Optical Properties of Vibronically Coupled Cy3 Dimers on DNA Scaffolds

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B: Biophysical Chemistry and Biomolecules

Optical Properties of Vibronically Coupled Cy3 Dimers on DNA Scaffolds Paul D. Cunningham, Young C. Kim, Sebastián A. Díaz, Susan Buckhout-White, Divita Mathur, Igor L. Medintz, and Joseph S. Melinger J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b02134 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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The Journal of Physical Chemistry

Optical Properties of Vibronically Coupled Cy3 Dimers on DNA Scaffolds Paul D. Cunningham,1* Young C. Kim,2 Sebastián A. Díaz,3 Susan Buckhout-White,3 Divita Mathur,3,4 Igor L. Medintz,3 Joseph S. Melinger1† 1

Electronics Science and Technology Division, Code 6800, U.S. Naval Research Laboratory,

Washington, DC 20375 2

Materials Science and Technology Division, Code 6300, U.S. Naval Research Laboratory,


Center for BioMolecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory,


College of Science, George Mason University, Fairfax, Virginia 22030, United States

*[email protected]

†[email protected]

ACS Paragon Plus Environment

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ABSTRACT We examine the effects of electronic coupling on the optical properties of Cy3 dimers attached to DNA duplexes as a function of base-pair separation using steady-state and timeresolved spectroscopy. For close Cy3-Cy3 separations, 0 and 1 bp between dyes, intermeditate to strong electronic coupling is revealed by modulation of the absorption and fluorescence properties including spectral bandshape, peak wavelength, and excited state lifetime. By use of a vibronic exciton model, we estimate coupling strengths of 150 cm-1 and 266 cm-1 for the 1 and 0 bp separations, which are comparable to those found in natural light-harvesting complexes. For the strongest electronic coupling (0 bp separation) we observe that the absorption band shape is strongly affected by the base-pairs that surround the dyes, where more strongly hydrogen bonded G-C pairs produce a red-shifted absorption spectrum consistent with a J-type dimer. This effect is studied theoretically using molecular dynamics simulation, which predicts an in-line dye configuration that is consistent with the experimental J-type spectrum. When the Cy3 dimers are in standard aqueous buffer the presence of relatively strong electronic coupling is accompanied by decreased fluorescence lifetime, suggesting it promotes non-radiative relaxation in cyanine dyes. However, we show that use of a viscous solvent can suppress this non-radiative recombination and thereby restore the dimer fluorescent emission. Ultrafast transient absorption measurements on Cy3 dimers in both standard aqueous buffer and viscous glycerol buffer suggest that sufficiently strong electronic coupling increases the probability of excited state relaxation through a dark state that is related to Cy3 torsional motion.


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Introduction

DNA scaffolds have proven to be a powerful way to organize matter on the nanoscale.1 A variety of inorganic and organic materials have been arranged by DNA scaffolds including quantum dots,2 metal nanoparticles,3, 4 nitrogen vacancy centers,5 enzymes,6 and a wide range of organic dye molecules.7 In particular, the spatial positioning of dye molecules can be as fine as 3.4-3.6 Å, which is the distance between base pairs (bp) along the double helix of the B form of double stranded DNA (dsDNA). While control of dye orientation with DNA scaffolds is challenging,8 the spatial positioning can be finer than the size of typical dye molecules, providing an opportunity to control intermolecular interactions for both fundamental studies and applications.

DNA scaffolds have the potential to organize dye molecules into artificial exciton networks that mimic those found in natural light harvesting (LH) complexes. It is well known that nature uses a protein scaffold to precisely position and orient chromophore molecules (chlorophylls) in a network that uses both strong and weak electronic coupling to transport photoexcitations with near unit quantum efficiency.9 A number of studies have explored using DNA scaffolds to transfer energy in both small7, 8, 10, 11 and large dye networks12-15 in the weak coupling limit via Förster resonance energy transfer (FRET). Some of these works have demonstrated rather sophisticated DNA scaffolds that focus photoexcitations to a common site in a way that is reminiscent of nature.12-14 On the other hand, our ability to use DNA scaffolds to create effective energy transfer structures when the dyes are closely spaced is less advanced.16 In this limit, relatively small changes in dye position and orientation can have large effects on absorption and fluorescence properties. Therefore, it becomes very important to better understand how to control


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both DNA scaffold properties, e.g. rigidity, and the local dye environment in order to create multi-dye systems with optimal energy transfer properties.

