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: firstname.lastname@example.org.
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 ?
which we passivated the particle surfaces with biocompatible (40)
low-molecular-mass PEGs. We then synthesized DNA-linked
30 and 60 bp by using a liquid phase DNA-programmed assembly
strategy [see supporting information (SI) Fig. 6].
Homogeneous samples of plasmon rulers were obtained by
gel-electrophoretic purification (SI Fig. 6). Sample homogeneity
(SAXS). The SAXS measurements were needed because interpar-
ticle distances in aqueous solution cannot be inferred from TEM
measurements. The pair distribution functions of isolated plasmon
rulers obtained by SAXS (SI Fig. 6) contain strong dimer peaks of
separated 40-nm gold particles and thus confirm that our synthetic
strategy leads to DNA tethered dimers (SI Fig. 6).
To increase the temporal resolution of the plasmon ruler mea-
surements, we investigated the dependence of the scattering inten-
sity on the interparticle distance. In previous applications of plas-
mon rulers, we used the distance dependence of the resonance
24). However, the spectral analysis of individual plasmon rulers
relies on the dispersion of light, forcing us to integrate photon
counts for several hundred milliseconds to obtain a detectable
signal in our set-up. This rate is too slow to resolve the dynamics of
biological processes such as enzymatic DNA bending and cleavage,
which typically occur on timescales of micro- to milliseconds.
temporal resolution than spectral information, because no disper-
sion is required. It can be shown that, like the plasmon resonance
wavelength, the scattering cross-section of a coupled pair of dimers
particle is treated as a simple dipole. Using this assumption, the
cross-section of two coupled particles with identical diameters is
following expression can be derived by averaging over longitudinal
and perpendicular plasmon modes (25):
1 ? 2?(R?RP)3?
1 ? ?(R?RP)3?, with ? ?
? ? ?m
? ? 2?m,
where ? is the wavelength-dependent dielectric function of gold
interparticle distance, and RP is the particle radius. We set the
dielectric constant of the medium to ?m? 1.6, as before (24). Eq.
1 is a highly simplified description of the relationship between
scattering cross-section and interparticle distance; among other
in an isotropic environment, and we are neglecting multipole
contributions. Therefore, we expect qualitative rather than quan-
titative agreement of Eq. 1 with experiment.
To validate this intensity-based approach, we recorded the
scattering spectra of EcoRV-catalyzed DNA cleavage reactions by
using an intensified CCD detector. The biological aspects of this
experiment are described below; here it is only important to know
that DNA cleavage by the EcoRV enzyme leads to plasmon ruler
disassociation. As the DNA tethering the particles is cut, the
interparticle distance suddenly increases, and consequently the
scattering wavelength blue-shifts (Fig. 1) in a well understood
manner (24). The particle plasmons cease to couple when the
dimers dissociate, and consequently their resonance wavelengths
and scattering cross-sections relax to the monomer values.
To correlate the spectral shifts with changes in scattering inten-
sity, we computed the total scattering intensities by integrating
photon counts on the spectrometer over all wavelengths for six
coefficient between integrated intensity and peak wavelength in
individual trajectories is r ? 0.92, confirming that color (peak
wavelength) and scattering intensity are correlated and that the
scattering intensity can be used to monitor interparticle distance.
Ultimately, we achieved temporal resolutions of 240 Hz in a 20 ?
20-?m field of view.
Highly Parallel Single EcoRV Restriction Digestion Assay. Having
enriched samples of surface passivated plasmon rulers and a
method for monitoring the ruler’s interparticle separation with
high temporal resolution, we set out to monitor the bending and
cleavage dynamics of single-EcoRV enzymes. We immobilized
plasmon rulers on the surface of a glass flow chamber via a
biotin–Neutravidin bond between the biotin functionalized par-
ticle and surface (Fig. 2a). After equilibrating the chamber with
10 mM Mg2?reaction buffer, we introduced EcoRV and mon-
itored the plasmon rulers’ color and intensity. Because of the
brightness of the plasmon rulers (scattering cross-section of
40-nm particles, Csca? 130 nm2) (42), we could simultaneously
monitor 500–1,500 plasmon rulers by using a low numerical
aperture and low-magnification ?40 objective. The complete
field of view in these experiments is limited to 150 ? 100 ?m by
our camera and magnification.
