Multistep Fluorescence Resonance Energy Transfer in Sequential

NOVEMBER/DECEMBER 2003 Volume 14, Number 6 © Copyright 2003 by the American Chemical Society

ARTICLES Multistep Fluorescence Resonance Energy Transfer in Sequential Chromophore Array Constructed on Oligo-DNA Assemblies Yuichi Ohya,* Kentaro Yabuki, Masafumi Hashimoto, Atsushi Nakajima, and Tatsuro Ouchi Department of Applied Chemistry, Faculty of Engineering & High Technology Research Center, Kansai University, Suita, Osaka 564-8680, Japan. Received February 28, 2003; Revised Manuscript Received July 24, 2003

Sequential arrays of chromophores at regulated distances were constructed on a noncovalent DNA molecular assembly system in aqueous media. Photoinduced fluorescence resonance energy transfer (FRET) behaviors were then observed. We designed a number of chromophore/oligo-DNA conjugates with varying sequences. The chromophores eosin (Eo), TexasRed (TR), and tetramethylrhodamine (Rho) were employed as the energy donor, acceptor, and mediator, respectively, based on overlapping excitation and emission spectra. The chromophores were attached via aminolinkers to the 5′-terminals of 10mer oligo-DNAs consisting of AT rich sequences. The arrangement of Eo-Rho or Rho-TR with 10-residue (1 pitch of duplex) distances was ensured by duplex formation of the conjugates with a 20mer matrix oligo-DNA composed of complementary sequences to the conjugates. Single-step FRET from Eo to Rho and from Rho to TR was confirmed on the duplex. The three chromophore conjugates were then mixed with longer matrix oligo-DNAs (30 or 40mer) consisting of complementary sequences to the conjugates, producing Eo-(Rho)n-TR (n ) 1 or 2) arrays with 10-residue distances. Multistep FRET from Eo to TR through the Rho mediator(s) was observed on the molecular assemblies. This photoenergy transmission system offers a good model for a photoenergy transmission system mimicking photosynthetic systems.

INTRODUCTION

In natural systems, the arrangement of functional molecules and groups with regulated distances, orders, and orientations provide highly efficient functionality. For example, in natural photosynthetic systems, the arrangement of porphyrin derivatives with regulated distance and geometry through noncovalent interaction provide highly efficient photoinduced energy transfer (13). To achieve noncovalent “homo” arrangement of one * Corresponding author. Phone +81-6-6368-0818, fax +816-6339-4026, e-mail [email protected].

kind of molecule or functional group, solid crystal, liquid crystalline systems or Langmuir-Blodgett film techniques may be useful. However, to achieve “hetero” and sequential arrangement of different kinds of molecules or functional groups, such systems are not effective. When constructing this type of system, numerous sets of specific noncovalent interactions should be provided (Figure 1). In natural and artificial systems, several types of noncovalent specific interactions, such as host-guest molecule, antigen-antibody, avidin-biotin, and saccharide-lectin, are known. However, it is difficult to include a wide variety of these specific interactions, and most of them require relatively large spaces.

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Figure 2. Schematic representation array constructed by mixing with complementary matrix oligo-DNA.

Figure 1. Arrangement of functional molecules or groups by self-assembly through specific interactions.

The biological tasks of nucleic acids (DNA and RNA) are genetic information storage and propagation. A single chain of poly- or oligo-nucleotide is able to interact specifically with its complementary counterpart chain through sequence-specific hydrogen bonding. By focusing on this property of nucleic acids, a wide variety of noncovalent binding pairs, binding donors, and binding acceptors, with high specificity and stability, can easily be provided simply by varying the oligo-DNA sequences. Oligo-DNA is therefore useful molecular glue for constructing molecular assembly systems. In fact, highmolecular-weight supramolecular polyassembly systems using complementary oligo-DNAs as binding groups have been reported both by our group (4) and Takenaka et al. (5). Seeman reported topological DNA assembly systems of various shapes and sizes (6-10). These characteristics of oligo-DNA facilitate the sequential arrangement of functional molecules. As described above, in natural photosynthetic systems, arrangement of porphyrin derivatives with regulated distances and geometry through noncovalent interaction provide highly efficient photoinduced energy transfer. Arrangement of chromophores (multichromophore array) would therefore provide a good model for artificial photosynthetic systems. Chromophore arrangements using noncovalent interactions have been studied as models for artificial photosynthetic systems (11-17). Arrangements of chromophores in polymeric systems and the energy or electron transfer have also been investigated as models for photon-harvesting systems (18-20); however, the flexibility of the polymer chains interfered with the distance-controlled spatial arrangement of the chromophores. Sisido et al. attempted to arrange chromophores along a relatively rigid polypeptide R-helix and reported energy or electron transfer in such systems (2123). The high rigidity and regularity of DNA duplex may prove to be more practical and applicable for ensuring distance-controlled spatial arrangement of chromophores. The DNA duplex contains a rich π-electron system comprising four bases stacked upon one another. A number of researchers have investigated the kinetics of electron transfer (ET) (24-29) and hole transport (3032) through the DNA duplex as well as conductivity of the DNA duplex (33-35). Labeling of oligo- and polynucleotides with fluorescent probes is a classical but still very important technique for analysis of DNAs and RNAs. Spectral changes of the probes and fluorescent resonance energy transfer (FRET) or ET between probes covalently attached to DNAs and RNAs have been investigated for detection of duplex or triplex DNA formations (36, 37), for structural analyses of DNA and