Recently, the absorption properties of closely spaced cyanine dyes attached to dsDNA scaffolds have been examined.17, 18 In these works, either H-like or J-like dimers where reported depending on the specific dyes that were used. However, a full theoretical description of the electronic coupling that includes electron-vibrational coupling was missing, which can potentially lead to overestimation of the electronic coupling strength. As both the absorption and fluorescence properties are affected by dye interactions, an understanding of how strong vibronic coupling influences the fluorescence properties and excited state lifetimes is still needed. Further, the effect of the local DNA environment (base pair sequence) on the dye orientation and coupling strength has not yet been explored. Such an understanding is crucial to the design of highly efficient exciton networks, similar to natural photosynthetic systems,19 that exploit strong electronic coupling.16

Here we show that the absorption and fluorescence of Cy3 dimers attached to dsDNA can be tuned by controlling the separation between the dyes, as well as by altering the base pairs that flank the dyes. When the Cy3 dyes are placed at the closest separations (0 bp and 1 bp cases, as in Figure 1a), we observe clear signatures of exciton formation through Davydov splitting of the low energy absorption band into J-like and H-like components as well as redistribution of oscillator strength into the higher energy vibrational bands due to exciton-vibration coupling that is consistent with vibronic exciton theory.20,

21

The relative weightings of J- and H-like

components depends on choice of bp sequence flanking the dimer. Molecular dynamics (MD)


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simulations confirm that the environment provided by flanking the dimer with more strongly hydrogen bonded G-C pairs allows the dyes to orient (on average) in a nearly end-to-end configuration, consistent with a J-type absorption spectrum. Furthermore, we observe that the strongest electronic coupling (0 bp and 1 bp separations) produces shortened fluorescence lifetimes when the dimers are in aqueous buffer solution but not when the dimers are in viscous glycerol/H2O buffer. Our measurements suggest that sufficiently strong electronic coupling increases the probability that excited Cy3 dimers relax through non-radiative channels that are related to torsional motion about the Cy3 polymethine bridge.

Methods Sample Preparation. Dye-labeled and unlabeled DNA strands were obtained from obtained from Integrated DNA Technologies (Coralville, IA, USA). DNA duplexes were formed by heating the complementary strands to 90 °C and then ramping the temperature down to 4 °C over the course of one hour. All experiments were performed with the series in the standard 2.5x PBS (342.5 mM NaCl, 25 mM phosphate, 6.75 mM KCl) unless otherwise noted. Steady state absorption and fluorescence measurements were performed on this series. Both room temperature and low temperature measurements, with the coldfinger at 78 K, were made on the 0 bp structures in 2:1 glycerol:H2O at 2.5x PBS. Absorption and Emission. Steady-state absorption spectra were measured for 150 µL samples in a 1 cm path length cuvette using an Agilent 8453 diode array UV-vis spectrophotometer. For low temperature measurements, absorption spectra were recorded using a Perkin-Elemer Lambda 750


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spectrophotometer and using a 1 mm path absorption cell mounted in a copper sample holder that was attached to the cold finger of a Janis ST-100 cryostat. Fluorescence spectra and fluorescence excitation profiles, corrected for the wavelength response of the lamp, grating, and detector, were measured using a Multifunction Microtiter Plate Reader (Tecan Infinite MR 1000 Pro). Molecular Dynamics Simulations. All molecular dynamics (MD) simulations were performed with Gromacs 5.1.4 package22 in the NPT ensemble, using the CHARMM 36 force field for DNA23 and Cy3 parameters.24 The longrange electrostatics were computed using the particle-mesh Ewald method with a real-space Coulomb cutoff of 1.2 nm. The van der Waals interactions were cut off at 1.2 nm. All bonds were constrained using the LINCS algorithm.25 The neighbor searching algorithm was used with a cutoff of 1.2 nm and the neighbor list was updated every tenth step. A time step of 2 fs was used for all simulations.

The starting Cy3-DNA structures were built using the UCSF Chimera software, production version 1.11.2. Rectangular periodic boundary conditions were used with box length of ~ 8.2 nm x 8.2 nm x 8.2 nm, ensuring a water layer of at least 1 nm between the DNA and the edge of the box. The systems were solvated in TIP3P water and Na+ and Cl- ions were added to satisfy the salt concentration of 350 mM. The final systems contained ~ 17000 water molecules. The systems were then energy-minimized using the steepest descent method for 1000 steps. The systems were first equilibrated for 200 ps at constant temperature of 300 K and pressure of 1 atm. Cy3-DNA and solvent was coupled separately to temperature baths of the reference temperature (300K) with a coupling time of 0.1 ps, while the pressure was kept constant to a bath of the


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reference pressure (1 atm) using a coupling time of 1.0 ps. The production trajectories of the Cy3-Cy3-DNA complexes were calculated for 1 µs keeping the number of particles, temperature and pressure constant. The velocity-rescale thermostat26 was used at 300 K with a coupling constant of 0.1 ps applied to Cy3-DNA and water separately. The pressure was maintained at 1 atm isotropically with the Parinello-Rahman barostat27 and a coupling constant of 1.0 ps. The coordinates were written every 10 ps for analysis.

The distance and orientation between two Cy3 dyes were calculated from a group of four carbon atoms (C6A, C6B, C7A, C7B; naming convention from Spiriti et al.24 ) from each dye. Specifically, the distance between two dyes was set equal to the distance between centers of mass of the four carbon atoms. The orientation between two dyes was calculated using the following vectors; a vector connecting the centers of mass and an average of two vectors, one from C6A to C6B and the other from C7A to C7B, from each dye.

