foundation (PHY-082261, Center for the Physics of Living Cells), the US National Institutes of Health (R21 RR025341 A) and the Howard Hughes Medical Institute. Y.R.C. is supported by Burroughs-Wellcome Fund Career Awards at the Scientific Interface.

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Authors and Affiliations

  1. Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Matthew J Comstock, Taekjip Ha & Yann R Chemla

  2. Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Taekjip Ha & Yann R Chemla

  3. Howard Hughes Medical Institute, Urbana, Illinois, USA

    Taekjip Ha

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  1. Matthew J Comstock
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Contributions

Y.R.C. and T.H. conceived the combined high-resolution trapping with single-molecule fluorescence detection instrument project. M.J.C., Y.R.C. and T.H. designed the instrument. M.J.C. built the instrument including all optics, electronics and control software. M.J.C. performed all experiments and analyzed all the data. M.J.C., Y.R.C. and T.H. wrote the paper.

Corresponding authors

Correspondence to Taekjip Ha or Yann R Chemla.

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The authors declare no competing financial interests.

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Supplementary Figures 1–7 and Supplementary Note 1 (PDF 2500 kb)

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Comstock, M., Ha, T. & Chemla, Y. Ultrahigh-resolution optical trap with single-fluorophore sensitivity. Nat Methods 8, 335–340 (2011). https://doi.org/10.1038/nmeth.1574

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