RNA (38-40), and for diagnostic detection of specific genes (41-43). Specific chromophores with overlapping excitation and emission spectra that are arranged in order of excitation energy level should form a “photoenergy transmission pathway”. Such systems would provide a good model for artificial photosynthetic systems and assist our understanding of the natural photosynthetic system. We previously utilized DNA duplex formation to construct sequential arrangements of the three chromophores fluorescamine, rhodamine B, and fluorescein to act as the photoenergy donor, acceptor and mediator, respectively. We then reported the FRET behavior in aqueous media (44). Multistep photoinduced energy transfer from fluorescamine to rhodamine B through one or more fluorescein mediators was actually observed. However, the thermodynamic stability of the system was less than ideal, as only five residues were used for complementarity-determining regions. FRET systems involving three chromophores on DNAs have also reported by other groups (45-48). In the present study, we designed a new series of chromophore/oligo-DNA conjugates with 10mer oligoDNA as complementarity-determining regions. Using these, we constructed a relatively stable sequential arrangement of chromophores separated by a regulated distance and produced a multistep FRET in a noncovalent molecular assembly system in aqueous media. Three different chromophores with overlapping excitation and emission spectra were utilized: eosin (Eo), TexasRed (TR), and tetramethylrhodamine (Rho), as the photoenergy donor, acceptor, and mediator, respectively. These were attached to the 5′-terminals of 10mer oligo-DNAs displaying differing sequences. The sequences and the structure of the conjugates synthesized and matrix oligoDNAs were shown in Table 1. The sequences of these oligo-DNAs were carefully chosen after considering several factors. We previously reported primitive study on energy transfer behavior in a similar system consisting of GC rich oligo-DNAs; however, the energy transfer efficiencies were not so high (49). Then we revealed that guanine residue(s) possessed a fluorescent quenching effect on chromospheres attached to oligo-DNA when guanine was located near the chromophore (50). Guanine residues have also been reported to exhibit charge transport ability through a hole transport mechanism (30, 31). To avoid such unexpected quenching phenomena, we ensured that only A-T pairs were located near the chromophores (terminal regions of oligo-DNAs) in the chromophore/oligo-DNA conjugate. Moreover, the sequences were chosen to avoid formation of unexpected three-dimensional structures by intramolecular hydrogen bonding. The resulting conjugates were mixed with matrix oligo-DNA (30 or 40mer) consisting of complementary sequences for the conjugates in order to construct a sequential Eo-(Rho)n-TR array (where n ) 1 or 2) in a DNA duplex with 10 residues separating each chromophore component (Figure 2). The most common

FRET in Chromophore Array on DNA

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Table 1. Sequences and Abbreviations of the Compounds Used

abbreviation

structure(sequenced)

E10a R10b R10c T10d 10a 10b 10c 10d m20a′b′ m20b′d′ m30a′b′d′ m40a′b′c′d′

Eo-5′TTTTCTGATA3′ Rho-5′ATTAGCTATT3′ Rho-5′ATTTCGAATT3′ TR-5′TATGTTCTAT3′ 5′TTTTCTGATA3′ 5′ATTAGCTATT3′ 5′ATTTCGAATT3′ 5′TATGTTCTAT3′ 5′AATAGCTAAT-TATCAGAAA3′ 5′ATAGAACATA-AATAGCTAAT3′ 5′ATAGAACATA-AATAGCTAAT-TATCAGAAA3′ 5′ATAGAACATA-AATTCGAAAT-AATAGCTAAT-TATCAGAAA3′

conformation for natural DNA duplex is B-form, where 10 residues form one turn. Chromophores on the DNA duplex were thus assumed to be arranged on the same side of the DNA duplex separated by one helical pitch (34 Å). The FRET behavior between the three chromophores was then investigated by monitoring fluorescence spectra in buffer solution. EXPERIMENTAL PROCEDURES

