Probe Position-Dependent Counterion Dynamics in DNA:Comparison of Time-Resolved Stokes Shift of Groove-Bound to Base-Stacked Probes in the Presence of Different Monovalent CounterionsSachin Dev Verma, Nibedita Pal, Moirangthem Kiran Singh, and Sobhan Sen*

Spectroscopy Laboratory, School of Physical Sciences, Jawaharlal Nehru University, New Delhi 110067, India

*S Supporting Information

ABSTRACT: Time-resolved fluorescence Stokes shifts (TRFSS) of 4′,6-diamidino-2-phenylindole (DAPI) inside the minor groove of DNA are measured in the presence ofthree different monovalent counterions: sodium (Na+), rubidium (Rb+), and tetrabutylam-monium (TBA+). Fluorescence up-conversion and time-correlated single photon counting arecombined to obtain the time-resolved emission spectra (TRES) of DAPI in DNA from 100 fsto 10 ns. Time-resolved Stokes shift data suggest that groove-bound DAPI can not sense thecounterion dynamics because the ions are displaced by DAPI far from the probe-site.However, when these results are compared to the earlier base-stacked coumarin data, the sameions are found to affect the nanosecond dynamics significantly. This suggests that the ionscome close to the probe-site, such that they can affect the dynamics when measured by base-stacked coumarin. These results support previous molecular dynamics (MD) simulation dataof groove-bound and base-stacked probes inside DNA.

SECTION: Biophysical Chemistry and Biomolecules

Water and ions play an important role in stabilizing theDNA double helical structure.1,2 Because DNA is a

negatively charged polyion, counterions interact strongly withDNA.2 It is found that almost 75% of DNA charge can becomeneutralized by the mobile counterions present in the“condensation” layer surrounding the DNA within radius of∼9 Å.3−5 Counterions can bind inside the grooves of DNA,4−7

sometimes inducing the groove-width8 and conformation ofDNA.9 Binding of proteins to DNA is also found to criticallydepend on the specific positioning of ions inside DNA−proteincomplexes as well as the displacement of ions by proteinresidues.10−14

Building the molecular picture of DNA−ion interaction isessential to understand how the dynamic ions around DNAstabilize its structure and control its interaction with proteinsand small molecules. Efforts have been made to understand thedirect and/or indirect role of ions on the DNA structure,4−9

DNA−protein complexation,10−14 and in the interactions ofDNA with small molecules.15,20,24,31,33 However, it is notknown whether the ion dynamics around DNA can changewhen the DNA interacts with proteins and small molecules.The question remains whether the ion-dynamics depend on theion-distributions around DNA when it forms complexes withproteins and small molecules.This Letter tackles these questions in the context of DNA

interaction with small molecules and shows that a groove-bound (probe) molecule can not sense the ion-dynamics as theions are displaced by the groove-bound molecule far from theprobe-site. However, a covalently attached base-stacked probe

inside DNA can sense the ion motions as this probe does notperturb the ion distributions around DNA.16

One way to observe DNA−ion interaction could be throughthe direct measurement of ion dynamics around DNA or by themeasurement of ions’ effect on the overall DNA motion. Time-resolved fluorescence Stokes shift (TRFSS) and relatedexperiments have the capability to measure the ion, hydration,and biomolecular dynamics directly on the time-scales of theirmotions.15−29 In general, TRFSS experiments follow thesolvation dynamics of a system by recording the change inelectrostatic interaction energy of a fluorescent probe with itssurrounding environment.29 Upon optical excitation, the dipolemoment of probe changes due to the perturbation in chargedistribution in the probe. In order to stabilize the excited probedipole, surrounding charged and dipolar molecules (in this casewater, ions, and DNA) reorient themselves by exerting reactionelectric field on the probe. This phenomenon decreases probe’sinteraction energy with its surroundings that leads to the shiftsin the fluorescence spectra of probe toward lower frequencies.Hence, the solvation dynamics is directly reflected in probe’stime-dependent fluorescence Stokes shifts.29

Even though DNA has much less structural diversity thanproteins, recent TRFSS experiments show that the dynamics inDNA depend strongly on the position of probes inside DNA,15

effects similar to those seen in proteins.25−27,37 Moleculardynamics (MD) simulations, on the other hand, provided the

Received: July 12, 2012Accepted: August 31, 2012Published: August 31, 2012

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details of contribution of water, ion, and DNA motions thatcouple together to control the total dynamics in DNA.30−32

However, disagreements are persistent about the relativecontributions of components to the total dynamics.32,34−36

