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Fluorescence resonance energy transfer FRET/ and competing processes in donor acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data
Anja Dietrich, Volker Buschmann, Christian Muller, Markus Sauer U
Physikalisch-Chemisches Institut, Uni ersitat Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany

Abstract We studied the uorescence resonance energy transfer FRET. efciency of different donor acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine TMR.; rhodamine 6G R6G.; and a carbocyanine dye Cy3. were covalently attached to the 5 -end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye Cy5., and a rhodamine derivative JA133. were attached at modied thymidine bases in the complementary DNA strand with donor acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donorracceptor DrA. distances and orientations. FRET efciencies have been calculated from donor and acceptor uorescence intensity as well as from time-resolved uorescence measurements of the donor uorescence decay. Dependent on the DrA pair and distance, additional strong uorescence quenching of the donor is observed, which simulates lower FRET efciencies at short distances and higher efciencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efciency, even at short DrA distances. In addition, the measured acceptor uorescence intensities and lifetimes also partly show uorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the DrA distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short DrA distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger DrA distances, we suggest that the -stack of the DNA double helix

Corresponding author. Tel.: q49-6221-548460; fax: q49-6221-544255. E-mail address: sauer@urz.uni-heidelberg.de M. Sauer..

1389-0352r02r$ - see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 9 - 0 3 5 2 0 1 . 0 0 0 3 9 - 3

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mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve uorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Fluorescence resonance energy transfer FRET.; Single-molecule spectroscopy; DNA mediated electron transfer

1. Introduction Electronic energy transfer between ground and excited states of chromophores play a key role in chemistry, biology and physics Mataga and Kubota, 1970; Turro, 1978; Agranovich and Galanin, 1982.. Generally these photophysical processes involve non-radiative transfer of electronic excitation from an excited donor, DU to a ground state acceptor molecule A, and occur on time scales from femtoseconds to milliseconds at distances ranging from a few to approximately 100 A. For donor acceptor distances within the weak coupling limit, i.e. donor acceptor distances ) 2 nm, Forster 1948, 1968. derived an expres sion for the rate constant k ET for dipole dipoleinduced energy transfer wEq. 1.x. k ET s 9000ln10 2 D 128 5 n4 NA D R 6

The efciency of FRET, E is then dened to be equal to: Es 1 1 q RrR 0 .

6

3.

H0

FD .

A 4

.d

1.

Eq. 1. expresses the rate constant for energy transfer in measurable spectroscopic quantities such as: the refractive index of the medium, n; the orientation factor 2 which is generally assumed to be 2r3 for random orientations Dale et al., 1979.; the uorescence quantum yield of the donor, D ; its uorescence lifetime, D ; Avogadros number, NA ; the normalized uorescence spectrum of the donor, FD .; the absorption spectrum of the acceptor, expressed by its extinction coefcient, A .; and the average transition frequency in cmy1 . Eq. 1. can be written in terms of the Forster critical transfer radius R 0 , the distance at which the transfer efciency equals 50% wEq. 2.x. k ET s 1
D

R0 R

2.

Forster type resonant energy transfer occurs for allowed singlet singlet transitions if the emission of DU and the absorption of A overlap signicantly. For such transitions the critical transfer radii range from 10 to 100 A Berlman, 1973.. In combination with the strong distancedependence, uorescence resonance energy transfer FRET. is ideally suited to obtain information about structure and structural changes of biologically important molecules Stryer and Haugland, 1967; Veatch and Stryer, 1977; Stryer, 1978; Stuhmeier et al., 1997; Tuschl et al., 1994; Parkhurst et al., 1996; Szollosi et al., 1998.. Dur ing the last few years, improvements in sensitivity and spatial resolution of conventional uorescence microscopy have led to an enforced practical application of FRET for review see Weiss, 1999, 2000; Selvin, 2000.. In combination with genetically encoded dyes, such as green uorescent protein GFP. and its relatives Tsien, 1998., FRET established the principal ability to monitor interactions and distances between molecules even in living cells. However, one should be aware of the fact that the distance range that can be efciently probed by FRET is limited. Due to the 1rR 6 distance dependence, distances in the range 0.5 1.5 R 0 , i.e. FRET efciencies, E in the range 0.98 0.10, are suitable for FRET measurements. At higher distances, the FRET efciency drops to zero, at shorter distances, the FRET efciency is close to unity, and distance changes result in very small changes in FRET efciency. Furthermore, the

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FRET methodology can be applied only for distances within the weak coupling limit. In a more general description, electronic energy transfer involves non-radiative transfer of electronic excitation energy from an excited donor DU to an acceptor molecule, A, independent of the distance. Only when long range coulombic interactions contribute weak coupling between donor and acceptor., the energy transfer process can be formulated in terms of dipole dipole interactions via Eq. 1. Speiser, 1996.. For intermediate or strong coupling between the donor and acceptor, short range exchange interaction, as formulated by Dexter 1953. can dominate the electronic energy transfer process, even when the relevant electronic transitions are forbidden. In addition, the uncertainty in the orientation factor 2 renders the application of FRET for the determination of absolute distances more difcult. Therefore, FRET is rather suited for the detection of dynamic distance changes. On the other hand, conformational changes such as folding or unfolding of a protein are difcult to reveal from ensemble measurements due to the lack of synchronization. Furthermore, subpopulations with slightly changed DrA distances or orientations are averaged out in ensemble experiments. Therefore, the spectroscopic observation of individual DrA pairs seems to be the method of choice to overcome the described problems. Ha et al. 1996a. rst demonstrated single pair uorescence resonance energy transfer spFRET. on double-labeled DNA strands adsorbed on a dry surface. Fluctuations in FRET efciencies have also been used to study conformational dynamics of: single immobilized SNase protein molecules during catalysis Ha et al., 1999a.; ligand-induced conformational changes in single RNA molecules Ha et al., 1999b.; folding dynamics of individual GCN4 peptides Jia et al., 1999.; and the effect of salt on the dissociation of the coiled coil dipeptide -tropomyosin Ishii et al., 1999.. However, care must be taken to ensure minimal perturbation from the immobilization of DNA, RNA, or proteins on modied glass surfaces Osborne et al., 2001.. Therefore, spFRET measurements on freely diffusing molecules seem to be a valuable approach. On the one hand,