General Methods. The UV-vis absorption and fluorescence spectra were recorded on UV-2500PC (Shimadzu, Japan) and F4010 (Hitachi, Japan) spectrophotometers, respectively. CD spectra were measured using a J-600 (JASCO, Japan). MALDI-TOF-MS experiments were performed on an AXIMA CFR (Shimadzu, Japan) [negative mode, matrix: 3-hydroxypicolic acid (H2O/ acetonitrile ) 7/3)] after samples were treated with 0.1 M diammonium hydrogen citrate solution. Reverse-phase HPLC was carried out using a Toso-8020 system with a TSKgel OligoDNA RP column. Materials. Eosin isothiocyanate (EITC), tetramethylrhodamine isothiocyanate (TRITC), and Texas Red sulfonyl chloride (TRS-Cl) were purchased from Molecular Probes, Inc. (USA). 5′-End-free fully protected oligo-DNAs on solid support (control pore glass, CPG), biotinyl-oligoDNA, and nonmodified oligo-DNAs were obtained from Hokkaido System Science Co Ltd. (Japan). Aminoethanol, ethyl trifluoroacetate, 1H-tetrazole/acetonitrile solution and anhydrous acetonitrile were purchased from Wako Pure Chemical Ind. (Japan). 2-Cyanoethyl-N,N,N′,N′tetraisopropylphosphorodiamidite was purchased from Aldrich Chemical Co. Inc. (Milwaukee, WI). Water was purified using a reverse-osmotic membrane. DMF and other organic solvents were purified by usual distillation methods. Other materials were of reagent grade and used without further purification. Synthesis of chromophore/ oligo-DNA conjugates was performed according to the methods reported previously (49). Structures and sequences of the oligo-DNAs and chromophore/oligo-DNA conjugates synthesized are shown in Table 1. Synthetic procedures and spectral data of the conjugates are available in the Supporting Information. Spectroscopic Studies. Concentration of unmodified oligo-DNA was calculated from the UV absorbance at 260 nm (A260), and extinction coefficients were determined using the nearest-neighbor approximation. Concentration

[Eo/oligo-DNA] [Rho/oligo-DNA(1)] [Rho/oligo-DNA(2)] [TR/oligo-DNA]

of each chromophore/oligo-DNA conjugate was determined using UV-vis absorbance at 525, 554, and 597 nm for Eo/oligo-DNA E10a, Rho/oligo-DNA R10b and R10c, and TR/oligo-DNA T10d, respectively. Conjugate concentration in solution was confirmed by the fact that their hypochromicity with complementary unmodified oligo-DNAs was maximal at 1/1 molar ratio. Hypochromicity measurement was performed by monitoring A260 of the solution containing the conjugate and complementary oligo-DNA in various ratios. Melting curves were recorded by starting at 60 °C sufficiently above Tm and reducing temperature at a rate of 10 °C/h to 10 °C sufficiently below Tm. Absorbance values were continuously recorded at intervals of 5 °C. CD spectra were measured on a JASCO J-600 using quartz cells of 0.5cm path length at 15 °C. Concentrations of each conjugate and matrix oligo-DNA were 1.0 × 10-5 M. Fluorescence measurements were performed on a Hitachi F4010, using a 1 × 1 cm quartz cell. Excitation wavelengths used were 525, 554, and 597 nm for Eo, Rho, and TR, respectively. Fluorescence spectra for the mixture of several chromophore/oligo-DNA conjugates and complementary matrix oligo-DNA were obtained by varying the mixing ratios. The following procedure is an example of the titration of E10a and m20a′b′ with R10b. “Solution A”, containing E10a (5.0 × 10-7 M) and m20a′b′ (5.0 × 10-7 M), and “Solution B” containing E10a (5.0 × 10-7 M), m20a′b′ (5.0 × 10-7 M), and R10b (40.0 × 10-7 M) were prepared. The fluorescence spectra of “Solution A” were measured under titration with “Solution B”. The final ratio of R10b to E10a was 4.0. All fluorescence experiments were performed at 5 °C in 0.05 M Tris‚HCl buffer (pH 7.5) containing 0.5 M NaCl. The fluorescence intensity of each chromophore in mixed systems containing two or more chromophores was calculated by decomposition of the obtained spectra to the emission spectra of each chromophore. Excitation spectra were measured on the equipment described above at an emission wavelength of 609 nm. QCM Measurement. Sequential duplex formation of chromophore/oligo-DNA conjugates with matrix oligoDNA was investigated by 27-MHz quartz crystal microbalance (QCM) analysis at 15 °C. Experiments were carried out using the same equipment and methods as previously reported (51). In this experiment, 30mer matrix oligo-DNA-3′-biotinylated through a tetraethylene


Ohya et al.