Nonetheless, these studies imply that the observed coupledmotion of water, ions, and biomolecule should depend on theposition of probe inside the biomolecule where the dynamics isbeing measured. Furthermore, the total dynamics shoulddepend on the relative motions of water, ions, and biomoleculethat the probe can sense. Thus, changing the probe-positioninside DNA would result in observable change in the iondynamics, which would depend on whether the probe can sensethe ion motions through modulation of ions’ electric-field at theprobe-site. Long-range electrostatic coupling among thecomponents, however, expects that the probe must sensesimilar ion dynamics, along with similar water and DNAdynamics, even if the position of probe is changed inside DNA.This paper will show that the former concept holds; that is, agroove-bound probe can not sense the ion motions as theprobe displaces the ions far from the grooves, whereas the sameions can affect the dynamics when measured by a base-stackedprobe that does not perturb the ion-distribution aroundDNA.16 This suggests that ions can access the base-stackedprobe-site inside DNA.TRFSS experiments are extensively used to study dynamics

in proteins.25−28 In DNA, the slow dynamics was first observedby following the TRFSS of a covalently attached base-stackedcoumarin, opposite an abasic-site, inside DNA.17 Subsequently,dynamics in DNA were measured using other base-stacked andgroove-bound probes.15,18−24 MD simulations were alsoperformed to gain molecular details of the dynamics andcompare MD results with TRFSS experiments.4−6,30−35

However, all these studies focused on DNA dynamics withonly Na+ as counterion. Sen et al. reported the first elaboratenanosecond TRFSS study on counterion dynamics in DNA byreplacing Na+ with eight other monovalent counterions.16 Thisstudy found that in the presence of small alkali ions (exceptRb+), the coumarin can sense similar ion dynamics that follow apower-law, but, for larger ions, the dynamics beyond ∼1 nsdepend on the hydrodynamic size of the ions.16

Here, we measure the TRFSS of a minor groove binder, 4′,6-diamidino-2-phenylindole (DAPI), in 14-mer DNA, d(5′-CGCGCAATTGCGCG-3′)2, in the presence of three differentmonovalent counterions of different sizes: sodium (Na+),rubidium (Rb+) and tetrabutylammonium (N(C4H9)4

+ orTBA+) (see Supporting Information for Materials andMethods). Time-resolved emission spectra (TRES) of DAPIin DNA are measured from 100 fs to 10 ns independently inthe presence of the three ions. Results show identical Stokesshifts of DAPI in the presence of all three counterions. Theseresults are then compared to the ion-dependent dynamicsmeasured earlier using a covalently attached base-stackedcoumarin, which replaces a base-pair inside DNA, in thepresence of the same three ions (and also other ions).16 Thedynamics measured by coumarin show significant difference inthe nanosecond time-range among the three ions.16 We chooseonly three ions for this study based on the fact that Sen et al.found maximum ion dependence for these three ions with base-stacked coumarin, where Na+ shows a power-law dynamics, Rb+

shows anomalous behavior, and TBA+ affects slow dynamicsthe maximum.16 Thus, it is expected DAPI would showmaximum ion dependence, if there is any, in the presence ofthese three counterions.

Figure 1 compares the excitation and emission spectra ofgroove-bound DAPI to base-stacked coumarin13 in the

presence of the three counterions. DAPI spectra are identical,which imply that there are no ion effects. However, appreciablechange is found in the spectral shapes and peak positions ofcoumarin in the presence of the same ions. The spectra in thepresence of Rb+ are broader compared to other ions, whereas,TBA+ spectra are blue-shifted compared to Na+.16

Although the dynamics of water is complete within a fewpicoseconds,29 in DNA, the dynamics is found to spread overfemtoseconds to nanoseconds.15−19,24 To capture this broaddynamic range (from 100 fs to 10 ns), we combine the TRES ofDAPI-DNA, constructed independently from the decaysmeasured in fluorescence up-conversion (UPC, TRES range:100 fs to 1 ns) and time-correlated single photon counting(TCSPC, TRES range: ∼30 ps to 10 ns) (SupportingInformation). Figure 2 shows three raw decays of DAPI (see

Figure 1. Normalized fluorescence emission and excitation spectra ofDAPI bound to minor groove of DNA (solid lines), and base-stackedcoumarin (dashed lines) in the presence of Na+ (blue), Rb+ (red), andTBA+ (N(C4H9)4

+) (green). Data show substantial ion effects oncoumarin spectra.

