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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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.
The authors declare no competing financial interests.
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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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