Upon addition of the enzyme, some plasmon rulers exhibited
sudden intensity drops (Fig. 2b and SI Movie 1). Green dots are
drops. Complete detachment of the dimer from the surface cannot
corresponding to a single surface-immobilized gold particle, re-
mains after the intensity drop. Rather, the ruler dissociation events
are due to enzymatic DNA cleavage (see below).
First, plasmon rulers constructed from DNA lacking the EcoRV
seen with the control DNA, whereas for dimers with the EcoRV
recognition site, hundreds of dimer dissociations were observed.
Second, the addition of Ca2?[an inhibitor of DNA cleavage by
EcoRV (43)] to the reaction buffer dramatically reduces the
number of intensity drops (Fig. 3b). The first-order rate constants
of the intensity drops decreased from 0.036 (no Ca2?) to 0.004 s?1
(2 mM Ca2?). Together, the observed sequence specificity and the
inhibition of cleavage reaction by Ca2?confirm that the observed
dimer dissociation is the result of DNA cleavage by EcoRV.
Plasmon rulers with 30-bp spacers exhibit a lower cutting rate
than those with longer spacers (Fig. 3a), probably due to steric
hindrance. According to the worm-like chain model for DNA (44)
(contour length L ? number of bases/10.5?3.4 nm, persistence
spacers is 9.4 nm compared with 12.4 and 18.3 nm for 40 and 60 bp,
respectively. The PEG brush (L ? 4.1 nm, P ? 2 nm) adds ?6 nm
the enzyme in the case of the 30-bp spacer compared with 6.4 nm
for the 40-bp spacer. It is known that the binding of one molecule
sity. Scattering wavelength (Left) and integrated intensity (Right) during
EcoRV-catalyzed DNA tether cleavage reaction recorded at 2 Hz. Dimer dis-
(Right). Spectral shift and change in total intensity are highly correlated
(Pearson correlation coefficient ? 0.9).
Correlation of plasmon resonance wavelength and scattering inten-
www.pnas.org?cgi?doi?10.1073?pnas.0607826104Reinhard et al.
of EcoRV covers ?15 bp ? 4.8 nm of DNA (38). Presumably, the
in the case of the 30-bp spacers, slightly reducing cleavage kinetics.
Magnitude of Intensity Changes Due to EcoRV-Catalyzed DNA Cleav-
age. After assigning the sudden intensity drops as dimer disasso-
ciation resulting from DNA cleavage by EcoRV, we analyzed the
resolution. According to the standard model of DNA cleavage by
52°). Based on an end-to-end distance from the worm-like chain
model and a 52° bend, the interparticle distances in our geometries
should decrease by 0.9 nm (?30bp), 1.0 nm (?40bp), and 1.3 nm
(?60bp) due to DNA bending by EcoRV. Judging from the inverse
pair polarizability–distance dependence given in Eq. 1, bending
should slightly increase the scattering intensity in all three cases.
the DNA spacer length, this is a challenging test case for the
sensitivity of the plasmon rulers.
Our search for intensity changes due to bending was greatly
facilitated by the large intensity drop upon dimer dissociation,
because it allowed us to locate the disassociation time with high
precision and thus also constrain the region that needed to be
analyzed for evidence of DNA bending. Using the dissociation
event as a fiduciary time-point, we calculated the average intensity
preceding the dissociation from all recorded trajectories. Although
we typically monitored the plasmon rulers’ intensity at 85 Hz to
increase the throughput with the larger field of view.
