Use of plasmon coupling to reveal the dynamics
of DNA bending and cleavage by single EcoRV
Bjo ¨rn M. Reinhard*†‡§, Sassan Sheikholeslami†§¶, Alexander Mastroianni†¶, A. Paul Alivisatos†¶, and Jan Liphardt*‡?
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)
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
over the 1- to 100-nm length scale in biology. Here, we substantiate
that plasmon rulers can be used to measure dynamical biophysical
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 moni-
tored 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 unlim-
ited 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.
ribozymes (4), and DNA helicases and binding proteins (5, 6) is an
important tool for biomedical research (reviewed in refs. 7–9). The
between individual organic donor and acceptor dye molecules (10,
11). FRET has proven to be an effective tool for revealing the
However, conventional organic dyes typically photobleach after
absorbing ?107photons and exhibit complex photophysics includ-
ing long-lived dark-states (12). Single-molecule FRET studies thus
remain challenging because of low signal/noise ratio, limited con-
tinuous observation time, limited accessible distance range, and
We recently reported an alternative method for dynamic
distance measurements on the nanometer scale by using pairs of
40-nm gold nanoparticles (13). Gold nanoparticles efficiently
scatter visible light and do not blink or photobleach. Their
optical properties are controlled by their plasmons, which are
wavelength can be tuned from blue into infrared (14, 15) by
varying their shape and structure (hollow/solid). The plasmon
wavelength is also sensitive to the proximity of other particles,
because plasmons couple (16–23) in a distance-dependent mat-
ter. With decreasing interparticle distance, the plasmon reso-
nance wavelength red-shifts (24) and the scattering cross-section
increases (25). Plasmon coupling can be used for colorimetric
detection of analytes in bulk, as pioneered by Mirkin and
The distance dependence of plasmon coupling can also be
used to monitor the spacing between two nanoparticles linked
by DNA (13). Individual pairs of biopolymer-linked noble
metal nanoparticles therefore act as ‘‘plasmon rulers.’’ The
he optical characterization of the function and dynamics of
single biomolecules such as molecular motors (1–3), RNA
inherent brightness of plasmon rulers makes them good can-
didates for highly parallel single-molecule assays able to reveal
the dynamics of biological processes and biopolymers. A
drawback of plasmon rulers compared with FRET methods is
the relatively large size of the nanoparticles compared with
organic fluorophores (?30–40 nm vs. ?1 nm).
One experimental geometry where the drawback of larger probe
dynamic range are studies of DNA bending by proteins. DNA
bending plays a crucial role in determining the specificity in
DNA-protein recognition (26), transcription regulation (27–31),
and DNA packaging (32, 33). Typically, these DNA-bending pro-
cesses are quite slow (millisecond timescales), and it is desirable to
monitor a particular DNA for extended durations (milliseconds to
days), so that the effects of enzyme concentration, ionic strength
and pH changes, and presence of cofactors can be explored.
Using plasmon rulers, we investigated the dynamics of the
EcoRV-catalyzed DNA cleavage reaction in a highly parallel,
high-bandwidth (up to 240 Hz) single-molecule assay. We picked
the EcoRV restriction enzyme because it is a member of the type
II restriction endonucleases, which are paradigms for the study of
its DNA substrate, and the bend angle is known from crystal
structures to be ?52° (38, 39). Using plasmon rulers, we were able
to follow certain steps in the catalytic cycle of EcoRV and directly
the standard model of how this enzyme works. By analysis of the
interparticle potentials, we were also able to see the softening of
the DNA resulting from its interactions with the enzyme before
Results and Discussion
plasmon rulers to monitor the dynamics of single enzyme–DNA
complexes. First, nonspecific protein–particle interactions needed
to be suppressed. Second, methods were needed to synthesize
temporal resolution needed to be significantly improved from its
previous value of ?1 Hz (13).
To eliminate nonspecific interactions between the gold surface
and the enzyme, we used a stepwise ligand exchange strategy in
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.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviation: SAXS, small-angle x-ray scattering.
§Present address: Department of Chemistry, Boston University, Boston, MA 02215.
?To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
February 20, 2007 ?
vol. 104 ?
no. 8 ?
processes. The ability of the ruler to operate over extended time
scales and to be effective in measuring distances over the 1- to
100-nm range make it a unique tool for studying macromolecular
General. Aqueous solutions of 40-nm gold particles with a concen-
CA). Freshly deionized water from a Millipore (Billerica, MA)
water system was used for all experiments. All buffers were filtered
with a 0.2-?m Millipore filter directly before use. Trithiolated
oligonucleotides were from Fidelity Inc. (Gaithersburg, MD).
Passivation and Bulk Assembly of Plasmon Rulers. We applied a
scheme of sequential ligand exchanges to functionalize and passi-
vate 40-nm gold particles (see SI Methods for details).
Axioplan2 upright microscope (Zeiss, Jena, Germany) with a
512 pixel chip; Andor, South Windsor, CT) on the imaging port of
the SpectraPro 2300i spectrometer (Acton, MA).