Time-resolved Fluorescence. The Cy3 photoluminescence dynamics were measured via time-correlated single photon counting (TCSPC) that has been detailed elsewhere.10 Briefly, the system was based on a 80 MHz 7 ps pulsed 532nm frequency-doubled diode-pumped Nd:YVO4 laser (High-Q picoTRAIN). The dye-labeled dsDNA in 2.5x PBS buffer/water solutions were places in 1 mm path quartz spectrophotometric cell. Sample fluorescence was collected and sent through a polarizer set to the magic angle28 and then filtered using a monochromator. A micro channel plate photomultiplier tube (Hamamatsu) was used to detect the fluorescence with a ~45 ps instrument response function (IRF) as measured using scattered 532 nm light.


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Ultrafast transient absorption. Transient absorption spectroscopy was used to measure the exited state dynamics of ~250 µL samples in a 1 cm stirring cuvette maintained at a temperature of 283 K to avoid potential sample degradation from laser induced heating. The experimental setup is detailed elsewhere.29 Briefly, a non-collinear visible optical parametric amplifier (NOPA, Clark-MXR) is used to produce tunable excitation pulses and a small amount of power from a Ti:sapphire amplifier (CPA 2101, Clark-MXR) is used to generate white light continuum probe pulses in a sapphire plate. Photoinduced changes in the sample transmission spectra were analyzed using a scanning monochromator. All measurements were performed with linearly polarized pump and probe pulses, with the pump polarization at magic angle with respect to the probe to eliminate the influence of depolarization dynamics.

Results and Discussion Cy3 pairs were assembled on dsDNA wire scaffolds to allow precise control of the inter-dye separation. Cy3 molecules were internally attached to the DNA strands during synthesis through double phosphate attachments, see Figure 1a, which are known to localize dyes and reduce the uncertainty in their absolute position.10, 30 The Cy3 molecule used here does not contain the sulfonate groups sometimes used in Cy3 to increase solubility. The placement of one of the Cy3 dyes was varied to produce a series of dsDNA constructs where the interchromophore distance has been systematically varied from 0 – 7 bp separation. Schematic examples are shown in Figure 1a for 7 bp, 1 bp, and 0 bp cases. The sequences used for all doubly (Table S1) and singly (Table S2) labeled DNA duplexes are shown in the Supporting Information (SI). In order to


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investigate the influence of the flanking DNA dp sequence on dye interaction, two types of 0 bp structures were synthesized that differ only by the base pairs flanking the dyes: AT or GC, see Figure 1a.

Absorption Properties

Figure 1. (a) Schematic of Cy3-Cy3 labeled dsDNA for 7 bp, 1 bp, and 0 bp separations between Cy3 molecules. The structure of the Cy3 double phosphate attachment chemistry is shown. Also shown are the two variations of the local base pair sequences that flank the Cy3 dyes for the 0 bp case. (b) Measured absorption spectra of Cy3-Cy3 labeled dsDNA in 2.5x PBS for dye separations of 0 - 7 bp normalized by the 0-0 peak. A-T pairs flank the Cy3 dyes for the 0bp spectrum. Inset shows the ratio of the 0-1 absorption feature amplitude with respect to the 0-0 absorption amplitude as a function of bp separation.


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The absorption spectra were measured in phosphate saline buffer (2.5x PBS) as a function of Cy3-Cy3 dye separation, see Figure 1b. We observe the vibrational progression typical of cyanine dyes with vibrational energy of approximately 150 meV (1200 cm-1) due to the C-C stretch of the polymethine skeleton.31 In the following discussion, we will refer to the lowest energy (longest wavelength) absorption band as the 0-0 transition, and the next two higher energy (shorter wavelength) bands as the 0-1 and 0-2 transitions, respectively. This simplification neglects couplings to other high frequency vibrations and to low frequency vibrations. As the bp separation decreases, the absorption spectrum is altered due to increased electronic coupling between the two Cy3 molecules, see Figure 1b. Specifically, there is an increase in the relative intensity and an accompanying blue shift of the 0-1 vibrational shoulder near 518 nm as the two dyes are brought closer together, Figure 1b inset. These alterations to the absorption spectrum are most pronounced for the 1 bp and 0 bp separations. This behavior has been previously observed in both homo- and hetero-dimers of cyanine dyes10,

17, 32, 33

These

observations cannot be explained by a simple H-dimer with energy coincident with the 0-1 transition, as the H-dimer energy depends on coupling strength and would therefore vary significantly with dye separation. There is also an increase in the relative intensity of the 0-2 vibrational shoulder near 475 nm. These spectral alterations are consistent with the borrowing of oscillator strength by weaker vibronic transitions, which can occur in dimers as a consequence of coupling and energy-level hybridization.20 For 0 bp separation, the 0-0 state absorption shows significant broadening that is consistent with Davydov splitting, which is where intermolecular coupling splits the 0-0 band due to both in-phase and out-of-phase interactions of the transition dipoles on the two interacting molecules.34