Figure 3. Excitation and emission spectra of Eo, Rho and TR in duplex form. Excitation spectra shown doted lines and emission spectra shown solid lines. The excitation wavelengths for emission spectra of Eo, Rho and TR were 525 nm, 554 nm and 597 nm, respectively. Emission wavelengths for excitation spectra of Eo, Rho and TR were 574 nm, 578 nm and 609 nm, respectively.

glycol spacer (bio-m30a′b′d′) was used as a matrix oligoDNA. Bio-m30a′b′d′ (400 pmol) was added to an avidinimmobilized QCM sensor tip immersed in 8 mL of 10 mM Tris‚HCl-1mM EDTA-200 mM NaCl buffer (pH 7.8). After frequency decreased to -150 Hz, the sensor tip was washed with water and 50 mM Tris‚HCl-500 mM NaCl buffer (pH 7.5) and dipped into 8 mL of 50 mM Tris‚HCl500 mM NaCl buffer (pH 7.5). A frequency decrease of 1 Hz corresponded to a mass increase of 0.61 ( 0.1 ng cm-2 on the QCM electrode (51-53). The frequency decrease of 150 Hz corresponded 91.5 ng cm-2 (1 pmol on 4.9 mm2) binding of bio-m30a′b′d′ to avidin immobilized on the sensor tip. Chromophore/oligo-DNA conjugates (E10a, R10b, T10d) were then sequentially added to the solution, and frequency changes were observed. RESULTS AND DISCUSSION

We selected Eo, TR, and Rho as the photoenergy donor, acceptor, and mediator, respectively, because of their overlapping excitation and emission wavelength ranges. The excitation and emission spectra of the chromophore/ oligo-DNA conjugates in duplex form are shown in Figure 3. The λmax values of excitation/emission spectra for Eo/ oligo-DNA, Rho/oligo-DNA, and TR/oligo-DNA were 525/ 547 nm, 554/578 nm, and 597/609 nm, respectively. The Tm value of the stoichiometric mixture of E10a, R10b, T10d, and m30a′b′d′ was determined to be ca. 37 °C in 0.05 M Tris‚HCl-0.5 M NaCl buffer (pH 7.5). The 30mer matrix oligo-DNA m30a′b′d′ was composed of 3 units of 10mer oligo-DNA, each one of which was complementary with E10a, R10b, or T10d. The melting curve seemed to display a single transition point and no obvious double or triple transition points. This indicates that each of the 10mer units had similar Tm values. The Tm value of the stoichiometric mixture of matrix oligo-DNA m30a′b′d′ and the three types of oligo-DNA 10a, 10b, and 10d, having the same sequences as E10a, R10b, and T10d, was ca. 37 °C. The mixture of conjugates and matrix oligo-DNA revealed that the chromophores attached to the 5′-teminals of oligo-DNA exerted almost no effect on formation of the duplex (thermal dissociation curves are available in the Supporting Information). On the basis of the results, further fluorescence measurements were performed at 5 °C, which is sufficiently lower than the Tm. Specific and nonspecific interactions of chromophores with single- and double-stranded DNA, such as intercala-

Figure 4. (a) Fluorescence spectra for titration of E10a and m20a′b′ complex with R10b in 0.05 M Tris‚HCl-0.5 M NaCl buffer (pH 7.5) at 5 °C. [m20a′b′] ) [E10a] ) 5.0 × 10-7 M, [R10b]/[E10a] ) 0-1.0. Excitation wavelength: 525 nm. (b) Schematic representation of procedure. (c) Plots of relative fluorescence intensity at 547 nm vs ratio of [Rho]/[E10a]. Square R10b; circle 1:1 mixture of 10b and free Rho. [m20a′b′] ) [E10a] ) 5.0 × 10-7 M. Excitation wavelength: 525 nm.

tion, were not observed in either UV or fluorescence spectra measurements. Before studying multistep FRET, we investigated single-step FRET behaviors between two chromophores. Experiments were performed using 20mer matrix oligoDNA, donor/oligo-DNA displaying complementarity with the 10 3′-terminal residues of the matrix oligo-DNA and acceptor/oligo-DNA displaying complementarity with the 10 5′-terminal residues of the matrix oligo-DNA. Figure 4a shows the fluorescence spectra for titration of E10a (donor) and the 20mer matrix oligo-DNA m20a′b′ with R10b (acceptor). Fluorescence spectra were measured at an excitation wavelength of 525 nm (λmax of Eo) in 0.05 M Tris‚HCl-0.5 M NaCl buffer (pH 7.5) at 5 °C. A schematic illustration of the experimental procedure is shown in Figure 4b. Quenching of fluorescence from donor chromophores (Eo) and increased fluorescence from acceptor chromophores (Rho) were observed on addition of R10b. Effective quenching and increased fluorescence of Rho were not observed when a mixture of free Rho and oligo-DNA 10b was added instead of R10b. Figure 4c shows relative fluorescence intensity of Eo at 547 nm vs the molar ratio of R10b to E10a. The donor (Eo) quenching was almost saturated when the molar ratio



Figure 5. (a) Fluorescence spectra for titration of R10b and m20b′d′ complex with T10d in 0.05 M Tris‚HCl-0.5 M NaCl buffer (pH 7.5) at 5 °C. [m20b′d′] ) [R10b] ) 5.0 × 10-7 M, [T10d]/[R10b] ) 0-1.0. Excitation wavelength: 554 nm. (b) Schematic representation of the procedure.