uorescence bursts from single molecules traversing the laser beam in solution are small, i.e. only a limited number of photon counts in the order of tens to hundreds can be detected from an individual molecule. On the other hand, detailed analysis of the photon bursts can provide invaluable information about the distributions of molecular properties, undisturbed by surface effects Deniz et al., 1999, 2000; Dahan et al., 1999.. Due to the availability of hundreds to thousands of events in only a few minutes, single molecule studies in solution can uncover easily subpopulations of analyte molecules in heterogeneous ensembles Sauer et al., 1998; Eggeling et al., 1998; Deniz et al., 1999.. Independent of the applied experimental conditions, great care must be taken in attributing the change of spFRET efciency to a distance change between donor and acceptor. In single molecule experiments there are several factors which inuence the measured FRET efciencies including a. digital photobleaching Ha et al., 1996a. and b. so called blinking due to: rotational jumps Ha et al., 1996b; Ruiter et al., 1997; Bartko and Dickson, 1999; Weston and Goldner, 2001.; intersystem crossing into long lived triplet states Veerman et al., 1999; Weston et al., 1999; English et al., 2000a,b.; spectral diffusion Lu and Xie, 1997; Yip et al., 1998; Weston and Buratto, 1998.; cis trans isomerization as in case of the indocarbocyanine dye Cy5 Widengren and Schwille, 2000.; and uctuations in the excited state kinetics Tinnefeld et al., 2000, 2001.. In addition, intermolecular quenching of the donor due to dynamic interactions with the biomolecule, e.g. DNA, has to considered. As Seidel et al. 1996. already reported, coumarin dyes are more or less efciently quenched by all four DNA nucleotides. Simultaneously, we found that most rhodamine and oxazine dyes are efciently quenched by the DNA base guanosine Sauer et al., 1995; Nord et al., 1997; Lieberwirth et al., 1998.. Covalent linking of these dyes to oligonucleotides containing guanosine residues results in a diminished uorescence quantum yield and decay time dependent on the distance between the guanosine residue and the uorescent dye. In addition, we demonstrated that the uorescence

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kinetics of the dye are inuenced mainly when the guanosine residue is located in close vicinity to the dye Sauer et al., 1998.. This quenching effect has been supported by several other groups Vamosi et al., 1996; Widengren et al., 1997. and ` has been used to study conformational uctuations in DNA oligonucleotides at the single molecule level by time resolved uorescence spectroscopy Edman et al., 1996; Jia et al., 1997; Eggeling et al., 1998; Sauer et al., 1998.. As in the case of stilbene labeled hairpin oligonucleotides with dC dG stems, a photo-induced electron transfer reaction from the guanine ground state to the excited rhodamine or oxazine singlet state provides a plausible mechanism for uorescence quenching Lewis et al., 1997.. The difference in behavior of neighboring dG compared with dA, dT, or dC bases can be attributed to the lower oxidation potential of dG vs. dA or the pyrimidine bases dT and dC Sauer et al., 1995; Seidel et al., 1996; Steenken and Jovanovic, 1997.. Besides the monitoring of the dynamical behavior of DNA oligonucleotides, the quenching inuence of guanosine residues on the attached reporter dye can also be used as a powerful tool to probe the local DNA sequence in double- or single-stranded DNA Knemeyer et al., 2000.. Furthermore, it should be noted that each of these processes inuences the measured FRET efciency to a different degree, and even more important, the dye structure of the donor and acceptor itself might control the contributions from non-distance change processes. Therefore, it is essential to choose suitable control samples and examine both single molecule and bulk measurements to fully understand the inuence of non-distance change processes. In addition, most problems might be circumvented by a careful spectroscopic study of the only donor and only acceptor labeled molecule and by changing the donor and acceptor dye and the coupling position. Motivated by these considerations we investigated and compared the FRET efciency of DNA molecules labeled with different donor and acceptor molecules at different positions in ensemble measurements as well as at the single molecule

level. As donor molecules we used two rhodamine derivatives: rhodamine 6G R6G.; and tetramethylrhodamine TMR., and a carbocyanine derivative, Cy3 coupled to the 5 -end of a 40-mer oligonucleotide. With a persistence length of ; 50 nm Bustamante et al., 1994., 40mer doublestranded DNA should represent an ideal FRET system with relatively xed DrA distance, inuenced only by the conformational exibility of the used linkers. The acceptor dyes, a carbocyanine derivative Cy5. and a rhodamine derivative JA133., were coupled to the complementary strand at different distances of 5, 15, 25 and 35 base pairs Fig. 1.. Since TMR and R6G are efciently quenched by guanosine residues in a close neighborhood, we used a specic oligonucleotide sequence containing no guanosine residues at the 5 -end. The FRET efciencies were calculated via uorescence intensity and time resolved data. Independent of the used method, the observed spectroscopic data indicate the presence of an additional quenching path. Our data evidence that other uorescence quenching processes like electron transfer reaction, either through space in case of the 5 base pair distance. or DNA-mediated via the -stack of the DNA bases Meggers et al., 1998; Kelley and Barton, 1999; Ye and Jiang, 2000; Lewis et al., 2001., have to be taken into account.

2. Results and discussion 2.1. Design of FRET constructs A set of differently labeled FRET constructs with varying DrA DrA. base pair separation was synthesized to investigate and compare the distance dependence and the inuence of the dye structure on the measured spectroscopic characteristics. To keep parameters such as diffusion rate and thermal stability constant, the different DNA constructs had the same sequence and a constant length of 40 base pairs. From intermolecular quenching experiments it is known that the two rhodamine derivatives R6G and TMR are efciently quenched by guanosine residues Sauer

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215

et al., 1995, 1998; Nord et al., 1997.. On the other hand, the rhodamine derivative JA133 and most indocarbocyanine dyes such as Cy5 are not quenched by DNA nucleotides Lieberwirth et al., 1998.. Due to these observations, we decided to use FRET constructs where guanosine residues are so far apart from the donor dyes that any quenching inuence of the DNA base guanine should be minimized. The selected 40mer oligonucleotide consists of six consecutive adenosine thymidine base pairs at the donor side of the construct Fig. 2.. The Forster radii, R 0 of the four investigated DrA pairs of: 63.5 A R6GrCy5.; 64.5 A Cy3rCy5.; and 59.0 A TMRrCy5.; 55.8 A TMRrJA133. were calculated from the spectral overlap of the separate absorption and emission spectra of the donor and acceptor only doublestranded oligonucleotides, and the uorescence quantum yields of the donor only constructs, respectively. Extinction coefcients of 1.1= 10 5 and 2.5= 10 5 l moly1 cmy1 for JA133 Sauer et al.,

1995. and Cy5, respectively, have been used. All dyes were assumed as free rotors. As can be seen from the data in Table 1, the donor dyes R6G and Cy3 exhibit monoexponential uorescence decay times attached at the doubled-stranded DNA. The TMR labeled oligonucleotide shows a second shorter uorescence decay time of 1.25 ns with a relatively small amplitude. Due to the exibility of the used C 6aminolinkers, the donor dyes can adopt different conformations with respect to the oligonucleotide, thereby preventing or promoting rotational mobility of some side chains such as the amino groups in case of TMR. Hence, the different rotational mobility is directly reected in the measured uorescence decay time and quantum yield Drexhage, 1977; Vogel et al., 1988.. However, together with the relatively high uorescence quantum yields, the data imply that the donor dyes are not quenched by DNA nucleotides. In other words, the donor only constructs exhibit relatively homogeneous spectros-

Fig. 1. Molecular structures of the used dyes. The donor dyes 5-carboxyrhodamine 6G R6G., 5-carboxytetramethylrhodamine TMR., and the indocarbocyanine dye Cy3 as well as the acceptor dye Cy5 were obtained as functionalized N-hydroxysuccinimidyl esters. As a second acceptor we used a rhodamine derivative, JA133.