Surface Immobilization of Plasmon Rulers. The flow chambers were
prepared by first incubating for 15 min with a solution of 1 mg/ml
BSA-biotin (Roche, Nutley, NJ) and then flushed with 300 ?l T50
(50 mM NaCl/10 mM Tris, pH 8). Next, the chambers were reacted
with a solution of 1 mg/ml Neutravidin (31000; Pierce, Rockford,
IL) for 15 min and flushed with 300 ?l of T50. The chambers then
were incubated with SuperBlock (37515; Pierce) for 45 min and
rulers was flushed into the chamber. The plasmon rulers bind only
with biotinylated particle to the Neutravidin-coated glass surface,
while the nonbiotinylated particle is free to move in solution.
Control experiments verified that nonbiotinylated, pegylated par-
ticles did not stick to the passivated glass surfaces. Finally, the
chamber was equilibrated with EcoRV reaction buffer (100 mM
Tris?HCl/100 mM NaCl/10 mM MgCl2/1 mM DTT, pH 7.9).
Single-Molecule Experiments. After equilibration of chambers with
reaction buffer, 40 units of EcoRV restriction endonuclease (New
England Biolabs, Beverly, MA) in 200 ?l of reaction buffer was
flushed into the chamber (?1.75 nM EcoRV). The scattering
intensities of the plasmon rulers in the field of view were contin-
uously monitored with a color camera (Coolsnap cf.; Roper Sci-
entific, Trenton, NJ) or for faster temporal resolution with the
Andor CCD detector. To correlate intensity and wavelength infor-
mation, we recorded the scattering spectra of individual plasmon
rulers during the EcoRV-catalyzed cleavage reaction. To prebind
EcoRV enzyme to plasmon rulers, we incubated plasmon rulers
with EcoRV (4.4 nM) for ?1 min in Mg2?free buffer (50 mM
Tris?Hcl/100 mM NaCl). Then we flushed the chamber with the
buffer to remove excess enzyme. Reaction buffer containing Mg2?
then was introduced, and we started recording.
Data Analysis. By inspecting the movies recorded with the Andor
CCD detector using software from the manufacturer, we detected
all dimers in the field of view that showed a sudden intensity drop.
The movies were then imported into Matlab (Mathworks, Natick,
MA), where the exposed pixels were manually marked with a box.
The CCD detector has a pixel size of 16 ? 16 ?m, and most of the
pixel. The intensity trajectories of individual plasmon rulers were
calculated as sequence of the most intense pixels in the defined
boxes. All recorded trajectories contained a clear drop in intensity
that was set to 0 ms. For analysis of average scattering intensity
trajectories for a given spacer length. First, we calculated the
average scattering intensities over defined intervals preceding
dimer dissociation for each trajectory (interval lengths: 1770.0,
1180.0, 885.0, 559.0, 472.0, 354.0, 236.0, 118.0, 59.0, and 23.6 ms).
Within each trajectory, the average intensity of the longest interval
was set to 1, and the normalized intensities for each interval were
averaged over all trajectories.
We thank Merek Siu, Stephanie Hsiao, and Harish Agarwal. This work was
supported in part by the University of California, Berkeley (J.L.), the
Hellman Faculty Fund (J.L.), the Sloan and Searle foundations (J.L.), and
the U.S. Department of Energy, Energy Biosciences Program and the
Director of Basic Energy Biosciences, Materials Science, and Engineering
Division Contract DE-AC02-05CH11231 (to A.P.A.).
1. Itoh H, Takahashi A, Adachi K, Noji H, Yasuda R, Yoshida M, Kinosita K (2004) Nature
2. Kural C, Kim H, Syed S, Goshima G, Gelfand VI, Selvin PR (2005) Science 308:1469–1472.
3. Yildiz A, Tomishige M, Vale RD, Selvin PR (2004) Science 303:676–678.
4. Zhuang XW, Kim H, Pereira MJB, Babcock HP, Walter NG, Chu S (2002) Science
5. Bianco PR, Brewer LR, Corzett M, Balhorn R, Yeh Y, Kowalczykowski SC, Baskin RJ
(2001) Nature 409:374–378.
6. Shivashankar GV, Libchaber A (1998) Biophys J 74:A242–A242.
7. Giepmans BNG, Adams SR, Ellisman MH, Tsien RY (2006) Science 312:217–224.
8. Weiss S (2000) Nat Struct Biol 7:724–729.
9. Xie XS, Lu HP (1999) J Biol Chem 274:15967–15970.
10. Weiss S (1999) Science 283:1676–1683.
11. Ha T, Enderle T, Ogletree DF, Chemla DS, Selvin PR, Weiss S (1996) Proc Natl Acad Sci
12. Dubois A, Canva M, Brun A, Chaput F, Boilot JP (1996) Appl Opt 35:3193–3199.
13. Sonnichsen C, Reinhard BM, Liphardt J, Alivisatos AP (2005) Nat Biotechnol 23:741–745.
14. Halas N (2005) MRS Bull 30:362–367.
15. Hirsch LR, Gobin AM, Lowery AR, Tam F, Drezek RA, Halas NJ, West JL (2006) Ann
Biomed Eng 34:15–22.