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In order to better establish the presence of multiple peaks within the broadened 0-0 band, we cooled the 0 bp samples in a cryostat with liquid nitrogen (LN2) to reduce homogeneous broadening of the absorption peaks. The 0 bp samples were prepared at 2 µM dsDNA in a 2:1 glycerol:H2O solvent containing 2.5x PBS (2:1 gly:H2O/PBS), which yields an optical quality glass at cryogenic temperature. Differences in the room temperature absorption spectra for the 0 bp samples in 2.5x PBS and in 2:1 gly:H2O/PBS suggest the Cy3-Cy3 relative orientations are somewhat different in each solvent, see Figure 2 a and b. The 0 bp samples were cooled until the temperature sensor on the cryostat cold finger reached 78 K. Because of the relatively poor thermal conductivity of the sample, it is likely that the sample reached an equilibrium temperature higher than 78 K. Even so, the absorption peaks become considerably narrower (by approximately a factor of two), exposing two components within the 0-0 band, Figure 2a and b. These components are the H-like (higher energy) and J-like (lower energy) contributions to the 0-0 Davydov splitting, see Figure 2c, and their relative intensity depends on the precise geometry of the dimer. It has recently been shown that, in addition to broadening the absorption features, temperature dependent disorder can also modulate the electronic coupling of Cy3 dimers on DNA scaffolds,35 and it should be noted that while qualitatively similar the precise geometry at low temperature is not necessarily the same as at high temperature. It is interesting to note that the H-like peak appears broader than the J-like peak, suggesting rapid relaxation times of those higher energy states.


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Figure 2. Absorption spectra of the 0 bp sample measured at 295 K (red) and cooled by LN2 (blue) in 2:1 gly:H2O/PBS. Room temperature spectra in 2.5x PBS (grey) are included for comparison. Absorption spectra were measured for both (a) A-T and (b) G-C flanking base pairs. The energy splitting of the 0-0 transitions are indicated by arrows based on fitting Gaussian peaks to the data. The absorption curves have been vertically shifted for clarity. (c) Schematic energy level diagram based on molecular exciton theory


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showing that dye coupling in dimers leads to Davydov splitting of the 0-0 transition. The allowed (dark) and forbidden (light) transitions depend on the dimer geometry where Hdimers allow only for the higher energy transition leading to a blue-shift and J-dimers only allow the lower energy transition leading to a red-shift

We speculated that the DNA sequence in the vicinity of the Cy3 dyes might affect the spacing and orientation of the dyes, which would be reflected in the observed absorption spectrum. For the 0 bp structure, with corresponding absorption spectra in Figure 1b and 2a, the flanking base pairs are both A-T. However, A-T base pairs are more weakly bonded and possess two hydrogen bonds while G-C pairs are more strongly bonded with three hydrogen bonds. If the A-T base pairs are instead replaced with G-C base pairs, the absorption spectrum near the 0-0 feature becomes dominated by the red-shifted (J-like) contribution and the Davydov splitting becomes less obvious, see Figure 2b. Further, the relative intensity of the vibronic peak is reduced, which is consistent with a J-like transition.20 In 2:1 gly:H2O/PBS at room temperature, the H-like peak is further suppressed and the whole spectrum is red-shifted by 4 nm (16 meV). When cooled as before using LN2 the 0-0 absorption bands narrow considerably and reveal a relatively intense Jtype band and a relatively diffuse H-type band. For a direct comparison between the 0 bp samples with A-T and G-C base pairs see SI Figure S1. Overall, the absorption spectra of the 0bp sample with G-C flanking base pairs are consistent with a more J-type, i.e. in-line, dye configuration. These observations suggest large differences in the orientation of the two Cy3 molecules are achieved by simply changing the surrounding DNA sequence.


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The 0-0 splitting can be estimated by fitting the absorption spectrum with the sum of four Gaussian peaks, see Figure S2. According to molecular exciton theory, the electronic coupling energy is approximately half the 0-0 splitting.34 This method estimates the splitting between the center wavelengths of these two components of the 0 bp (A-T) absorption spectrum as 45 meV (366 cm-1) at low temperature, which is somewhat smaller than the estimated 64 meV (512 cm-1) splitting in 2.5x PBS buffer at 295 K. For the 0 bp (G-C) sample, the splitting between the center wavelengths is similarly estimated as 49 meV (397 cm-1) at low temperature, which is somewhat smaller than the estimated 57 meV (464 cm-1) splitting in 2.5x PBS buffer at 295 K. The broad 01 feature, when cooled with LN2, is likely a result of multiple transitions associated with electron-vibration (i.e. vibronic) coupling within the dimer.