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Fig. 2. a. Schematic diagram of the optical setup. For efcient excitation of the donor dyes TMR, R6G and Cy3 we used a frequency-doubled Nd:YAG laser emitting at 532 nm. The collimated laser beam was directed into an inverted microscope and coupled into the microscope objective with high numerical apertures oil immersion, 100 = , NA 1.4. via a dichroic beam splitter. Within the microscope objective, the beam was focused into the sample to detect freely diffusing FRET constructs. The uorescence light was collected through the same objective and imaged onto a 100- m pinhole to reject out-of-focus light. The transmitted uorescence light is then split by a dichroic mirror and focused onto the active areas of two avalanche photodiodes, APDs SPCM AQR-14.. To further isolate the donor and acceptor signal we used additional band pass lters in front of the APDs 570DF60 and 675RDF50.. The signals of both APDs were coupled to a counting board and a personal computer. Sample solutions 10y1 1 M. were prepared from 10y6 M stock solutions by several dilution steps. For diffusion measurements, the average excitation power at the sample was adjusted to be 325 W. b. Model of the DrA DNA constructs with varying distance: D-N.5 -A; D-N.15 -A; D-N. 25 -A; and D-N. 35 -A. The 40mer complementary oligonucleotides: i. 5 -UATA TAA GCT ATG CAA TGC TAT GGT AAC GTA TCG AAT CGT A-3 ; and ii. 5 -T ACG ATU T CGA TAC GTTU ACC ATA GCA TU TG CAT AGC TTUA TAT-3 were custom synthesized. All donor dyes were coupled to the 5 end of oligonucleotide i. via 5 -aminomodier C 6 . Acceptor dyes were also coupled to C 6 amino-modied thymidine bases at four different positions in the complementary oligonucleotide ii. resulting in DrA distances of 5, 15, 25 and 35 base pairs, respectively. Since 10 base pairs make one turn in B form double helix, the relative positions of both dyes are similar in all FRET constructs. To ensure a dened and comparable environmental inuence independent of the DrA distance, we used similar sequences nearby the acceptor positions T U .. Coupling reactions were carried out in 250 mM carbonate buffer, pH 9.3, at room temperature for 2 h. The labeled oligonucleotides were puried by reversed phase RP18-column. HPLC using a gradient of 0 75% acetonitrile in 0.1 M aqueous triethylammonium acetate. As conrmed by absorption spectroscopy, this method yields 100% labeled DNA. The constructs are referred to as Donor-N. n-Acceptor, where n indicates the base pair separation between the dyes, e.g. TMR-N. 25 -Cy5 for tetramethylrhodamine labeled oligonucleotide i. hybridized to Cy5 labeled oligonucleotide ii. with a DrA distance of 25 base pairs.

A. Dietrich et al. r Re iews in Molecular Biotechnology 82 (2002) 211 231 Table 1 Ensemble spectroscopic characteristics of the different FRET constructs in aqueous buffer containing 1 M NaCl
D ab s em f,rel

217

ED

EA

D av

nm. TMR-N. TMR-N.35 -Cy5 TMR-N.25 -Cy5 TMR-N.15 -Cy5 TMR-N.5-Cy5 R6G-N. R6G-N.35 -Cy5 R6G-N.25 -Cy5 R6G-N.15 -Cy5 R6G-N.5-Cy5 Cy3-N. Cy3-N.35 -Cy5 Cy3-N.25 -Cy5 Cy3-N.15 -Cy5 Cy3-N.5 -Cy5 TMR-N. TMR-N.35 -JA133 TMR-N.25 -JA133 TMR-N.15 -JA133 TMR-N.5-JA133 557 557r650 557r650 557r650 552r652 534 534r650 534r650 534r650 529r652 551 551r650 551r650 551r650 549r651 557 558r620 559r621 558r622 554r625

nm. 581 584r663 584r666 584r667 584r667 557 558r663 557r663 556r665 548r668 565 565r664 565r664 565r664 564r667 581 581r 581r634 581r638 581r639 1.00 0.78 0.63 0.31 0.11 1.00 0.91 0.70 0.34 0.24 1.00 0.87 0.75 0.49 0.29 1.00 0.77 0.69 0.36 0.20

ns.ra1 0.22 0.37 0.69 0.89 0.09 0.22 0.53 0.63 3.55r0.89 3.50r0.86 2.85r0.88 3.01r0.55 3.26r0.82 4.28r1.00 4.14r0.96 3.77r0.96 3.77r0.74 3.82r0.91 0.90r1.00 1.42r0.52 1.33r0.48 1.34r0.25 1.43r0.20 3.55r0.89 3.48r0.89 3.39r0.78 3.01r0.61 3.31r0.78

ns.ra2 1.25r0.11 1.27r0.14 0.81r0.12 0.79r0.45 0.62r0.18

D av

ns. 3.30 3.19 2.61 2.01 2.78 4.28 4.00 3.65 3.07 3.56 0.90 0.86 0.75 0.51 0.44 3.30 3.21 2.96 2.22 2.83

0.03 0.21 0.39 0.16

0.09 0.30 0.66 0.76

0.04 0.12 0.53 0.45

0.57r0.04 0.84r0.04 1.09r0.26 0.98r0.09

0.07 0.15 0.28 0.17

0.13 0.25 0.51 0.71

0.06 0.14 0.29 0.51

0.25r0.48 0.22r0.52 0.23r0.75 0.19r0.80 1.25r0.11 1.06r0.11 1.45r0.22 0.99r0.39 1.11r0.22