of R10b to E10a was 1. The same experiment was performed for the combination of R10b and T10d. Figure 5a shows the fluorescence spectra for titration of R10b (donor) and a 20mer matrix oligo-DNA m20b′d′ with T10d (acceptor). Fluorescence spectra were measured at an excitation wavelength of 554 nm (λmax of Rho) in 0.05 M Tris‚HCl-0.5 M NaCl buffer (pH 7.5) at 5 °C. A schematic illustration of the experimental procedure is shown in Figure 5b. Quenching of fluorescence from donor chromophores (Rho) and increased fluorescence from acceptor chromophores (TR) was observed on the addition of T10d. Effective quenching and increased fluorescence of TR were not observed when a mixture of free TR and oligo-DNA 10d was added instead of T10d. These results suggest that single-step FRET from Eo to Rho and from Rho to TR occurred along the 20bp DNA duplex. Single-step FRET was successfully observed between two chromophores, and we therefore investigated twostep energy transfer from Eo (donor) through Rho (mediator) to TR (acceptor) on a duplex with a 30mer matrix oligo-DNA m30a′b′d′. As an initial step, E10a displaying complementarity with the 10 3′-teminal residues of the matrix oligo-DNA was mixed with matrix oligo-DNA m30a′b′d′ in a 1:1 ratio. The complex formed was then titrated with an equivalent amount R10b displaying complementarity with the 10 central residues of the m30a′b′d′. The obtained solution of E10a, R10b and m30a′b′d′ (1:1:1 ratio) was then titrated with T10d displaying complementarity with the 10 5′-teminal residues of m30a′b′d′. Figures 6a and 6b show the fluorescence spectra of the two-step titration for the complex of m30a′b′d′ and E10a with R10b, and subsequent titration with T10d. Fluorescence spectra were measured at an excitation wavelength of 525 nm (λmax of Eo) in 0.05 M Tris‚HCl-0.5 M NaCl buffer (pH 7.5) at 5 °C. A schematic illustration of the experimental procedure is shown in Figure 6c. Initially, quenching of Eo and

Figure 6. Fluorescence spectra for titration of E10a and m30a′b′d′ complex with R10b (a) and T10d (b) in 0.05M Tris‚ HCl-0.5M NaCl buffer (pH 7.5) at 5 °C. [m30a′b′d′] ) [E10a] ) 5.0 × 10-7 M. [R10b]/[E10a] ) 0-1.0. [T10d]/[E10a] ) 0-2.0. Excitation wavelength: 525 nm. (c) Schematic representation of the procedure.

increased fluorescence from Rho were observed on addition of R10b, as shown in Figure 4. Quenching of Rho and increased fluorescence from TR were then observed on addition of T10d, as shown in Figure 5. The fact that obvious fluorescence from TR was observed at an excitation wavelength of 525 nm indicates that the photoenergy transferred from Eo to Rho was further transferred to TR by two-step FRET. We then investigated multistep FRET from Eo (donor) through two Rhos (mediators) to TR (acceptor) on a duplex with a 40mer matrix oligo-DNA m40a′b′c′d′ containing 20 complementary residues with R10b and R10c in the central region. Initially, R10b (complementary with residues 21-30 of m40a′b′c′d′) was added to the 1:1 ratio mixture of m40a′b′c′d′ with E10a (complementary with the residues 31-40 of m40a′b′c′d′) to a final ratio of 1:1:1. The complex formed was then titrated with an equivalent amount R10c (complementary with residues 11-20 of m40a′b′c′d′). This complex, E10a, R10b, R10c and m40a′b′c′d′ (1:1:1:1 ratio), was titrated


Ohya et al.

Figure 7. Fluorescence spectra for titration of E10a and m40a′b′c′d′ complex with R10b (a), R10c, (b), and T10d (c) in 0.05M Tris‚HCl-0.5M NaCl buffer (pH 7.5) at 5 °C. [m40a′b′c′d′] ) [E10a] ) 5.0 × 10-7 M. [R10b]/[E10a] ) 0-1.0, [R10c]/[E10a] ) 0-1.0, [T10d]/[E10a] ) 0-4.0. Excitation wavelength: 525 nm. (d) Schematic representation of the procedure.

Figure 8. Excitation spectra for complexes (a) (m30a′b′d′ + E10a + 10b + 10d), (m30a′b′d′ + 10a + R10b + 10d), (m30a′b′d′ + E10a + R10b + 10d), and (b) (m30a′b′d′ + E10a + 10b + 10d), (m30a′b′d′ + 10a + R10b + 10d), (m30a′b′d′ + 10a + 10b + T10d), (m30a′b′d′ + 10a + R10b + T10d), (m30a′b′d′ + E10a + R10b + T10d) in 0.05 M Tris‚HCl-0.5 M NaCl buffer (pH 7.5) at 5 °C. Emission wavelength: 609 nm. [E10a] ) [R10b] ) [T10d] ) [10a] ) [10b] ) [10d] ) [m30a′b′d′] ) 5.0 × 10-7 M.