Figure 2. Time-resolved fluorescence decays (with fits) of DAPIbound to the minor groove of DNA in the presence of Na+ (blue), Rb+

(red), and TBA+ (N(C4H9)4+) (green). (A) Data collected in the UPC

technique (up to 1.5 ns), and (B) data collected in TCSPC. Decays atthree wavelengths are compared here to minimize clumsiness. SeeSupporting Information for other decays.

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Supporting Information for all decays). The decays are almostidentical, which suggest that the relative ion-dependence isindistinguishable among the ions. The sums of 3−4exponentials are used to fit the decays. TRES were constructedusing the fitted parameters and steady-state fluorescence, suchthat the TRES at common time points in UPC and TCSPCmatch (Supporting Information). Figure 3 plots the TRES of

DAPI in the presence of Rb+. Similar data are obtained for Na+

and TBA+ (Supporting Information). Log-normal fits to TRESare used to calculate the time-dependent Stokes shifts. Theshifts are reported in terms of mean (1st moment) frequencies(Supporting Information).Figure 3 includes the time-zero spectrum of DAPI-DNA with

Rb+ in frozen glass, measured in dry ice/acetone mixture at −78°C. Glass spectrum represents time-zero position, because inglass all diffusive dynamics in sample are frozen, althoughvibrational and phonon-like inertial motions can persist.15−19

The mean frequency of glass spectrum, thus, gives the time-zero position from where the diffusive solvation dynamics startsat room temperature. Hence, the dynamics is reported in termsof “absolute” Stokes shifts as S(t) = vglass − v(t) (see Figure 4).Figure 4 compares the Stokes shift dynamics of groove-

bound DAPI and base-stacked coumarin in the presence ofNa+, Rb+, and TBA+. The plots immediately reveal that theDNA dynamics measured with DAPI are identical (within errorlimit) in the presence of all three counterions. Figure 4Acompares only the slow part of the dynamics from 40 ps to 10ns (base-stacked coumarin data in Rb+ and TBA+ did notextend below 40 ps).16 This plot shows substantial iondependence in the nanosecond dynamics when measuredwith coumarin,16 but not with DAPI. The absolute Stokes shiftsof coumarin at 40 ps can vary within a large range from 1150cm−1 to 1425 cm−1 among the ions, whereas for DAPI the shiftsare similar (∼1530 cm−1). Coumarin TRFSS in the presence ofRb+ and TBA+ did not extend below 40 ps;16 hence, the directcomparison of coumarin and DAPI data in faster time-scales isnot possible. Nonetheless, the large difference in the absoluteStokes shifts of coumarin at 40 ps suggests that there aresubstantial differences in the subpicosecond and femtoseconddynamics among the three counterions.To obtain a quantitative estimation of the slow ion-

dependent dynamics, we analyzed the absolute Stokes shiftdata of DAPI and coumarin in the long time-range from 40 psto 10 ns (see Supporting Information). The sum of two

exponentials is used to fit the DAPI data. All three data arefitted with similar (exponential) time constants (τ1 = 260 ps, τ2= 5 ns). However, the coumarin data in Na+ could be fittedwith a single power-law (of exponent 0.15).16,18,19 TBA+ andRb+ data could only be fitted with a power-law plus anexponential (Figure 4A).16 Fits to TBA+ data shows an initialpower-law dynamics, similar to that in Na+, and switching ofthis dynamics to exponential relaxation (τ = 3.2 ns) beyond ∼1ns.16 Dynamics in Rb+, however, is seen to follow a differentpower-law (of exponent 0.23) initially, and then switch toslower exponential relaxation (τ = 7.3 ns) compared to TBA+

(see Supporting Information). Berg and co-workers extendedthe TRFSS measurement of base-stacked coumarin in DNA inthe presence of Na+ over six decades of time from 40 fs to 40ns.18,19 They found that the dynamics follow single power-law(with exponent 0.15) over the entire six decades from 40 fs to40 ns.18,19 However, within the time-range of 40 ps to 10 ns,TBA+ and Rb+ data initially follow power-law, but beyond acertain time the dynamics switch to exponential type. Theseresults suggest that there is substantial ion dependence on thedynamics when they are measured by a probe that isincorporated as part of DNA and does not perturb the iondistributions around DNA.16

Previous X-ray,38 NMR,39−41 and MD simulation4,8 studiesfound that ions can bind differently to different base-sequences,

Figure 3. TRES constructed from UPC (circles) and TCSPC (stars)decays in the presence of Rb+. Matching of TRES from UPC andTCSPC at a common time point (100 ps) is shown. Dashed or solidlines through points denote log-normal fits. Red solid curve is time-zero glass spectrum measured in a dry ice/acetone mixture at −78 °C.