0.04 0.17 0.43 0.51

0.23 0.31 0.64 0.80

0.03 0.08 0.25 0.04

0.03 0.10 0.33 0.14

D Absorption and emission maxima of the donor and acceptor: relative uorescence quantum yield, f,rel ; and uorescence lifetime, D of the donor; and FRET efciencies calculated from the donor decrease, E D ; donor decrease and acceptor increase, D E A , and from the donor average uorescence lifetime, E av .. Absolute uorescence quantum yields of donor only labeled D oligonucleotides, f s 0.40 for TMR-N., 0.70 for R6G-N. and 0.20 for Cy3-N., were calculated using rhodamine 6G in ethanol as standard with a quantum yield of 0.90 Arden-Jacob, 1992.. The instrument response function required for deconvolution of the uorescence decays was obtained from a scattering solution. The quality of the decay ts was assessed by means of the reduced chi-squared statistical parameter 2 .. In most cases a multiexponential t was necessary to describe the measured decays satisfactorily I t . s ai expyt y1 ... Here ai are pre-exponential factors that describe the ratio of the excited species ai s 1., i and i denote their lifetimes, respectively. Average uorescence lifetimes av were calculated via av s ai irai i . All uorescence decays were tted with 2 values - 1.2. Fluorescence anisotropies at the emission maxima, r for the dyes were calculated from the polarization of the emission components I VV , I VH , I HV and I HH where the subscripts denote the orientation of the excitation and I VV y GI VH . emission polarizers. as r s where Gs I VH rI HH . R 0 values were calculated from the overlap of the donor I VV q 2GI VH . conjugate emission spectrum and the acceptor conjugate absorption spectrum in 1 M NaCl assuming a refractive index, n of 1343.

copic characteristics, which is at least important, if not the prerequisite, for successful FRET experiments. 2.2. Distance dependence of FRET The absorption spectra of the hybridized double labeled FRET oligonucleotides show the expected three peaks of donor absorption, acceptor absorption and the absorption of the oligonu-

cleotide at approximately 260 nm. Without any direct interaction between the dyes in the ground state, the absorption spectra of the FRET constructs should equal the sum of the absorption spectra of donor and acceptor only labeled oligonucleotides. As can be seen from Table 1, this behavior can be observed for FRET constructs with larger DrA distances of 15, 25 and 35 base pairs. In strong contrast, the absorption maxima of the donor and acceptor in the ve base

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pair constructs are always slightly shifted to the blue and red, respectively, independent of the donor or acceptor dye used Table 1.. The emission spectra of the FRET constructs with DrA distances of 15, 25 and 35 base pairs show the expected decrease in donor uorescence and increase in acceptor uorescence Fig. 3a d.. In addition, the emission curves intersect in one point, which demonstrates a direct correlation between the decrease of donor uorescence and

increase of acceptor uorescence. However, again the constructs with ve base pair separation exhibit striking behavior. Although the donor intensity decreases, the energy is not completely transferred to the acceptor, i.e. with exception of the Cy3rCy5 pair, the acceptor uorescence intensities of all other pairs are lower than for the constructs with 15 base pair separation. In addition, the 5-bp constructs show slightly shifted emission maxima implying weak dye dye interac-

Fig. 3. Fluorescence emission spectra of the different FRET constructs in aqueous buffer containing 1 M NaCl 25 C, 10y6 M.. a. TMRrCy5 excited at 520 nm; b. R6GrCy5 excited at 480 nm; c. Cy3rCy5 excited at 520 nm; and d. TMRrJA133 excited at 520 nm. For comparison the emission spectra of the donor and acceptor only labeled constructs, donor-N. and N.-acceptor, respectively, are given. Complementary combinations of donor and acceptor labeled oligonucleotides i. and ii. were mixed 1:1 at room temperature in 100 mM Tris borate, pH 8.3, containing 1 M NaCl. The completeness of hybridization was veried by addition of small amounts of the acceptor labeled oligonucleotide while monitoring the donor uorescence intensity. For complete 1:1 hybridization, the donor uorescence intensity should be independent on any further addition of acceptor labeled oligonucleotide. To exclude polarization effects, the uorescence was recorded under the magic angle 54.7 ..

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219

Fig. 4. Ensemble uorescence decays of the TMRrCy5 labeled DNA constructs 10y6 M. in aqueous buffer containing 1 M NaCl. In addition, the uorescence decay of the only donor labeled double-stranded DNA is shown. Excitation at 495 nm, emission at 570 nm, 37 psrchannel. Ensemble uorescence lifetimes were determined from 10y6 M solutions of the conjugates at the emission maxima using either a pulsed LED center wavelength: 495 nm. or a diode laser emitting at 635 nm as excitation source and time correlated single photon counting TCSPC..

tions even in the excited states. Only the Cy3N.5-Cy5 construct exhibits at least a decrease and increase in donor and acceptor uorescence, respectively, compared to the uorescence properties of the 15-bp construct. With exception of the 5-bp constructs, the uorescence decay functions Fig. 4. show the same trend, i.e. with decreasing DrA distance, the lifetime decreases. All donor uorescence decays measured from FRET constructs could only be described satisfactorily with at least a biexponential model. Even at the shortest DrA distance of 5 bp, the donor uorescence decay still exhibits a long uorescence component with relatively high amplitude. Interestingly, the 5-bp Cy3rCy5 construct exhibits the expected shorter decay time. Table 1 summarizes the relative uorescence quantum yields and uorescence decay times of the donor dyes obtained from ensemble measurements. The data strongly support the idea that another effect has to be taken into account at short DrA distances. In addition, the biexponential uorescence decays with the longer compo-

nent, always comparable to the lifetime of the donor only labeled construct, imply a strong conformational heterogeneity, e.g. at least two extreme conformations with different orientations of the dipole moments. We calculated the FRET efciency, E D from the decrease in donor uorescence intensity via Es 1 y I DA rI D , where I DA , and I D denote the measured integrated uorescence intensity of the donor in the presence and absence of the acceptor in the wavelength range 540 600 nm, respectively Table 1.. FRET efciencies calculated from excitation spectra of the acceptors show similar results data not shown.. In addition, we calculated the FRET efciency, E A from the quantum yield and cross-talk corrected acceptor intensity, IA between 640 and 700 nm, and the donor intensity, I D between 540 and 600 nm via E A s IA r I D q IA .. This method is generally applied to calculate the FRET efciency from single molecule data. Furthermore, Table 1 shows the FRET efD ciencies, E a . obtained from the average uorescence lifetimes measured for the donor dyes in the presence and absence of the acceptor. Independent of the method used, the FRET efciencies measured at short DrA distances 5 bp. appear to be much to low. For long distances 35 bp., unexpectedly high FRET efciencies were observed for all four constructs if the efciency is calculated solely from the donor intensity. Comparison of the differently calculated FRET efciencies implies that an additional efcient quenching pathway has to be taken into account to explain the observed strong deviations of the experimental FRET data from theory. The overall lower FRET efciencies calculated from the average uorescence lifetimes indicates the presence of a short decay component which we can obviously not resolve with our experimental time resolved equipment. Hence, even the time resolved data although in an indirect way. imply that a fraction of the donor dyes for example a conformational subpopulation . is efciently quenched by another mechanism. 2.3. Anisotropy and distance distributions Fluorescence anisotropy measurements show

