Use of plasmon coupling to reveal the dynamics of DNA bending and cleavage by single EcoRV restriction enzymes

Departments of *Physics and
^†Chemistry, University of California, Berkeley, CA 94720; and
^‡Divisions of Physical Biosciences and
^¶Materials Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA 94720

Edited by Donald M. Crothers, Yale University, New Haven, CT, and approved December 20, 2006 (received for review September 6, 2006)

Abstract

Pairs of Au nanoparticles have recently been proposed as “plasmon rulers” based on the dependence of their light scattering on the interparticle distance. Preliminary work has suggested that plasmon rulers can be used to measure and monitor dynamic distance changes over the 1- to 100-nm length scale in biology. Here, we substantiate that plasmon rulers can be used to measure dynamical biophysical processes by applying the ruler to a system that has been investigated extensively by using ensemble kinetic measurements: the cleavage of DNA by the restriction enzyme EcoRV. Temporal resolutions of up to 240 Hz were obtained, and the end-to-end extension of up to 1,000 individual dsDNA enzyme substrates could be simultaneously monitored for hours. The kinetic parameters extracted from our single-molecule cleavage trajectories agree well with values obtained in bulk through other methods and confirm well known features of the cleavage process, such as DNA bending before cleavage. Previously unreported dynamical information is revealed as well, for instance, the degree of softening of the DNA just before cleavage. The unlimited lifetime, high temporal resolution, and high signal/noise ratio make the plasmon ruler a unique tool for studying macromolecular assemblies and conformational changes at the single-molecule level.

Footnotes

^‖To whom correspondence should be addressed. E-mail: liphardt{at}physics.berkeley.edu
Author contributions: B.M.R., A.P.A., and J.Y.L. designed research; B.M.R., S.S., and A.M. performed research; B.M.R. and S.S. contributed new reagents/analytic tools; B.M.R., S.S., and A.M. analyzed data; and B.M.R., S.S., A.P.A., and J.L. wrote the paper.
↵ ^§Present address: Department of Chemistry, Boston University, Boston, MA 02215.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0607826104/DC1.
↵ ‖ In the absence of divalent metal ions, EcoRV endonuclease binds nonspecifically to DNA, with no preference for its recognition site over any other (47). It is known from crystal structures that in noncognate DNA–EcoRV restriction enzyme complexes the DNA retains a B configuration-like structure and is not bent (38). This was confirmed by bulk FRET measurements that did not show an intensity change upon addition of EcoRV without divalent ions (46).
↵ ** The gold probes might also slow the dynamics of the enzymatic reaction by perturbation of the two rate-determining steps, DNA hydrolysis and product release. Based on the Stokes-Einstein approximation, the diffusion coefficient of a free 40-nm particle in water at 298 K is ≈1.1 × 10⁻¹¹ m²·s, and it has an rms displacement of 4.7 nm/μs. For estimation of the force exerted on the DNA by the nonimmobilized particles, we assume that displacement occurs completely along the interparticle axis. In that case a pertubating force of ≈1.6 pN could act on the enzyme–DNA complex. Wuite and coworkers (50) investigated the efficiency of DNA cleavage by EcoRV under tension and showed that cleavage efficiency is not affected by forces significantly below 30 pN.
Abbreviation:

SAXS,

small-angle x-ray scattering.