Figure 4. (A) Comparison of absolute Stokes shift in DNA in thetime-range of 40 ps to 10 ns probed by groove-bound DAPI (circles),and base-stacked coumarin (stars)16 in the presence of Na+ (blue),Rb+ (red), and TBA+ (green). Lines through points show the fits todata (see Supporting Information and text for the fitted results). (B)Comparison of absolute Stokes shifts from 100 fs to 10 ns of DAPI inDNA in the presence of Na+, Rb+, and TBA+ with that of coumarin inthe presence of only Na+. (C) Comparison of the solvation correlationfunction C(t) of DAPI and coumarin, constructed from the Stokesshifts in panel B.

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and binding of ions to DNA can be ion-specific. Ion-inducedgroove-width and conformational change in DNA is alsoreported.8,9 MD simulations in native DNA with Na+ as thecounterion found a very slow ion convergence around DNA.5,6

Beveridge5 as well as Furse and Corcelli6 independently foundthat the granularity of Na+ distribution around DNA can reachconvergence only in hundreds of nanoseconds, althoughrelating this granularity to the observed ion dependence inTRFSS experiments is not straightforward. The dynamiccontributions of different components (in this case water, ion,and DNA) can be obtained through decomposition of MDtrajectories.30−32,35 In fact, decomposition of total electric-field(or energy) fluctuations and correlations into individualcomponents show large Na+−water anticorrelation, whichsuggest that solvent water shields the ion-charges throughdielectric screening.30,32,35 Thus, a probe embedded insideDNA can only sense the screened average field from the ions.Bagchi and Hynes found large anticorrelation between Na+ andwater in simulation.30 Correlating the individual components,they predicted that slow dynamics in native DNA is mainlycontrolled by the ion and water motions, and not DNAmotion.30 Sen et al. analyzed MD trajectories of native DNAgenerated by Beveridge’s group and found excellent agreementbetween simulation and TRFSS results of coumarin in thepresence of Na+.32 Applying polarization model, theydecomposed the total electric-field and found that water isresponsible for the power-law dynamics in DNA.32 Moreimportantly, they also found large Na+−water anticorrelationover the entire 46 ns trajectory and a measurable contributionof intrinsic Na+ motion of ∼6% into the total dynamics.32

These results imply that measurable ion effects are expected ifthe dynamics is probed by a molecule that does not perturb theion configuration in and around DNA. Recently, Furse andCorcelli simulated DNA with base-stacked coumarin (same asused in experiment) in the presence of Na+ and compared theion distributions of coumarin-DNA with native DNA.6 Theyfound ion distributions in the first and second solvation shell aswell as in the major groove and backbone region of coumarin-DNA are very similar to those in native DNA, though minimaleffects are seen for minor groove-bound ions.6 Thus, theobserved ion effects in TRFSS experiments on base-stackedcoumarin are not unexpected. However, it is still not clear howthe motion of different counterions couple with the water andDNA motions to control the total dynamics that are seen inexperiments.16 TRFSS results of coumarin in the presence ofnine different counterions find that the slow ion-dynamics canbe correlated to free diffusion (i.e., hydrodynamic size) of theions.16 Thus, one possibility could be that, because Na+ has lowbinding affinity to DNA grooves,39 it can travel in-and-out ofthe grooves whose motion can couple with the water motion tocontrol the power-law dynamics in DNA.32 However, becausethe size of TBA+ is much larger than Na+, the crowding ofTBA+ around DNA can influence the slow dynamics, which isdirectly related to the free diffusion of the ion near DNA.16 Onthe other hand, Rb+ showed anomalous dynamics that neitherfollow the same power-law, as in Na+, nor show any correlationwith hydrodynamic size. This anomalous behavior may arisedue to the specific binding and long-term localization of Rb+

inside DNA grooves, because preferential binding of Rb+ insideDNA grooves is actually found in NMR and X-ray studies.8,38,40

Nonetheless, further MD simulations in the presence ofdifferent counterions are needed to rationalize the experimentalresults, although proper parametrization of ionic force fields is

still an issue.42 Such simulations may have the predictive abilityto show that intrinsic ion dynamics is slowed in proportion tothe ions’ diffusion constants (i.e., hydrodynamic size), whichthen reassemble with water and DNA components to make thefull signal that is similar to what is found in TRFSSexperiments.16