with T10d displaying complementarity with the residues 1-10 of m40a′b′c′d′. Figures 7a-c show changes in fluorescence spectra for the three-step titration of complex of m40a′b′c′d′ and E10a with R10b, R10c and the subsequent titration with T10d. Fluorescence spectra were measured at an excitation wavelength of 525 nm (λmax of Eo) in 0.05 M Tris‚HCl-0.5 M NaCl buffer (pH 7.5) at 5 °C. A schematic illustration of the experimental procedure is shown in Figure 7d. Quenching of Eo and increased fluorescence from Rho were observed on addition of R10b, as shown in Figures 4 and 6. Slightly quenching of Eo and somewhat increased fluorescence from Rho were observed on addition of R10c. Furthermore, quenching of Rho and increased fluorescence from TR were observed on addition of T10d, as shown in Figures 5 and 6. The fact that obvious fluorescence from TR was observed at excitation of 525 nm indicates that the photoenergy transferred from Eo through the two Rhos was further transferred to TR. These results suggest that energy migration between the two Rhos occurred. To determine the energy transfer efficiency, excitation spectra were measured for the chromophore/oligo-DNA

assembly systems in 0.05 M Tris‚HCl-0.5 M NaCl buffer (pH 7.5) at 5 °C. The emission wavelength was 609 nm (λmax of emission of TR) for all excitation spectra. Figures 8 and 9 show typical excitation spectra for the 30mer and 40mer assembly systems, respectively. Combinations of the conjugates, control oligo-DNAs and matrix oligo-DNA are illustrated in the figures. Figure 8a suggests FRET from Eo to Rho occurred. Eo (E10a + 10b + 10d + m30a′b′d′) showed very little excitation, because Eo does not show effective emission at 609 nm. Rho (10a + R10b + 10d + m30a′b′d′) showed some excitation around 560 nm, corresponding with absorbance by Rho. However, the Eo-Rho system (E10a + R10b + 10d + m30a′b′d′) showed double the maximum excitation as each of Rho and Eo showed. The difference between (E10a + R10b + 10d + m30a′b′d′) and (10a + R10b + 10d + m30a′b′d′) was larger than that for (E10a + 10b + 10d + m30a′b′d′). This suggests that effective energy transfer from Eo to Rho in (E10a + R10b + 10d + m30a′b′d′) occurred. In Figure 8b, FRETs from Rho to TR and from Eo to TR were shown. TR (10a + 10b + T10d + m30a′b′d′) showed substantial excitation around 600 nm corresponding with absorbance by TR. The Rho-TR



ing of the donor chromophore was calculated using the following equation:

Q(%) ) (q - q0)/(1 - q0) × 100 where q ) 1 - I/I0, and represents the quenching with acceptor/oligo-DNA conjugate, q0 is the quenching with free chromophore and oligo-DNA without chromophore, I is the fluorescence intensity, and I0 is the fluorescence intensity of donor/oligo-DNA conjugate. Energy transfer (T(%)) based on excitation spectra was calculated using the following equation (54):

Tapp(%) )

Figure 9. Excitation spectra for complex (m40a′b′c′d′ + E10a + 10b + 10c + T10d), (m40a′b′c′d′ + E10a + R10b + 10c + T10d), (m40a′b′c′d′ + E10a + 10b + R10c + T10d) and (m40a′b′c′d′ + E10a + R10b + R10c + T10d) in 0.05 M Tris‚ HCl-0.5 M NaCl buffer (pH 7.5) at 5 °C. Emission wavelength: 609 nm. [E10a] ) [R10b] ) [R10c] ) [T10d] ) [10a] ) [10b] ) [10c] ) [10d] ) [m40a′b′c′d′] ) 5.0 × 10-7 M.

system (10a + R10b + T10d + m30a′b′d′) showed double the maximum excitation as each of Rho and TR showed. The difference between (10a + R10b + T10d + m30a′b′d′) and (10a + 10b + T10d + m30a′b′d′) was larger than that for (10a + R10b + 10d + m30a′b′d′). This indicates that effective energy transfer from Rho to TR in (10a + R10b + T10d + m30a′b′d′) occurred. Furthermore, for the Eo-Rho-TR system (E10a + R10b + T10d + m30a′b′d′), a peak at around 520 nm, corresponding with Eo, was observed in addition to the spectra seen with (10a + R10b + T10d + m30a′b′d′). The difference between these systems was larger than that for (E10a + 10b + 10d + m30a′b′d′). This suggests that absorption of Eo contributed to fluorescence at 609 nm: photon-energy absorbed by Eo was effectively transferred to TR. In Figure 9, FRETs in the quadraplex system on 40mer matrix oligo-DNA. The Y-axis of Figure 9 is the same as Figures 8a and 8b. Eo-(blank)-(blank)-TR system (E10a + 10b + 10c + T10d + m30a′b′c′d′) showed almost same excitation spectra as TR (10a + 10b + 10c + T10d + m30a′b′c′d′). No effective energy transfer from Eo to TR separated 30 residues. Eo-Rho-(blank)-TR system (E10a + R10b + 10c + T10d + m30a′b′c′d′) and Eo-(blank)-Rho-TR system (E10a + 10b + R10c + T10d + m30a′b′c′d′) showed excitation peak around 560 nm. The intensity was larger in the latter, which had shorter distance (10 residues) between Rho and TR. In Eo-Rho-Rho-TR system, larger excitation peak for Rho was observed because this system two Rhos. In addition to the Rho excitation peak, small peak corresponding with Eo was observed around 520 nm. This suggest some energy transfer from Eo to TR separated 30 residues. Quantitative for all excitation spectra below. On the basis of the titration fluorescence spectra and excitation spectra, we were able to estimate quenching efficiency (Q(%)) and energy transfer efficiency (T(%)). Quenching efficiency (Q(%)) based on fluorescence quench-