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Figure 3. Measured absorption spectra of Cy3-Cy3 labeled dsDNA for dye separations of (a) 7 bp, (b) 1 bp, (c) 0 bp (A-T), (d) 0 bp (G-C) (dots) and simulated spectra (lines). The individual transitions of the simulated spectra are indicated by narrow Gaussian peaks of different colors. For all simulations S = 0.58 and = 145 meV. The normalized coupling strength, η, and phase, ϕ, are described in the SI.

The coupling strength can also be estimated by modeling the absorption spectra of the Cy3 dimers using the vibronic exciton model within the two-particle approximation, following the theoretical framework developed by Kühn, Renger, and May,20,

21, 36

for details see SI. The

model includes two vibrational quanta in both the ground and excited states. We use Eqn S12 to


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simulate the absorption spectra of the Cy3-Cy3 system for 7, 0, and 1 bp separations. For 7 bp separation, we treat the two Cy3 molecules as having insufficient coupling to produce excitonic effects associated with dimerization. Therefore, for 7 bp separation the absorption spectrum can be used to determine the Huang-Rhys factor (S) that describes the electron-vibration coupling in Cy3. Estimating the 0-1 transition to be = 145 meV (1170 cm-1) above the 0-0 transition, the absorption spectrum is well described by electron-vibration coupling with S = 0.58, Figure 3a. The Gaussian widths were assumed to be 0.33 for the 0-0 peaks, and 0.39 for the higher vibrational peaks. Keeping S fixed, we simulate the experimental absorption spectra of the 1 bp, 0 bp (A-T) and 0 bp (G-C) samples by varying the coupling strength, η, and phase factor, ϕ, in Eqn S6. The best agreement with the measured absorption spectrum was found for 1 bp separation using η = 0.23 and ϕ = 0.40 π (Figure 1b), for 0 bp (A-T) using η = 0.41 and ϕ = 0.48 π (Figure 1c), and for 0 bp (G-C) using η = 0.43 and ϕ = 0.56 π (Figure 1d). For the 0 bp samples, the dipole coupling strength is approaching the value of the electron-vibration coupling (i.e. η ~ S), indicating the dye interaction is in the intermediate coupling regime.20 The resulting

electronic coupling between the 0-0 states, , , is estimated from Eqn. S6 as 19 meV (150 cm1

), 33 meV (266 cm-1), and 35 meV (282 cm-1) for the 1 bp, 0 bp (A-T), and 0 bp (G-C)

separations. This is much smaller than the value of ~ 75 meV (~600 cm-1) arrived at by assuming the strong 0-1 peak is due to a subpopulation of H-dimers coincident with the 0-1 transition energy, and demonstrates the importance of accounting for vibronic coupling.


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Figure 4. Molecular dynamics simulations of the Cy3-Cy3 dye separation and relative orientation of the 0 bp separation sample for (a) A-T and (b) G-C as the flanking base pairs. Representative structural and ribbon models (c) – (g) are shown for the higher probability configurations that are indicated in (a) and (b).

We performed MD simulations in order to gain more insight into how changes to the flanking base pair sequences affect the geometrical configurations of the Cy3 dyes and the DNA scaffold, which, in turn, affect the absorption spectrum.

The simulations were performed at room

temperature and in the presence of water with 350 mM NaCl, and run for 1 µsec. The first 50 ns of the simulation is considered an equilibration time, where the dye/dsDNA configuration relaxes from the input structure, and is not used as representative dye configurations. The dye-


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dye center-to-center separation (R) and dye orientation were determined at each subsequent time step. The dye orientation factor is calculated as = ̂ ∙ ̂ − 3 ∗ ̂ ∙ ∗ ∙ ̂ ,

(1)

where is the unit vector pointing from the donor transition dipole moment to the acceptor transition dipole moment. ̂ and ̂ are the donor and acceptor transition dipole moment unit vectors respectively and are taken to coincide with the long axis of Cy3.

The configurations of the dyes are found to depend on the flanking base pairs, Figure 4a and b. For A-T pairs the variations in R and are relatively large, Figure 4a, where R varies from 1030 Å and varies over nearly the full range of values from -2 to 2. The broad distribution of configurations also contains three peaks that indicate preferred configurations. The largest peak is centered at R ~ 21 Å and ~ 0.8. Two smaller peaks occur at R ~ 11 Å and ~ 0.9, and R ~ 11 Å and ~ 1.3. Inspection of the simulation showed that the dyes tend not to intercalate into the DNA base stack. Instead, the dyes undergo motions outside the DNA base stack region. As examples, Figure. 4c-e shows representative snapshots of the simulation for the three highest probability configurations in Figure 4a. The flanking A-T pairs move into the region spanned by the dyes, thus preventing the dyes from intercalating into the base stack. We interpret the exciton splitting of 512 cm-1 (Fig 2a) to arise from configurations where the dyes come closer than 20 Å. While the MD simulations qualitatively produce the oblique relative orientations of the dyes expected for Davydov splitting, this is only a partial picture of the dye configurations. The 1 µsec time frame may be insufficient to yield a full free-energy landscape. To achieve this, more sophisticated sampling methods such as an umbrella sampling with the weighted histogram analysis method (WHAM)37 or the replica exchange molecular dynamics (REMD)38 are required.