Contrary to coumarin data, the same ions are found not toaffect the dynamics when measured by groove-bound DAPI. Ina recent report, Pal et al. compared the dynamics of DAPI withcoumarin in the presence of only Na+ over the time range of100 fs to 10 ns.15 Results show that both DAPI and coumarinfollow the same power-law from 100 fs to ∼100 ps, but DAPIdynamics deviate from the power-law beyond ∼100 ps andconverge rapidly to equilibrium showing exponential typerelaxation.15 In the present study, we compare the absoluteStokes shifts (Figure 4B) and solvation correlation functions(C(t) = (S(∞) − S(t))/S(∞)) (Figure 4C) of DAPI in thepresence of Na+, Rb+, and TBA+ with that of coumarin in thepresence of only Na+ (coumarin data in Rb+ and TBA+ did notextend below 40 ps). As can be seen, the DAPI dynamics in allthree counterions merge within the entire time range, showingsimilar power-law dynamics before ∼100 ps and deviation fromthis power-law beyond ∼100 ps.Furse and Corcelli simulated DNA with groove-bound

Hoechst in the presence of Na+.31,34,35 They decomposed theoverall energy correlation function following the linear responseapproach and found that Na+ motion contributes merely (<1%)to the total dynamics (compared to 6% in case of nativeDNA).31 They also found that the overall slow dynamics ismainly controlled by the DNA motion, and not the ion orwater motions.31 The present DAPI data support thissimulation result, which suggests that ions do not affect theDNA dynamics if it is measured by a groove-bound probe.Thus, if Na+ can not access the probe-site inside the groove,then it is expected that other bigger ions like Rb+ and TBA+

may also not access the same, which leads to theindistinguishable ion dynamics in the case of groove-boundprobes. These results can be reconciled by following the relativebinding constants of groove-bound probes and ions to nativeDNA. We measured the binding constants of DAPI to DNA inthe presence of three ions independently (SupportingInformation). The binding constants (Kb) of DAPI are foundto vary within ∼106 − 107 M−1 among the ions. However, usingcapillary electrophoresis, Stellwagen and co-workers found thatmost of the alkali and alkylammonium ions bind to native DNAwith a binding constant of ∼103 M−1.43,44 The higher bindingaffinity of DAPI to DNA, compared to the ions, does confirmthat ions are displaced by a probe molecule upon its binding toDNA groove. Hence, it is conclusive that even if the motion ofions can strongly correlate with motions of water and DNA, theions mostly stay far from the groove-bound probe-site so thatthey can not influence the overall dynamics in DNA.The fact that mobile ions are displaced by minor groove

binders far from the probe site so that they can not influencethe total dynamics is an important finding, because it directlyrelates to the drug and protein binding to DNA. Ions and watercan interact strongly with native DNA, giving rise to substantialion effects in the total dynamics. However, the questionremains how the ions interact with their surrounding entitieswhen these ions are directly or indirectly involved in controllingthe interaction of DNA with proteins and small molecules. Thesame questions can be imposed to proteins as well. Our resultssuggest that, despite the long-range electrostatic interaction

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among ions, water, and DNA, the motional effects of thedisplaced ions remain negligible at the probe-site insidegrooves. Hence, it is implicative that there is a strongrelationship between the ion-distribution around the inducedDNA-structure and the ion-dynamics near that DNA. Thepresent data also confirm that the measured dynamics can getmodulated at different probe positions inside DNA based onthe extent of access the probe can have to sense the ion motion,together with water and DNA motions. Many questions remainregarding counterion dynamics in DNA, and whether thedynamics observed in small oligonucleotides can be translatedto the real genomic DNA or DNA inside cells. In fact, Banerjeeand Pal compared the slow dynamics of DAPI in oligonucleo-tide and genomic DNA, and found very similar results.24

Nonetheless, further studies are needed where combination ofTRFSS experiments and MD simulations can serve as effectivetools to answer these questions.

■ ASSOCIATED CONTENT*S Supporting InformationMaterials, methods and supporting data. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail address: sens@mail.jnu.ac.in.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThanks to the Department of Science & Technology (DST),Govt. of India, for financial support (SR/FTP/PS-16/2007 andDST-FIST Phase-II). We thank Prof. Mark Berg (USC) formany helpful discussions, and Dr. P. Mukhopadhyay (JNU) forthe use of fluorimeter. UPC and TCSPC data were collected atAIRF, JNU. S.D.V. thanks UGC, and N.P. and M.K.S. thankCSIR for fellowships.

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The Journal of Physical Chemistry Letters Letter

dx.doi.org/10.1021/jz300934x | J. Phys. Chem. Lett. 2012, 3, 2621−26262626