The average intensities as a function of time are shown in Fig. 4
b–d. For both the 30- and 40-bp DNA spacers, there is a significant
increase in average scattering intensity of up to 1.5% immediately
before dimer dissociation. The measured average intensity changes
[1.5 ? 0.8% (30 bp), 1.6 ? 0.9% (40 bp)] are in reasonable
glass surface through biotin–Neutravidin chemistry. The ho-
modimeric EcoRV enzyme binds nonspecifically to DNA bound be-
tween the particles (I), translocates and binds to the target site (II),
bends the DNA at the target site by ?50° (III), cuts the DNA in a
blunt-ended fashion by phosphoryl transfer (54) (IV), and subse-
quently releases the products (V). (b) A 150 ? 100-?m field of view
with surface immobilized plasmon rulers. Individual dimers are
visible as bright green dots. Dimer dissociation upon EcoRV-
catalyzed DNA cleavage leads to a strong change in scattering
intensities. The dimers are converted into monomers as shown for
in SI Movie 1.
Highly parallel single EcoRV restriction enzyme digestion
0 100200 300
0 30 60 90 120 150 180 210 240 270 300 330
10 mM Mg , no Ca
k = 0.036 s
k = 0.010 s
k = 0.004 s
, 1 mM Ca
, 2 mM Ca
tion of plasmon rulers. (a) The cleavage reaction is
highly specific. For all DNA spacers with an EcoRV
restriction site, hundreds of cutting events are ob-
served, and in the control experiments (60-bp dimer)
without a restriction site, the cutting efficiency is
almost zero. Each histogram contains the combined
results of two independent cutting experiments per
spacer length performed with similar surface cover-
age. (b) Percentage of plasmon rulers that have been
cleaved as function of time for increasing Ca2?con-
centrations. First-order kinetic fits are shown as
continuous lines. EcoRV requires Mg2?as a natural
cofactor to catalyze DNA cleavage. Ca2?can replace
Mg2?and facilitates formation of the enzyme–DNA
complex, but the resulting complex does not
catalyze the phosphodiester bond cleavage. Ca2?inhibits the cleavage reaction in the presence of Mg2?. A Mg2?concentration of 10 mM was
Statistical analysis of EcoRV restriction diges-
Reinhard et al.
February 20, 2007 ?
vol. 104 ?
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agreement with the order-of-magnitude calculation [Eq. 1, 2.4%
(30 bp), 1.6% (40 bp)]. For the 30-bp spacer, the predicted value is
slightly above the experimental error margin. For the rulers with
largest initial interparticle separation (60 bp), no statistically sig-
?0.5 ? 0.8%), consistent with the known inverse relationship
between sensitivity and spacer length (24) and suggesting that the
rulers’ sensitivity was decreasing faster as predicted by Eq. 1. Given
assumptions that went into the estimates obtained from Eq. 1,
the end-to-end extension of the DNA, the thickness of the PEG
brush, the uncertainty in the dielectric function, and the omission
of multipole terms, the agreement between theory and experiment
is considerably better than expected.
Is DNA bending by the EcoRV restriction enzyme the most
probable explanation for observed intensity increases before the
cut? The scattering intensity also depends, next to the interparticle
distance, on the dielectric constant of the surrounding medium. It
is therefore conceivable that enzyme binding (Fig. 2a, step II) and
not DNA bending (step III) causes the observed intensity change
by a local change of the refractive index. Therefore, we decoupled
binding from bending by letting EcoRV react with 40-bp plasmon
rulers in the absence of divalent metal ions (KDin the absence of
free buffer. Finally, we introduced Mg2?, allowing bound enzymes
to cleave their DNA substrate. As shown in SI Figs. 7 and 8, these
trajectories exhibit intensity jumps preceding dimer dissociation,
confirming our interpretation of the intensity jumps as DNA
to steps III–IV in Fig. 2a in which the DNA is bent by 52°.
Individual traces show large variations in bending times. The
trajectory shown in Fig. 4e exhibits a spike in intensity before the
dimers dissociate. In this trajectory, only the very last frame before
dissociation has an increased intensity, whereas in Fig. 4 f and g the
dwell times are on the order of a few seconds (f) and a few tens of
seconds (g) respectively. Student’s t tests show that in 76% of all
high- and low-intensity states differ significantly (P ? 0.05). In 4%
of all collected trajectories, the large drop in intensity due to the
DNA cleavage is preceded by a smaller intensity drop as shown in
Fig. 4h. This behavior suggests two subsequent DNA cutting events
in dimers that have two instead of one DNA tether. The cleavage
of the first tether does not lead to the dissociation of the particles
but only to a slight increase in equilibrium distance. The low
probability of this behavior suggests that most of the dimers are
indeed tethered by only one DNA molecule.