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that none of the investigated donor and acceptor dyes can be regarded as a free rotor. For the 15, 25 and 35 bp constructs, and donor or acceptor only labeled oligonucleotides anisotropy, values vary between: 0.16 and 0.20 for R6G and TMR; 0.10 and 0.15 for Cy3; 0.24 and 0.28 for Cy5; and 0.26 and 0.29 for JA133 in 1 M NaCl. Closer examination of the donor anisotropies shows a systematic increase with decreasing DrA distance due to faster FRET. Interestingly, the anisotropy of all dyes increases considerably in the 5-bp distance constructs, e.g. for Cy5 we measured an anisotropy of 0.38 in TMR-N.5-Cy5; 0.31 in R6G-N.5-Cy5; 0.35 in Cy3-N.5-Cy5; and for JA133 we obtained r s 0.38 in TMR-N.5-JA133. These data clearly indicate nearly xed dipole moments of the donor and acceptor dye. As has been recently shown by several groups Sauer et al., 1995; Vamosi et al., 1996; Seidel et ` al., 1996; Nord et al., 1997; Widengren et al., 1997; Lieberwirth et al., 1998., rhodamine and oxazine dyes attached covalently to oligonucleotides have the tendency to interact with DNA bases. The degree of this aggregation is strongly controlled by the water solubility of the dye structure and the exibility of the used linker arm. Therefore, relatively high anisotropy values might appear. However, single molecule studies revealed the existence of several different conformational states with respect to the oligonucleotide which interchange in the microsecond to millisecond range Edman et al., 1996; Wennmalm et al., 1997; Eggeling et al., 1998; Sauer et al., 1998.. This conformational motion is slow compared to the emission lifetime of the dye. Hence, the measured anisotropy values might indicate xed rotors, which in fact change their transition dipole on a slower time scale. In addition, our uorescence data demonstrate strong heterogeneity of FRET efciency, which might be controlled by different orientations of the transition dipoles rather than by distance changes. This is supported by the observation that most uorescence decays contain more than 50% of a long uorescence component Table 1. which is similar to the uorescence lifetime measured for the donor only labeled oligonucleotides, independent of the DrA distance.

2.4. spFRET measurements To compare ensemble data with single molecule measurements, 10y1 1 M solutions of the constructs were excited at 532 nm and the donor and acceptor emission were detected separately Fig. 5.. As previously pointed out Dahan et al., 1999; Deniz et al., 1999., the ability of single molecule detection to measure distributions implies that inhomogeneous populations of molecules can be studied. However, the time resolution of the measurements controls whether the inhomogeneity appears as static or dynamic. For example, as already mentioned above, the conformational dynamics of dyes attached to DNA occur on a microsecond to millisecond time scale. Although the dyes may adopt all kinds of possible conformations with different orientations of the transition dipole moment, the anisotropy originating from the rotational mobility of the molecule during its excited state lifetime of a few nanoseconds. is high, which indicates strongly hindered rotational mobility of the chromophores attached to DNA. The typical transition time of a ds 40mer oligonucleotide in the detection volume ; 1 femtoliter. is approximately 1 ms. In other words, this is within the time range of these conformational changes. Hence, by choosing an integration time of 1 ms per bin, subpopulations exhibiting differ-

Fig. 5. Typical uorescence trajectories monitored on the donor gray. and acceptor black. channel of a 10y1 1 M solution of TMR-N. 25 -Cy5 in aqueous buffer 1 M NaCl..

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ent FRET efciencies should be revealed. Fig. 5 shows an example of a 1-ms integration time uorescence burst trajectory for a 10y1 1 M solution of TMR-N. 25 -Cy5 showing clearly correlated photon bursts. Because direct excitation of the acceptor and leakage of the donor emission into the acceptor channel is very small, uorescence bursts on the acceptor channel were assigned to acceptor molecules excited via FRET. With an average excitation energy of 325 W, typical background count rates of 0.62 kHz for the green and 0.47 kHz for the red channel were measured. To discriminate dye uorescence efciently from background noise, only time bins containing in total green and red channel . more than 30 counts 30 kHz. were used to calculate the FRET efciency. This criteria of thresholding selects only those uorescent bursts where donor andror acceptor emission are strong Ying et al., 2000..

The single pair FRET spFRET. efciencies, E sp were calculated from the background corcorr rected uorescence intensities of the donor I D , corr w and acceptor IA Eq. 4.x. E sp s
corr IA y C corr corr IA y C q I D .

4.

The cross-talk, C between the donor and acceptor channel was calculated from ensemble emission spectra and the transmission of the lter set 8.7% for TMR; 8.1% for Cy3; and 3.2% for R6G.. Fig. 6 shows the spFRET efciency histograms that were generated from the single molecule uorescence intensity data of: TMR-N. x-Cy5; R6G-N. x-Cy5; and Cy3-N. x-Cy5 constructs, and of only donor labeled oligonucleotides measured in aqueous buffer containing 1 M NaCl. In

Fig. 6. FRET histograms extracted from single molecule data of 10y1 1 M solutions of the differently labeled DrA constructs and corresponding Gaussian ts.

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absence of an acceptor, the donor only labeled oligonucleotides show only one peak with zero FRET efciency. For the 5-bp separation constructs, two peaks are evident, one centered at approximately zero efciency and a second at very high ) 0.95. efciency. With increasing separation length, the second peak clearly shifts to lower FRET efciency, as expected for Forster energy transfer. Due to the overlap with the zero peak, it was impossible to separate out the FRET peaks for the 35-bp constructs which should exhibit theoretically very low FRET efciencies 0.10.. It is generally assumed that the large peak in zero energy transfer efciency arises from non-hybridized, single-stranded donor labeled oligonucleotides, and premature photobleaching of Cy5. As has been recently shown Grunwell et al., 2001., the use of oxygen scavengers can drastically decrease the photobleaching and oxidative damage of Cy5, thereby decreasing the observed amplitude of the zero peak. In addition, it should be pointed out that single molecule analysis is always subjected to a kind of selection, in that single molecule experiments are only performed on the subset of all molecules that are sufciently bright to be detected and investigated under the applied burst recognition procedure. For example, dim or dark acceptor molecules may also intrinsically exist, which contribute to the large zero peak. Especially in the case of Cy5 as acceptor, the formation of a nonuorescent cis state has to be taken into account. It has been shown recently Widengren and Schwille, 2000., that irrespective of the excitation rate, 50% of the Cy5 molecules are in an essential non-uorescent cis state. The rate of interchange between the trans and cis state was found to be proportional to the excitation rate. Very recently Widengren et al., 2001., this behavior has been used to extract the FRET efciency by analyzing the acceptor uorescence Cy5. using uorescence correlation spectroscopy. Furthermore, the excitation spectra of the cis and trans forms overlap signicantly, i.e. the donor might transfer its energy to the acceptor in the cis state. Hence, the donor intensity is reduced but the reduction is not reected as an increase of acceptor uorescence, as expected for FRET. Since,