16. Kelly KL, Coronado E, Zhao LL, Schatz GC (2003) J Phys Chem B 107:668–677.
17. Khlebtsov B, Melnikov A, Zharov V, Khlebtsov N (2006) Nanotechnology 17:1437–1445.
18. Elghanian R, Storhoff JJ, Mucic RC, Letsinger RL, Mirkin CA (1997) Science 277:1078–1081.
19. Nordlander P, Prodan E (2004) Nano Lett 4:2209–2213.
20. Maier SA, Brongersma ML, Kik PG, Atwater HA (2002) Phys Rev B 65:193408.
21. Rechberger W, Hohenau A, Leitner A, Krenn JR, Lamprecht B, Aussenegg FR (2003) Opt
22. Prodan E, Radloff C, Halas NJ, Nordlander P (2003) Science 302:419–422.
23. Haynes CL, McFarland AD, Zhao LL, Van Duyne RP, Schatz GC, Gunnarsson L, Prikulis
J, Kasemo B, Kall M (2003) J Phys Chem B 107:7337–7342.
24. Reinhard BM, Siu M, Agarwal H, Alivisatos AP, Liphardt J (2005) Nano Lett 5:2246–2252.
25. Kreibig U, Vollmer M (1995) Optical Properties of Metal Clusters (Springer, Berlin).
26. Bloomfield VA, Crothers DM, Tinoco J, Ignacio (2000) Nucleic Acids, Structures, Properties,
and Functions (University Science Books, Sausalito, CA).
27. Chen L, Glover JNM, Hogan PG, Rao A, Harrison SC (1998) Nature 392:42–48.
28. Love JJ, Li XA, Case DA, Giese K, Grosschedl R, Wright PE (1995) Nature 376:791–795.
29. Gartenberg MR, Crothers DM (1988) Nature 333:824–829.
30. Ansari AZ, Bradner JE, Ohalloran TV (1995) Nature 374:371–375.
31. Rappas M, Schumacher J, Beuron F, Niwa H, Bordes P, Wigneshweraraj S, Keetch CA,
Robinson CV, Buck M, Zhang XD (2005) Science 307:1972–1975.
32. Travers A, Drew H (1997) Biopolymers 44:423–433.
33. Smith DE, Tans SJ, Smith SB, Grimes S, Anderson DL, Bustamante C (2001) Nature
34. Perona JJ (2002) Methods 28:353–364.
35. Pingoud A, Jeltsch A (1997) Eur J Biochem 246:1–22.
36. Wilcox DE (1996) Chem Rev 96:2435–2458.
37. Vipond IB, Halford SE (1993) Mol Microbiol 9:225–231.
38. Winkler FK, Banner DW, Oefner C, Tsernoglou D, Brown RS, Heathman SP, Bryan RK,
Martin PD, Petratos K, Wilson KS (1993) EMBO J 12:1781–1795.
39. Perona JJ, Martin AM (1997) J Mol Biol 273:207–225.
40. Zheng M, Davidson F, Huang XY (2003) J Am Chem Soc 125:7790–7791.
41. Johnson PB, Christy RW (1972) Phys Rev B 6:4370–4379.
42. Yguerabide J, Yguerabide EE (1998) Anal Biochem 262:157–176.
43. Vipond IB, Baldwin GS, Halford SE (1995) Biochemistry 34:697–704.
44. Kratky O, Porod G (1949) Recueil Des Travaux Chimiques Des Pays-Bas-Journal of the Royal
Netherlands Chemical Society 68:1106–1122.
45. Smith SB, Cui YJ, Bustamante C (1996) Science 271:795–799.
46. Hiller DA, Fogg JM, Martin AM, Beechem JM, Reich NO, Perona JJ (2003) Biochemistry
47. Taylor JD, Badcoe IG, Clarke AR, Halford SE (1991) Biochemistry 30:8743–8753.
48. Waters TR, Connolly BA (1994) Biochemistry 33:1812–1819.
49. Baldwin GS, Vipond IB, Halford SE (1995) Biochemistry 34:705–714.
50. van den Broek B, Noom MC, Wuite GJ (2005) Nucleic Acids Res 33:2676–2684
51. Singh-Zocchi M, Dixit S, Ivanov V, Zocchi G (2003) Proc Natl Acad Sci USA 100:7605–7610.
52. Zocchi G (2001) Biophys J 81:2946–2953.
53. Bustamante C, Bryant Z, Smith SB (2003) Nature 421:423–427.
54. Sam MD, Perona JJ (1999) Biochemistry 38:6576–6586.
www.pnas.org?cgi?doi?10.1073?pnas.0607826104Reinhard et al.