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Fully quantitative simulations of the dye-DNA system that predict the observed absorption spectra are beyond the scope of this study.

In contrast, the variations of R and κ are much smaller for the case of flanking G-C pairs, Figure 4b. Here, the distribution has its primary peak centered at ~ 17 Å and ~ 1.9. There is a second smaller peak centered near R ~ 11 Å and ~ 1.2. Representative snapshots of the simulation for each of the peak probability configurations are shown in Figures 4f and g. For the majority peak, both dyes intercalate in a configuration that is nearly in-line (i.e. end-on-end). This configuration enhances the relative oscillator strength in the J-type transition of the absorption spectrum. For the smaller peak in the distribution, the dyes remain intercalated but with transition dipoles approximately oblique, which produces oscillator strength in both J- and H-type transitions. These configurations are consistent with the experimental absorption spectra in 2.5x PBS and 2:1 gly:H2O/PBS, see Figure 2b, which show enhanced J-like contributions, suppressed H-like contributions, and reduced intensity of the vibronic band.

Partial intercalation of Cy3 dyes doubly attached to DNA has been previously observed in MD simulations by Stennett et al.30 That work considered Cy3 monomers flanked by GC pairs. The dye attachment chemistry used in that work involved removal of the nucleobase at the dye attachment point, which creates a small gap in the DNA base pair sequence. Their MD simulations showed that the dye configuration fluctuates between regions that are outside the base stack, as well as partially intercalated structures. We have employed similar attachment chemistry, with the main difference being that for 0 bp separation a pair of Cy3 dyes attach at the same location along the DNA. This creates a large enough gap for both dyes to intercalate into


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the base stack region. Our MD simulations suggest when the flanking base pairs are G-C pairs then the favored structure maintains this gap, and the two Cy3 dyes insert into the gap in a near in-line configuration. At first glance it is tempting to attribute the predominance of this structure (Figure 4b) to the stronger hydrogen bonding between G-C pairs (three hydrogen bonds) compared to A-T pairs (two hydrogen bonds). The G-C pairs may thereby stabilize the DNA duplex as evidenced by the higher melt temperature for the 0 bp (G-C) sample compared to the 0 bp (A-T) sample, for details see Table S1. However, base stacking is thought to play the dominant role in DNA stability,39, 40 though predicting the rigidity of a DNA strand based on its sequence remains a difficult problem.41 Therefore, what determines the positioning and relative orientation of the Cy3 dyes is likely to be more complex, involving an interplay of base stacking interactions, the − electron interactions between the Cy3 dyes and the nucleobases, and −electron interactions between the two Cy3 dyes. Particularly relevant to the results presented here is the work of Norman et al.,42 which showed that Cy3 dyes attached to the 5’ end of DNA duplexes tend to align with the terminal G-C base pairs.

It is important to contrast our results with previous works that have investigated linear optical properties of cyanine dimers on DNA scaffolds.17, 18, 43 Though not the focus of the work, Nicoli et al.17 measured a qualitatively similar absorption spectrum of a 0 bp Cy3 dimer, using the same double attachment scheme used here, flanked by A-T base pairs. However, the nature of the absorption or emission spectra was not elaborated on. Canon et al.43 and Markova et al.18 studied Cy5 dimers using the same double attachment chemistry and observed J-like absorption spectra, which indicated that both Cy5 dyes intercalated into the DNA to form an in-line configuration. One important difference in both of these works was that a G-C pair was placed on one side of


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the dimer and A-T pair on the other side, i.e., G-C/A-T. Apparently, this local base pair configuration also allows both dyes to intercalate for the case of Cy5. Very recently, 0 bp Cy3 dimers were similarly constructed with a G-C pair on one side and A-T pair on the other side, in which the effect of temperature dependent disorder on the electronic coupling strength was investigated.35 The reported absorption spectra were similar to those we observe for 0 bp Cy3 dimers flanked by A-T pairs.

Fluorescence Properties

The fluorescence spectra were measured as a function of Cy3-Cy3 dye separation. A much smaller increase in the vibrational shoulders are seen in the emission spectrum for 0 bp separation, see Figure S3, as compared to the absorption spectrum, see Figure 1b. This is quite different from the large alterations to the fluorescence spectra reported for high concentrations of Cy3 dyes confined within micelles.44 We also do not observe the red shifted emission expected for a cyanine H-dimer.45 No significant differences are present in the emission spectra for 1-7 bp separations.