the isomerization rates are fast compared to the observation time in our experiments 1 msrbin. the cis trans uctuations are smeared out. Hence, dependent on how FRET efciencies are calculated, lower values might result. However, experiments with the rigid rhodamine derivative JA133 as acceptor showed comparable FRET efciencies, and large zero peaks data not shown.. This indicates that the cis trans isomerization of Cy5 molecules is not responsible for the observed deviations from theoretically predicted efciencies. The appearance of two real maxima in most of the FRET distribution histograms instead of broad distributions implies that the conformational uctuations between sub-states with different orientations or DrA distances are on average slower than the measurement time 1 ms.. If the conformational uctuation rates were faster than the measurement time which equals the diffusion time., the high FRET peak would at least be smeared out or gradually shifted to lower FRET efciency. Table 2 gives the calculated spFRET efciencies, E sp , and distribution widths, w, obtained from Gaussian ts. There are several effects that contribute to the observed peak broadening. Especially the low signal intensity obtained from single molecule measurement raises strong ucTable 2 Single-pair FRET efciencies, E sp , and standard deviations, w, revealed from Gaussian ts of the spFRET distributions Esp TMR-N.40 TMR-N.5-Cy5 TMR-N.15 -Cy5 TMR-N.25 -Cy5 R6G-N.40 R6G-N.5-Cy5 R6G-N.15 -Cy5 R6G-N.25 -Cy5 Cy3-N.40 Cy3-N.5 -Cy5 Cy3-N.15 -Cy5 Cy3-N.25 -Cy5 0.01 0.99 0.58 0.21 0.01 0.97 0.52 0.19 0.02 0.95 0.48 0.24 w 0.13 0.11 0.49 0.32 0.07 0.10 0.54 0.13 0.17 0.11 0.38 0.35

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tuations in the FRET efciency calculated from intensity ratios. Another source of broadening is uctuations in DrA distance and uctuations in 2 , the orientation factor. Although a Gaussian t is not optimal, at least for very low and high FRET efciencies, it does not lead to major discrepancies. Surprisingly, spFRET efciencies differ substantially from the ensemble values. In particular, the 5-bp constructs exhibit a drastically increased spFRET efciency, i.e. as expected for such a short DrA distance. On the other hand, ensemble measurements show overall lower efciencies at short distances which we ascribed to an additional efcient quenching of the donor. These data clearly demonstrate the power of spFRET measurements to reveal subpopulations from heterogeneous ensembles. 2.5. Donorr acceptor distance model To compare the measured FRET efciencies with Forster theory, the bp separation of donor and acceptor was converted into distances using the model introduced for DNA-FRET efciencies by Clegg et al. 1993.. According to this model, the DrA distances R DA A. for two dyes attached at a B form double helix in solution can be estimated from the structural properties of the dyes and the vector sum of two components: one parallel; and one perpendicular to a cylindrical B form DNA model with a typical diameter of 20 A, rise and 36 per base pair: 3.4 A R DA s

However, if only one of the dyes is close to the helix, e.g. the dye adheres to the DNA indicated by high anisotropy values, this is no longer the case, and the modulation disappears. Based on the structural properties of the dyes, the tethers and the linkers, we used two extreme start posi tions: a. fully stretched linkers with Ks 4.0 A, L TM R s 22.0 A, L R6G s 22.0 A, LCy3 s 29.2 A, LCy5 s 29.2 A, LJA133 s 26.8 A and s 306 ; and b. Ks 4.0 A, L TM R s 12.0 A, L R6G s 12.0 A, LCy3 s 12.0 A, LCy5 s 12.0 A, LJA133 s 12.0 A and s 306 . This assumes that all chromophores refold and adhere to the DNA. Furthermore, we used three different R 0 values for all DrA pairs R 0 as calculated from ensemble spectra "10%. to calculate the expected FRET efciencies for the series of constructs. Fig. 7 shows the theoretically expected FRET efciencies for differently labeled DNA constructs assuming different R 0 values and different linker conformations. However, obviously no model can describe the measured ensemble values accurately. If any, then the spFRET efciencies can be described approximately by a model assuming the dyes adhered to the DNA. The most striking difference from theory is the relatively high FRET efciencies measured for the 25- and 35-bp construct. On the other hand, the FRET efciencies retrieved from single molecule measurements with the 5-bp construct match nearly ideally the theoretical predicted values. 2.6. Competing energy transfer mechanisms Table 3 shows the ensemble uorescence properties of the acceptor dyes in the FRET constructs excited at 635 nm direct excitation.. Interestingly, the Cy5 uorescence intensity decreases upon binding of the donor, independent of the donor dye used. While for the 35-, 25- and 15-bp constructs comparable quenching efciencies are observed, quenching increases considerably in the 5-bp constructs. In contrast, the average uorescence lifetimes increase slightly. On the other hand, the uorescence of the acceptor dye JA133 is not inuenced by the donor dye at longer distances. Once again at short distance

' Kq 3.4

N . q L2 q L2 y 2 L D LA D A cos q 36 N . . 5 .

Here, N represents the base pair separation; and K the distance between donor and acceptor along the helical axis for N s 0; L D and LA are the normal distances of the donor and acceptor chromophore to the helical axis, respectively; and is the inter-dye angular separation for N s 0. Using this helix model, i.e. describing the double-stranded DNA as a cylinder, the distance between the donor and acceptor is smaller when both chromophores are on the same side of the helix, and longer when they are on opposite sides.

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Fig. 7.