A(X) A(100)

× 100 )

{

}

φDFD (1 - T) + T × 100 φAFA

Tapp is the apparent energy transfer, A(100) is the emission spectrum, A(X) is the excitation spectrum of measurement system, φD and φA are the quantum yields of donor and acceptor chromophore, and FD and FA are the fluorescence of donor and acceptor chromophore/area of the emission spectrum. The Q(%), Tapp(%), and T(%) obtained for the molecular assemblies of chromophore/oligo-DNA conjugates with matrix oligo-DNAs summarized in Table 2. The efficiencies for one-step FRET from Eo to Rho and from Rho to TR were 32.3% and 36.3%, respectively. Direct energy transfer from Eo to TR in Eo-(blank)-TR system was 0%, because the 20 residue distance and small overlap of absorbance and emission spectra. In contrast, the EoRho-TR system showed effective energy transfer (23.7% efficiency). The effect of Rho as a mediator was clear. The two-step energy transfer efficiency from Eo to TR though Rho was smaller than that of the one-step FRETs, but larger than their product. When comparing the Eo-Rho system and the Eo-(blank)-Rho system, energy transfer efficiency decreased depending the distance. This is not exactly in line with the Fo¨rster equation, probably due to the flexibility of the spacer groups and friction of the chromophores. Single Rho mediator systems, Eo-Rho(blank)-TR system and Eo-(blank)-Rho-TR system, showed 13.8% and 8.5% of energy transfer efficiencies, respectively. On the other hand, the Eo-Rho-Rho-TR system having two Rho mediators showed larger energy transfer efficiency (21.6%), which was similar to that of the Eo-Rho-TR system (23.7%). These results indicate energy migration between two Rho mediators. Sequential and quantitative duplex formation of chromophore/oligo-DNA conjugates with matrix oligo-DNA was investigated by QCM analysis. The following Sauerbrey equation was for the AT-cut shear mode QCM (55),

∆F ) -

2F02

∆m AxFqµq

where, ∆F is the measured frequency change (Hz), F0 the fundamental frequency of the QCM (27 × 106 Hz), ∆m is the mass change (g), A is the electrode area (4.9 mm2), Fq is the density of quartz (2.65 g cm-1), and µq is the shear modulus of quartz (2.95 × 1011 dyn cm-2). Previous calibration for the 27 MHz QCM showed that a frequency decrease of 1 Hz corresponded to a 0.61 ng cm-2 mass increase (53). Typical results for the assembly of E10a, R10b, and T10d with m30a′b′d′ are shown in Figure 10. First, 80 pmol of E10a was added to m30a′b′d′ bound to avidinimmobilized QCM sensor tip (total concentration of E10a


Ohya et al.

Table 2. Quenching and Energy Transfer Efficiencies

a Quenching efficiency of donor. b Apparent energy transfer estimated from excitation spectra. c Energy transfer efficiency calculated from the following equation: Tapp(%) ) A(X)/A(100) × 100 ) {φDFD/φAFA(1 - T) + T} × 100 where A(100), emission spectrum; A(X), excitation spectrum of measurement system; φD(A), quantum yield of donor (acceptor) chromophore; FD(A), fluorescence of donor (acceptor) chromophore/ area of emission spectrum. d Calculated from quenching of Rho.

Figure 10. Typical time dependencies of QCM frequency for immobilized m30a′b′d′ responding to addition of E10a, R10b, and T10d in 0.05 M Tris‚HCl-0.5M NaCl buffer (pH 7.5) at 15 °C.