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Figure 5. Comparison between absorption spectra (black) and fluorescence excitation profiles (red) collected for 600 nm emission for (a) 2 bp, (b) 1 bp, (c) 0 bp (A-T pairs) in 2.5x PBS, (d) 0 bp (G-C pairs) in 2.5x PBS, (e) 0 bp (A-T pairs) in 2:1 Gly:H2O/PBS, and (f) 0 bp (A-T pairs) in 2:1 Gly:H2O/PBS.

Fluorescence excitation profiles (FEPs) were measured as a function of bp separation for Cy3 pairs in 2.5x PBS, Figure 5. Separations larger than 1 bp, show near complete agreement between FEP and absorption, Figure 5a. The 1 bp absorption spectrum shows significantly higher relative intensity of the 0-1 vibronic peak compared to the FEP, see Figure 5b. The profile of the 0 bp absorption spectrum with flanking AT base pairs, see Figure 5c, shows some disagreement with the FEP for both the 0-0 and 0-1 peaks. In the 0-0 region, the FEP peak is both slightly blue shifted by 2 nm and somewhat narrower than the absorption. The observation that the FEP does not fully agree with the absorption spectrum in the 0-0 region may be an indication that the Cy3 dyes in the ensemble that have the strongest electronic coupling are either weakly or non-fluorescent. In contrast, the 0 bp structure with G-C flanking base pairs shows


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good agreement between the FEP and absorption spectrum in the 0-0 region, see Figure 5d. This may be an indication of a more homogeneous arrangement of dye configurations, such that the dyes tend not to come so close as to produce fully quenched emission. From the lower relative intensity of the FEP near 0-1 transition, we conclude that the increased contribution of vibronic states may be accompanied by increased non-radiative relaxation.

We also compared the absorption spectra and FEPs for the 0 bp structure in 2:1 gly:H2O/PBS. For both A-T, see Figure 5e, and G-C base pairs, see Figure 5f, flanking the Cy3 dyes the absorption spectra and FEPs agree well in the 0-0 region. For A-T pairs, the relative intensity of the FEPs in the 0-1 region is lower than the absorption. However, for G-C the difference between FEP and absorption spectrum is small (few percent). These data suggest that the more viscous glycerol:H2O solvent suppresses the non-radiative relaxation, implying this relaxation channel involves a change in molecular geometry.


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Figure 6. (a) Fluorescence decay dynamics measured as a function of bp separation between Cy3s. Inset shows the average fluorescence lifetime calculated from multi-exponential fits to the decay dynamics. The color scale of the inset matches that in the main figure. (b) A comparison of the fluorescence decay dynamics in 2.5x PBS buffer and 2:1 gly:H2O/PBS (gly) for the 1 bp and 0 bp (A-T) samples.

Fluorescence lifetimes were measured by time-correlated single photon counting for the 0 – 7 bp series, see Figure 6a. The average lifetime is determined by fitting the experimental curves to a sum of up to three exponential decay functions and then computing an amplitude average, see Table S3. For comparison, fluorescence lifetimes were also measured for three Cy3 monomerlabeled dsDNA, represented as Cy3-x, corresponding to a single Cy3 dye located at the 0 bp, 4


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bp, and 5 bp positions. In these duplexes x represents a nucleobase located opposite the Cy3 dye (see SI). Average lifetimes for these monomer structures are also shown in Table S3. While the 1 – 7 bp samples have lifetimes that vary between 0.8 and 1.3 ns, which is near the monomer fluorescence lifetime of 1.1 ns, the fluorescence lifetime is significantly faster for the 0 bp sample. The average lifetime of the 0 bp sample (with A-T pairs) is 0.4 ns, 2.8 times shorter than the Cy3 monomer lifetime. A similar decrease in lifetime was observed for the 0 bp structure with G-C pairs, Figure S4. These results provide further evidence that non-radiative recombination increases as the electronic coupling strength increases.

When placed in more viscous 2:1 gly:H2O/PBS, the fluorescence lifetime of the 1 bp and 0 bp (A-T) Cy3 dimers substantially increased, Figure 6b. Similar changes in fluorescence lifetime are observed for both the 0 bp dimers flanked by A-T and G-C base pairs, see Figure S4. The 0 bp Cy3 dimer lifetime increased 4-fold to about 1.3 ns (both for A-T and G-C structures; see Table S4), whereas the 1 bp Cy3 dimer lifetime increased nearly 2-fold to about 1.5 ns.

One possible explanation for these observations is that sufficiently strong electronic coupling strength in the Cy3 dimer increases the transition rate of the Cy3 bright state to a dark state that is related to the torsional motion about the polymethine bridge. The viscous solvent suppresses this torsional motion. This is consistent with previous work that has shown evidence of longer cyanine fluorescence lifetimes and increased fluorescence quantum yields in more viscous environments.46-48 It is also consistent with previous work showing an increase in Cy3 dimer emission quantum yield for high dye concentrations in micelles where photoisomeriation is sterically hindered.44


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Figure 7. Transient absorption spectra of the (a) 0 bp Cy3-x, and 0 bp (G-C) dimer in (c) 2.5x PBS and (e) in 2:1 gly:H2O/PBS. Corresponding excited state dynamics measured for the GSB (blue) and SE (red) of the (b) 0 bp Cy3-x, and 0 bp (G-C) dimer in (d) 2.5x PBS and (f) in 2:1 gly:H2O/PBS.