A. Dietrich et al. r Re iews in Molecular Biotechnology 82 (2002) 211 231 Table 3 Ensemble uorescence properties relative uorescence quantum yield, upon direct excitation at 635 nm
A f,rel 1 A

225

A f,rel

and uorescence lifetime,

A.

of the acceptor dyes

A av

ns.ra1 1.62r0.86 1.65r0.59 1.72r0.67 1.67r0.62 1.83r0.75 1.78r0.49 1.82r0.59 1.88r0.48 1.80r0.78 1.65r0.73 1.69r0.83 1.72r0.73 1.88r0.77 3.99r1.00 3.91r1.00 3.89r1.00 3.89r1.00 3.91r0.87

ns.ra2 0.62r0.14 0.81r0.41 0.74r0.33 0.74r0.38 0.75r0.25 0.91r0.51 0.82r0.41 0.94r0.52 0.62r0.22 0.67r0.27 0.50r0.17 0.76r0.27 0.69r0.23

ns. 1.56 1.43 1.54 1.47 1.70 1.48 1.58 1.55 1.69 1.52 1.62 1.59 1.76 3.99 3.91 3.89 3.89 3.80
D f s 0.40

N.35 -Cy5 TMR-N.35 -Cy5 TMR-N.25 -Cy5 TMR-N.15 -Cy5 TMR-N.5-Cy5 R6G-N.35 -Cy5 R6G-N.25 -Cy5 R6G-N.15 -Cy5 R6G-N.5-Cy5 Cy3 Cy3 Cy3 Cy3 N.35 N.25 N.15 N.5 Cy5 Cy5 Cy5 Cy5

1.00 0.83 0.83 0.86 0.66 0.67 0.77 0.71 0.50 0.84 0.85 0.91 0.68 1.00 1.00 1.00 1.00 0.33

N.35 TMR TMR TMR TMR

JA133 N.35 JA133 N.25 JA133 N.15 JA133 N.5 JA133

1.16r0.13

D f s 0.80

Absolute quantum yields of the acceptor only labeled oligonucleotides were determined to be for N. 35 -JA133.

for N. 35 -Cy5 and

5 bp. the quantum yield is drastically reduced. Here a shorter uorescence lifetime component appears in the time-resolved data Table 3.. However, the small amplitude 13%. of the shorter component of 1.16 ns can not explain the reduction of the uorescence quantum yield of 67%. Comparison of the measured intensity and lifetime data of the acceptor uorescence Table 3. suggest that, at least in the 5-bp construct, an additional very efcient quenching process has to be taken into account. Together with the observed shifts in the absorption and emission maxima the data strongly

imply direct ground and excited state interactions between the donor and acceptor molecules, enabled by the short separation distance and the linker exibilities. The idea of direct interactions, e.g. aggregation of donor and acceptor, is additionally supported by the measured high anisotropy values for the dyes in the 5-bp constructs. It is well known, that ionic dyes tend to aggregate, even in diluted aqueous solutions, to form nonuorescent dimers Drexhage, 1977; Kemnitz et al., 1986; Liang et al., 1997.. Comparison of the molecular structures of the donor and acceptor dyes Fig. 1. indicates that the indocarbocyanine

Fig. 7. Theoretically expected FRET efciencies for the FRET pairs: a. TMRrCy5; b. R6GrCy5; and c. Cy3rCy5 attached to a double stranded DNA. Distances are calculated by the model shown above. Based on the structural properties of the dyes and the linkers two extreme start positions were used: left side. fully stretched linkers; and right side. collapsed linkers. Furthermore, three different R 0 values have been used R 0 as calculated from ensemble spectra "10%.. Open circles represent the FRET efciencies E D obtained from the decrease of donor intensity in ensemble measurements; open triangles represent E A , obtained from ensemble donor and acceptor intensities; and the black squares are the FRET efciencies calculated from single molecule data. The error bars represent the standard deviation.

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dyes Cy3 and Cy5 exhibit the most pronounced water solubility. Due to the additional negatively charged sulfonate groups, intermolecular interactions in water such as formation of dimers are reduced. Hence, the observed additional quenching process should be reduced in the Cy3rCy5 pair. This idea is strongly supported by the data shown in Table 1 and Table 3. The FRET efciencies for the Cy3rCy5 constructs calculated via the two different methods exhibit the smallest differences. In the other FRET pairs TMRrCy5, R6GrCy5, TMRrJA133., intermolecular interactions between the donor and acceptor dye are more likely. The absorption and emission maxima shift slightly, and provide additional quenching pathways such as dimer formation in the case of the 5-bp constructs. This reduces the observed quantum yield for both the donor and acceptor. Due to the fact that dimers are essentially nonuorescent, the measured ensemble uorescence lifetimes are not reduced. Hence, the donor energy cannot be transferred completely to the acceptor via FRET. Since the subpopulation of non-uorescent or only weakly uorescent dimers are not detected in single molecule experiments due to thresholding, higher FRET efciencies are calculated. The formation of non-uorescent intermolecular ground state complexes cannot explain the reduced uorescence quantum yield of the acceptor Cy5 at longer separation distances. Several reports have indicated Gasper and Schuster, 1997; Norman et al., 2000; Schuster, 2000. that in 5 -labeled double-stranded DNA, the chromophores are associated with DNA by end capping, i.e. the chromophore is stacked onto the end of the helix, in a manner similar to that of an additional base pair. Therefore, efcient charge transfer via the base pairs of DNA might be promoted, even at longer distances. The possibility that the -stacked base pairs of DNA might mediate charge transfer was suggested over 30 years ago Eley and Spivey, 1962.. Experimental investigations and theoretical treatments of photo-induced charge transfer in DNA have revealed the occurrence of at least two mechanisms: a single step superexchange mechanism which is strongly distance-dependent; and a

multi-step hole hopping mechanism which is only weakly distance-dependent Jortner et al., 1998; Bixon et al., 1999; Lewis et al., 2001.. In the hopping mechanism, a hole which is generally assumed to be a guanosine radical cation generated photochemically via electron transfer to an excited chromophore can reversibly hop from one guanosine to another until it reaches a trap site more than four subsequent ArT base pairs.. The energetics of photo-induced charge separation and charge recombination processes can be estimated by using Wellers equation wEq. 6.x, Gcs s Eox y Ered y E0,0 q C 6.

where Eox and Ered are the rst one-electron oxidation potential of the donor and the rst one-electron reduction potential of the acceptor in the solvent under consideration. E0,0 is the energy of the zero zero transition to the lowest excited singlet state of the excited partner, and C is the solvent-dependent Coulombic attraction energy Weller, 1982.. The value of C in highly polar solvents like water is sufciently small that it can be neglected. Electrochemical measurements were made for R6G, JA133 and Cy5 in acetonitrile solution at a glassy carbon electrode. Table 4 gives the redox potentials measured vs. the saturated calomel electrode SCE. and the corresponding transition energies of the dyes. The
Table 4 First one-electron oxidation and reduction potentials, Eox and Ered , of R6G, Cy5 and JA133 in acetonitrile and corresponding zero zero transition energies, E0,0 Eox VrSCE. R6G Cy5 JA133 1.39 0.82 1.20 Ered VrSCE. y0.95 y0.88 y0.71 E0,0 eV. 2.27 1.88 1.96

The redox potentials were determined by cyclic voltammetry CV. in dry acetonitrile purchased from Aldrich. Tetra-nbutylammonium hexauorophosphate wTBAxPF6 . was used as the supporting electrolyte. 0.1 M solutions of the electrolyte were puried and dried using neutral aluminum oxide ICN alumina N, super I.. Measurements were performed in a three electrode arrangement in a single cell at 20 C. The scan speed was 100 mVrs. The positive and negative voltage limits of the system are 2.4 V and y2.8 V vs. SCE in acetonitrile.

