Bioconjugate Chem. 2007, 18, 621−627
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DNA-Directed Assembly of Supramolecular Fluorescent Protein Energy Transfer Systems Florian Kukolka,† Oliver Schoeps,‡ Ulrike Woggon,*,‡ and Christof M. Niemeyer*,† Fachbereich Chemie Biologisch-Chemische Mikrostrukturtechnik and Fachbereich Physik, Universita¨t Dortmund, Otto-Hahn Str. 6, D-44227 Dortmund, Germany. Received May 31, 2006; Revised Manuscript Received January 16, 2007
Fluorescent proteins with a wide variety of physicochemical properties have evolved in the past few years. The use of these proteins for applications in biomolecular nanosciences requires their precise positioning at the nanometer length scale. To address this challenge, we report here on the self-organization of DNA-tagged fluorescent probes to construct a set of photofunctional supramolecular complexes which include the enhanced yellow fluorescent protein (EYFP). The optical functionality is based on the strongly distance dependent fluorescence resonance energy transfer (FRET), occurring between the donor (EYFP) and an acceptor fluorophore, i.e., the fluorescent dye Atto647. The photophysical properties of four bimolecular FRET complexes, each possessing a well-defined donor-acceptor distance defined by the length of the interconnecting DNA backbone, are investigated by twodimensional photoluminescence excitation spectroscopy (2D-PLE).
INTRODUCTION The major goal of the development and investigation of artificial light-harvesting systems concerns the mimicry of the initial steps of incredibly efficient photon energy uptake and funneling toward a reaction center during photosynthesis. In addition to the general understanding of the fundamental principles of such complex reaction cascades, there is currently also a great interest in the development of photonic energy transfer systems, because such devices hold promise for applications in several research areas of nanotechnology, such as molecular photonics and bioelectronics (1). Several organic chromophores have already been used for photon energy uptake in artificial light-harvesting complexes, such as porphyrin derivatives (2), polypyridine complexes of d6 metal ions (2, 3), or perylenes (5-8). However, in order to fabricate well-defined arrangements of different chromophores, one major problem in the realization of complex superstructures concerns the precise control of the spatial arrangement of the various components within the functional unit (1). To address this problem, the biomimetic self-assembly of the individual compounds appears to be straightforward. For example, the extraordinary specific molecular recognition of complementary ssDNA sequences has already been utilized for the spatially defined organization of DNA-tagged molecular and colloidal components (9-14). The photophysical characterization of such arrangements of synthetic fluorophores assembled along short double-stranded (ds)DNA molecules has been the subject of current research. For example, up to five different organic fluorophores have been organized at the nanometer length scale by means of DNA hybridization to form a photonic wire (15-19). These systems revealed a unidirectional energy transport based on fluorescence resonance energy transfer (FRET) along an energy cascade. * Prof. Dr. U. Woggon, Universita¨t Dortmund, Fachbereich Physik, Otto-Hahn Str. 6, D-44227 Dortmund (Germany). E-mail:
[email protected]. Prof. Dr. C. M. Niemeyer, Universita¨t Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, D-44227 Dortmund (Germany). Telefax: Int. + 49 (0)231/755 7082. E-mail:
[email protected]. † Fachbereich Chemie Biologisch-Chemische Mikrostrukturtechnik. ‡ Fachbereich Physik.
We have recently suggested utilizing fluorescent proteins for the synthesis and investigation of artificial light-harvesting antennae systems (20). The most prominent example of fluorescent proteins is the naturally occurring green fluorescent protein from the north Pacific jellyfish Aequorea Victoria (aVGFP), which has been used extensively as a biological marker (21). A full range of fluorescent proteins with emission maxima ranging from blue to red are nowadays available. These are either mutants of the aVGFP or derivatives of similar proteins from other marine organisms (22-25), and the ongoing discovery of novel proteins with different physicochemical characteristics suggests that the range of these interesting proteins is by far not fully exhausted yet. For example, fluorescent proteins with the ability of being reversibly photoswitched between a fluorescent and a nonfluorescent state have recently attracted attention (26-28). In addition, proteins, in general, can be tailored with a large variety of synthetic moieties, such as non-natural amino acids (29), to modulate their spectral properties and binding capabilities. Hence, one may anticipate that this panel of natural fluorophores should be well-suited as building blocks for artifical supramolecular antennae systems or as components of nanoscaled optical data storage devices. To initiate this novel approach, we recently reported on the synthesis of a semisynthetic covalent DNA-fluorescent protein conjugate, as a first step to the DNA-directed assembly of supramolecular fluorescent protein FRET systems (20). We report here on the assembly and spectroscopic characterization of various unique FRET systems (FS), which were constructed by means of DNA hybridization using the enhanced yellow fluorescent protein (EYFP) as the donor (D) and the organic fluorescent dye Atto647 as the acceptor (A) for energy transfer. In these systems, the interchromophore distance of donor and acceptor can be conveniently changed by variation of the binding sites of the fluorophores at the DNA “carrier” helix. In contrast to another study based on single-molecule spectroscopy, in which donor and acceptor were assembled by hybridization of a DNA-dye and DNA-EYFP conjugate along a complementary DNA scaffold (30) to obtain two different complexes, we report here on the assembly of four different FRET systems in a more direct, bimolecular hybridization, thus allowing us to gradually change the distance between A and D.