Ultrafast Spectroscopy To better understand the effects of electronic coupling and of solvent viscosity on the Cy3 dimer relaxation we used ultrafast transient absorption (TA) spectroscopy. Here, the excited state dynamics of the 0 bp (G-C) dimer was compared to a single Cy3 dye-labeled dsDNA (i.e. 0 bp Cy3-x). The TA spectra of 0 bp Cy3-x in 2.5x PBS in Figure 7a is composed of ground state bleaching (GSB) between 500 – 550 nm, stimulated emission (SE) between 550 – 650 nm, and excited state absorption (ESA) for wavelengths shorter than 500 nm. We note that the GSB and SE decay dynamics, Figure 7b, are essentially the same over the 2 ns measurement window. The TA spectra and associated GSB and SE decays for the 0 bp (G-C) dimer in 2.5x PBS are shown


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in Figure 7c and 7d, respectively. When compared to the monomer, the dimer TA spectrum shows an additional ESA feature near 550 nm, see Figure 7c. Furthermore, the SE emission decays much faster than the GSB for the 0 bp (G-C) sample in 2.5x PBS, see Figure 7d. This suggests that a dark, non-radiative excited state is accessed in the dimer and persists longer than the bright states that decay directly back to the ground state. This is consistent with the reduced fluorescence lifetimes and quantum yields that we observe in the 0 bp dimers. Interestingly, when the 0 bp (G-C) sample is placed in 2:1 gly:H2O/PBS, there are no significant changes in the qualitative shape of the TA spectra, see Figure 7e, however the disagreement between the GSB and SE decay rate is much smaller, see Figure 7f.

Taken together, the results of the FEPs, fluorescence lifetimes, and TA experiments suggest that strong electronic coupling between Cy3 dyes increases the probability of non-radiative relaxation through a dark state that is related to the torsional motion about the Cy3 polymethine bridge. The most compelling evidence comes from the TA experiment where the longer-lived GSB that decays more slowly than the SE implies that the dark exited state is more easily accessed in the dimers. That the GSB and SE dynamics become nearly equivalent when the Cy3 dimer is placed in the more viscous gly:H2O/PBS environment is consistent with suppression of torsional motion about the polymethine bridge. If the decreased lifetime of strongly coupled Cy3 dyes is due primarily to another mechanism, such as intersystem crossing, then placing the Cy3 dimer in a more viscous environment would not be expected to produce the observed near 3-fold increase in fluorescence lifetime for the 0 bp structure. Further, if the strong electronic coupling mainly enhanced the internal conversion rate then nearly equivalent GSB and SE decay dynamics would be expected.


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Conclusions We have examined the optical properties of Cy3 dimers on dsDNA scaffolds. For dyedye separations of less than 2 bp, we observe exciton delocalization characterized by Davydov splitting of the 0-0 absorption band due to strong electronic coupling and redistribution of oscillator strength into higher energy vibrational bands due to vibronic coupling. The ability to tune the electronic coupling strength and vibronic character of the excited state is crucial to forming exciton networks like those in nature that exploit coherent phenomena.19

Our

measurements and MD simulations show that the local dsDNA sequence affects the dimer geometry. Choosing G-C base pairs to flank the dyes produces a nearly in-line 0 bp dimer that has enhanced J-like character to the absorption spectrum. The resulting control of dye orientation and reduction of inhomogeneity may prove important in the design of dye networks that utilize coherent energy transport to achieve high quantum efficiency. Strong electronic coupling between Cy3 molecules appears to lead to increased non-radiative excited state relaxation through torsional motion about the polymethine bridge. This non-radiative loss can be mitigated through use of viscous solutions that increase the fluorescence yield several fold. This result provides a path to simultaneously tune electronic coupling between cyanine dyes in light harvesting networks while minimizing loss of emission.

Supporting Information Available: details concerning the DNA sequences, comparison between 0 bp G-C and A-T absorption spectra, Gaussian fits to 0bp G-C and A-T absorption spectra, vibronic exciton model, fluorescence spectra as a function of bp separation, fluorescence


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decay dynamics of 0 bp for G-C and A-T pairs, and fluorescence decay dynamics fit results. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgement: This work was supported by the Office of Naval Research (ONR) via the U.S. Naval Research Laboratory (NRL) Nanoscience Institute as well as by the Office of the Assistant Secretary of Defense for Research and Engineering (OSD R&E) via the Laboratory University Collaborative Initiative (LUCI) program in support of the Vannevar Bush Faculty Fellowship (VBFF) program.

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Optical Properties of Vibronically Coupled Cy3 Dimers on DNA Scaffolds

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