Dynamics of EcoRV-Catalyzed DNA Cleavage as Measured with Plas-
mon Rulers. Having assigned the intensity jumps to DNA bending
by the EcoRV enzyme and determined the average intensity
changes before DNA cleavage/particle pair disassociation, we built
and 40-bp dimer trajectories. The results of the dwell time analyses
are shown in Fig. 5a. The cumulative dissociation probability is
plotted in Fig. 5b. In these plots the percentage of dimers that have
been cleaved is plotted against the time they spend in the high-
intensity state. Exponential fits to the decay curves in Fig. 5b give
rate constants of k30? 0.50 s?1(30 bp, R2? 0.94) and k40? 0.46
s?1(40 bp, R2? 0.91). The similarity of the two rate constants is
in agreement with the assertion that the trajectories comprise
molecular events like DNA bending, DNA hydrolysis, and product
release, but not DNA binding, which is expected to be much slower
in case of the 30-bp spacer because of steric hindrance.
The catalytic cycle of EcoRV with short (7–14 bp) substrate
DNAs has been investigated in bulk by using FRET, stopped-flow
fluorescence, fluorescence anisotropy, and quench-flow methods
by rates of DNA hydrolysis and product release (Fig. 2a, steps IV
and V), which are approximately equivalent in magnitude (46),
whereas DNA bending is nearly simultaneous with binding. It is
thus possible to compare k30and k40with kcat. The determined k30
Mg2?and are closer to the bulk kcatfor 4 mM Mg2?(49). Our rate
constants are very close to the first-order rate constant of 0.4 s?1as
obtained by FRET for the later stages of the reaction cycle (46).
Thus, although deviations from the bulk rates are to be expected in
our experiments (in our case, the DNA substrate is tethered on the
surface and has less configurational freedom; the gold probes may
slow the dynamics of the enzymatic reaction, and there may be a
small nonzero force between the two particles**), such deviations
are therefore small (not more than 0.2 s?1).
Besides elucidating the dynamics of DNA bending and cleavage
by EcoRV, the scattering trajectories can also be used to estimate
the interaction potential ? between the two DNA-linked nanopar-
ticles. Just as the probability P of a DNA-tethered microsphere to
be at a certain height h above a surface depends on the potential
between the surface and the microsphere (51, 52), the probability
of an individual plasmon ruler to be in a certain intensity state I is
related to the interparticle potential ?. The interparticle potential
? determines the equilibrium interparticle distance. We used the
relationship P(I) ? exp(??(I)/kT) to construct the potential ? in
the bent and unbent states for the 40-bp plasmon rulers. P(I) is
obtained from scattering intensity histograms of the high- and
low-intensity states from single scattering trajectories with ?100
frames in the high-intensity state (Fig. 5c). The resulting average
potentials for the bent and unbent states are shown in Fig. 5d. The
repulsive part of the interparticle potential is mainly determined by
the surface coating of the particles and thus is identical for the bent
and unbent states. Consequently, the arbitrary additive constant of
the potentials has been chosen so that the repulsive parts of the
potentials coincide. Comparing the two potentials reveals that the
bent state is energetically disfavored by ?? ? 1.2kT.
This interpretation of the intensity distributions (Fig. 5c) must
take various error sources into account. Specifically, the measured
intensity distributions are convolutions of longitudinal and trans-
verse fluctuations of the top particle with respect to the surface-
bound particle and experimental noise. We are interested only in
the variations of the interparticle separation, corresponding to the
end-to-end distance of the tethering DNA. Assuming that the
transverse fluctuations and the experimental noise are free from
systematic errors, then their combined effect will be to broaden the
intensity distributions. In consequence, the intensity distributions
of the longitudinal fluctuations, and the potentials shown in Fig. 5d
are lower bounds constraining the shape of the unknown and not
directly measurable true potentials. Our ?? of 1.2kT is therefore a
lower bound on the true energy difference between the bent and
?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).
of the two rate-determining steps, DNA hydrolysis and product release. Based on the
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.
www.pnas.org?cgi?doi?10.1073?pnas.0607826104Reinhard et al.
straight states. Indeed, the worm-like chain predicts an energy
difference of 1.6kT for a 52° kink in a 40-bp DNA molecule (53),
exceeding our experimental value by 25%.