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rst one-electron oxidation potential for deoxyguanosine Eox s 1.25 V vs. SCE in acetonitrile was taken from Seidel et al. 1996.. The free energy changes Gcs for photo-induced electron transfer from a ground state guanosine residue to the excited dye are: y0.07 eV for R6G; 0.00 eV for JA133; and q0.25 eV for Cy5. These thermodynamic data support the experimentally observed behavior, that among the dyes used in this study, only R6G and the relatively similar dye TMR are quenched by guanosine residues Sauer et al., 1995; Nord et al., 1997; Eggeling et al., 1998; Lieberwirth et al., 1998.. Therefore, we used DNA constructs in which the rst GrC base pair is located 6 bp away from the 5 -terminus where the donor dyes R6G, TMR and Cy3 were attached. The acceptor seems to be quenched due to the presence of the donor at the DNA and vice versa due to another additional quenching process. From the measured redox potentials Table 4., Cy5 is the strongest electron donor among the dyes used. The free energy change Gcs for photo-induced electron transfer from Cy5 to R6G is y0.50 eV if R6G is excited, and y0.11 eV if the Cy5 chromophore is excited. Therefore, efcient charge transfer might occur at least at short separation distance like in the 5-bp constructs via direct through space interactions Tierney et al., 2000.. If so, the uorescence of the excited donor would be quenched supplementary to the uorescence resonant energy transfer to the acceptor. The uorescence of the excited acceptor could be quenched in the same way, via charge transfer to the ground state donor. The free energy changes for charge transfer between a rhodamine donor TMR, R6G. and a rhodamine acceptor JA133. are only slightly exergonic, if at all. Nevertheless, at short distances, strong uorescence quenching of the acceptor occurs Table 3.. This might be explained by strong intermolecular interactions between the two rhodamine dyes due to their relatively hydrophobic structure. At longer distances 15, 25, 35 bp., the acceptor JA133 exhibits nearly uninuenced uorescence characteristics. In strong contrast, the acceptor Cy5 exhibits a reduced uorescence quantum yield even at longer DrA distance Table 3.. Here we assume that a subpopulation of DrA constructs adopts a

conformation in which both donor and acceptor interact strongly with the -stack of the DNA, either by end capping or intercalation. Hence, the DNA might facilitate photo-induced electron transfer from Cy5 to the rhodamine. As already discussed by Kijima et al. 1998., excited electrons of the donor might interact with the acceptor dye covalently bound to the opposite end of a DNA by transfer across over 1000 base pairs. Dependent on the conformation of the dyes with respect to the -stack of the DNA, uorescence of the excited donor or acceptor might be efciently quenched. In single molecule experiments, these subpopulations cannot be detected due to the low uorescence quantum yields of these states and the application of a threshold for data collection. Hence, the spFRET efciencies obtained from the 5-bp construct are in accordance with the theoretically expected FRET efciencies Table 2; Fig. 7.. In ensemble measurements, the only weakly uorescent subpopulation which undergoes efcient electron transfer and dimerization in combination with the opening of new non-radiative deactivation channels inuences the calculated FRET efciencies in a way that the FRET efciencies appear to be to low for short DrA distances. On the other hand, at longer DrA distances the different extent of the electron transfer efciencies for the donor and acceptor also falsify the calculated ensemble FRET efciencies. Since the free energy change for photo-induced electron transfer between rhodamines and Cy5 is more negative for the excited donor than the excited acceptor, the FRET efciency calculated from the ensemble uorescence intensities, might indicate higher FRET efciencies. With the assumption that the charge transfer process occurs only in subpopulations with optimal conformations of the dyes with respect to the -stack of the DNA base pairs, very efcient charge separation can result. From the reduced uorescence intensities of the acceptor Table 3., on average approximately 10 30% of all FRET constructs exhibit such a favorable conformation in which fast electron transfer mediated by the DNA seems to be possible. Therefore, short uorescence

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lifetimes might appear which, however, are only poorly resolved with standard spectroscopy. Due to thresholding and low uorescence intensities, this subpopulation appears as dim or non-uorescent in single molecule experiments and is not detected. Therefore, single molecule experiments reveal more accurate FRET efciencies unperturbed by additional quenching effects.

3. Conclusions We studied the FRET efciencies of four different DrA pairs covalently attached to a double-stranded 40 base pair oligonucleotide by ensemble and single molecule spectroscopy in aqueous solution. All ensemble measurements revealed that, especially at short DrA distances, an additional uorescence quenching pathway for both the donor and acceptor has to be taken into account. Our data demonstrate that due to the conformational exibility of the linkers, the donor and acceptor dye can directly interact in the 5-bp constructs to form partly non-uorescent or only weakly uorescent complexes. Furthermore, we could show that the extent of the process is strongly controlled by the water solubility of the dyes. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; in particular the 5-bp constructs exhibit unusually high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide but on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of DrA distances and orientations. Comparison of the FRET efciencies obtained from ensemble and single molecule experiments demonstrates that the technique of spFRET experiments in solution is a powerful technique to uncover subpopulations. spFRET also facilitates correct interpretation of ensemble measurements. In single molecule experiments only those uorescence signals that exhibit a uorescence intensity above the signal threshold used contribute to the measured FRET efciency. Therefore, strongly quenched populations are not detected. The measured redox properties of the dyes imply

the possibility of a photo-induced electron transfer reaction between Cy5 and rhodamine chromophores. Hereby, the carbocyanine derivative acts as a strong electron donor, independent of whether the donor or acceptor is excited. This effect might seriously affect the FRET efciencies at a short DrA distance 5 bp.. For larger DrA distances we assume that dependent on the conformations of the dyes with respect to the DNA, an efcient charge transfer via the -stack of the base pairs occurs. These subpopulations, which appear strongly quenched and undetected in single molecule experiments, control the measured FRET efciencies in ensemble measurements. Our results and considerations demonstrate that not only direct intermolecular interactions between the donor and acceptor at short separation distance, but also long-range DNA mediated interactions have to be taken into account. Furthermore, our data show that spFRET experiments are only suited to resolve adequately uorescent subpopulations in heterogeneous mixture. Information about strongly quenched subpopulations gets lost. Consideration of the described competing quenching processes is important for any application of the FRET technology. Acknowledgements The authors thank J. Wolfrum for fruitful cooperation and stimulating discussion, and K.H. Drexhage and J. Arden-Jacob for the generous disposal of the rhodamine derivative JA133. Financial support by the Volkswagen-Stiftung Grant Ir74 443. and the Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie Grant 11864 BFA082. is gratefully acknowledged. References
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