) 10 nM). A decrease in frequency was observed and a further 160 pmol of E10a was added. After this second addition of E10a, a larger decrease in frequency was observed (twice the decrease observed with the first addition. Following this, a further 160 pmol of E10a was added, and slight decreases in frequency and saturation of frequency change were observed. These results indicate that 400 pmol of E10a was an excess for immobilized m30a′b′d′ and that the m30a′b′d′ on QCM sensor tip quantitatively bound to E10a. The decrease in frequency was 59.5 Hz after total E10a addition (400 pmol). Second, 400 pmol of R10b was added to the solution, and a similar decrease in frequency (62.2 Hz) as seen with E10a (400 pmol) was observed. After confirmation of no further R10b binding by addition of excess R10b (800 pmol), 400 pmol of T10d was added to the solution. A slightly higher but similar decrease in frequency (75.0 Hz) as observed with E10a (400 pmol) was observed. No further T10d binding was confirmed by addition of excess T10d (800 pmol). These results indicate that almost the same amounts of E10a, R10b, and T10d bound to m30a′b′d′, and each 10-residue region of m30a′b′d′ was quantitatively by E10a, R10b and T10d. From these

Figure 11. CD Spectrum for equivalent mixture of m40a′b′c′d′, E10a, R10b, R10c, and T10d (solid line), and equivalent mixture of m40a′b′c′d′, 10a, 10b, 10c, and 10d (dashed line) in 0.05 M Tris‚HCl-0.5 M NaCl buffer (pH 7.5) at 15 °C. Concentration of each conjugate and oligo-DNA ) 1.0 × 10-5 M.

results, formation of the expected assembly of chromophore/oligo-DNA conjugates with matrix oligo-DNA, as shown in Figure 2, was strongly suggested. In order to obtain information about the conformation of the assembly, CD spectra were measured for equivalent mixtures of chromophore/oligo-DNA conjugates and matrix oligo-DNA at 15 °C. Figure 11 shows a typical example for equivalent mixtures of E10a, R10b, R10c, T10d, and m40a′b′c′d′, which were used in the experiment shown in Figure 7. Strong negative and positive cotton effects were observed around 250 and 280 nm, respectively. This pattern displays the typical characteristics of a B-form DNA duplex and is almost the same as that seen for the equivalent mixture of 10a, 10b, 10c, 10d, and m40a′b′c′d′. The assembly system of the four kinds of chromophore/oligo-DNA conjugates with the 40mer matrix oligo-DNA therefore displayed a B-form double helical conformation. Other systems, such as E10a, R10b, T10d, and m30a′b′d′, showed similar results. Figure 12 shows geometrical illustrations for assemblies of the three types of chromophore/oligo-DNAs and matrix oligo-DNA (30 or 40mer) assuming B-form double helical conformation. As the B-form DNA duplex has 10 residues per turn (34 Å), the system was believed


Figure 12. Schematic illustration geometry of chromophore arrays constructed on oligo-DNA assemblies. (a) 1:1:1:1 mixture of E10a, R10b, T10d, and m30a′b′d′, (b) 1:1:1:1:1 mixture of E10a, R10b, R10c, T10d, and m40a′b′c′d′.

to have all chromophores on the same side of the duplex at one-pitch distance (34 Å), as shown in Figure 12. CONCLUSIONS

Three chromophores (Eo, Rho, and TR) were attached to the 5′-terminal ends of 10mer oligo-DNAs in order to produce chromophore/oligo-DNA conjugates. Programmed sequential arrangements of chromophores separated by regulated distances of about one pitch of DNA duplex (34 Å) on noncovalent molecular assembly systems in aqueous media were then constructed by mixing conjugates with longer matrix oligo-DNAs. Multistep FRET from Eo to TR through one or two Rho mediators was observed on the molecular assemblies. The results suggested that photon-energy transfer occurred from Eo to TR separated by a long distance (three helical pitches, 104 Å) through Rho mediators via a multistep FRET on the molecular assemblies. The information obtained from these systems will be useful in constructing a photon-collecting antenna for an artificial photosynthetic system, as well as in enhancing our understanding of the mechanisms of energy transfer behavior in noncovalent molecular assembly system, such as natural photosynthetic systems. In subsequent research, details of the FRET behaviors of chromophores on oligo-DNA assemblies should be investigated using measurements of fluorescence lifetime and transition state absorption spectra of the chromophores. ACKNOWLEDGMENT

A part of this study was financially supported by a Grant-in-Aid for Scientific Research on Priority Areas from The Ministry of Education, Science, Sports and Culture, Japan. The authors express their thanks to Prof. Yoshio Okahata, Department of Biomolecular Engineering Tokyo Institute of Technology, for his assistance in QCM analysis. Supporting Information Available: Experimental procedures for synthesis of chromophore/oligo-DNA conjugates, spectral data for conjugates, melting curves of assemblies, and illustration of QCM analysis. This material is available free of charge via the Internet at http:// pubs.acs.org/BC. LITERATURE CITED (1) McDermott, G., Prince, S. M., Freer, A. A., Hawthornthwaite-Lawless, A. M., Papiz, M. Z., Cogdell, R. J., and Isaacs,

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BC034028M

Multistep Fluorescence Resonance Energy Transfer in Sequential

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