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Nat Methods. 2014 Apr;11(4):456-62. doi: 10.1038/nmeth.2854. Epub 2014 Feb 23.

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

Author information

  • 11] Department of Applied Physics, Stanford University, Stanford, California, USA. [2] Department of Bioengineering, Stanford University, Stanford, California, USA.
  • 21] Department of Applied Physics, Stanford University, Stanford, California, USA. [2] Department of Bioengineering, Stanford University, Stanford, California, USA. [3].
  • 3Department of Bioengineering, Stanford University, Stanford, California, USA.
  • 41] Department of Molecular and Cell Biology, University of California, Berkeley, California, USA. [2].
  • 51] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Structural Biology, Stanford University Medical Center, Stanford, California, USA.

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.

PMID:
24562422
PMCID:
PMC4211898
DOI:
10.1038/nmeth.2854
[PubMed - indexed for MEDLINE]
Free PMC Article
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