A second quantity, the relative change of the width of the two
potentials, is less subject to various experimental errors because
they should equally affect both intensity distributions and the
assumption of coincidence of the repulsive parts of the potentials is
not needed. Parabolic fits ? ? 1 ? bI2to the superimposed
potentials in Fig. 5e reveal that bbentis 27% smaller than bunbent
(bbent? 0.016, bunbent? 0.022). The wider potential for the bent
DNA state indicates that the interaction of the DNA with the
?? and has mechanistic implications. By reducing the DNA stiff-
ness, the enzyme decreases the energetic bias of the bent state and
reduces the activation energies of the rate-determining steps,
phosphate backbone hydrolysis and DNA dissociation, both of
which occur in the bent state (Fig. 2a).
The plasmon ruler is a previously undescribed method for
measuring dynamical distance changes in single-molecule biophys-
ics experiments. In this work, we have taken an important step in
validating the use of this type of ruler, by performing an in-depth
plasmon ruler experiments recapitulate what is known from prior
ensemble kinetic studies and single-molecule FRET experiments,
indicating that the Au particles do not significantly perturb the
studying fluctuations in bending of DNA, which can be used to
determine the potential energy change between the straight and
bent DNA states. The plasmon ruler is now sufficiently well
developed to be used as a tool to study a wide range of biological
age trajectory recorded at 240 Hz by using an intensified CCD detector. (b–d)
Average scattering intensities at defined intervals preceding the dimer dissocia-
tion (0 ms) for 30 (b), 40 (c), and 60 (d) bp plasmon rulers (raw data recorded at
85 Hz). We set the time of the plasmon ruler dissociation equal to 0 ms and
calculated average intensities for shortening time intervals between [?1,770 to
plasmon coupling is too weak to observe the bending (see text). The slight drop
in the last data point of d is due to some uncertainty in defining the moment of
dissociation. In some trajectories, points during or shortly after the dissociation
were picked, leading to an overall lower scattering intensity. (e–h) Individual
scattering trajectories for 40-bp plasmon rulers recorded at 85 Hz. The raw data
are plotted in blue; sliding averages [over 10 (e), 25 (f), and 50 (g and h) frames]
are included in red to guide the eye. A common feature observed in many
(f) of milliseconds to tens of seconds (g). (h) In ?4% of the recorded trajectories,
we first observed a slight drop in intensity before dimer dissociation. This result
is consistent with subsequent cleavage event in plasmon rulers with multiple
Single DNA cleavage events monitored by plasmon coupling. (a) Cleav-
norm. Intensity (a.u)
10 121416 18 20
-20246810 12 14 16 18 20
k = 0.46 s
norm. Intensity (a.u)
norm. Intensity (a.u)
a bin size of 100 ms for 30 (Upper) and 40 (Lower) bp plasmon rulers. (b)
included as dashed lines (k30? 0.50 s?1, k40? 0.46 s?1). (c) Histogram of normal-
ized intensities for 21 individual cleavage trajectories of plasmon rulers with
intensities are normalized to the maximum of the intensity distributions in the
to higher-intensity values compared with the straight state (red). (d) Average
interaction potentials of the particles in plasmon rulers with bent (black) and
unbent (red) DNA spacer obtained from c. The potential of the bent state is
by ?? ? 1.2 kT. This energetic offset corresponds to the energy required for
bending the DNA. The histogram shows the intensity distribution of the penul-
timate frames before dimer dissociation for all 21 trajectories. (e) Superposition
Kinetic and thermodynamic analysis of single-molecule data. (a and b)
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