10.1021/bc060143w CCC: $37.00 © 2007 American Chemical Society Published on Web 03/23/2007
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The different FRET systems were characterized by twodimensional photoluminescence excitation spectroscopy (2DPLE). 2D-PLE is a powerful method to study electronic couplings and energy transfer between donor and acceptor states in FRET systems. The emission is monitored over a whole range of wavelengths instead of a narrow detection window, as in conventional, one-dimensional PLE. This enables one to display the full landscape of photoluminescence (PL) intensity as a function of the excitation energy and to analyze energy transfer within a multicomponent system. The mutual correlations between donor and acceptor states can be qualitatively identified from the magnitude of the corresponding off-diagonal signal in the spectrally resolved 2D intensity maps. From the experimental 2D contour plots, we were able to derive energy transfer efficiencies and to calculate the Fo¨rster radius R0 for the EYFPAtto647 system.
EXPERIMENTAL PROCEDURES DNA-Chromophore Conjugates. Recombinant CysEYFP was covalently coupled to the 5′-end of a 24mer oligonucleotide (sequence A24: 5′-TCC TGT GTG AAA TTG TTA TCC GCT3′) yielding conjugate CysEYFP-A24 similar to the method previously described (20). Because the two cystein groups of the native EYFP are not accessible for covalent coupling with DNA oligomers (20), a CysEYFP mutant was created by sitedirected mutagenesis bearing an extra cysteine residue at the C-terminal end, which is located at the protein’s surface. The protein was expressed in E. coli and purified by affinity chromatography. The amino-modified oligonucleotides were first derivatized with the heterobifunctional cross-linker sulfosuccinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (sSMCC), which comprises a maleimide and a N-hydroxysuccinimide (NHS) ester group, that are reactive toward thiols and primary amines, respectively (Scheme 1). The maleimidecontaining DNA oligomers were then reacted with the CysEYFP, and the conjugates were purified by anion-exchange chromatography and analyzed by UV/vis spectroscopy. The ratio of
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absorbances measured at 260 and 280 nm, respectively, was found to be 1.50, and this value was in good aggreement with a control solution comprising equimolar amounts of DNA A24 and the EYFP (1.53). In contrast, control samples containing either of the two components in a 2-fold excess, i.e., DNA/ EYFP ) 2:1 or DNA/EYFP ) 1:2, revealed values of 1.61 and 1.41, respectively, thus confirming the 1:1 stoichiometric ration of DNA/EYFP in the CysEYFP-A24 conjugate. Moreover, analysis of the CysEYFP-A24 conjugate by SDS-PAGE showed a single band with a molecular weight of approximately 39 kDa, which is in good aggreement with the expected mass of a 1:1 conjugate (data not shown). The conjugates were stored in TETBS buffer (20 mM Tris-buffered saline containing 150 mM NaCl, 5 mM EDTA, 0.005% Tween-20, pH 7.5). Atto647-DNA conjugates were purchased lyophilized from TIB MOLBIOL (Berlin, Germany), dissolved in H2O at a concentration of 100 µM, and stored at -20 °C until use. In these conjugates, the Atto647-fluorophore is attached through a C6-linker to the 3′-end, through a C7-linker to the 5′-end, or through a C2-linker at two different thymine bases dT (italicized) of the 24mer oligonucleotide cA24 (sequence: 5′-AGC GGA TAA CAA TTT CAC ACA GGA-3′), which is fully complementary to sequence A24, leading to four different conjugates: 3′-Atto-cA24, 5′-Atto-cA24, dT1-Atto-cA24, and dT2-AttocA24 (Scheme 2). According to manufacturer’s information, the DNA oligomers contained one Atto647 dye per DNA oligomer. Analysis of the Atto-labeled oligonucleotides by MALDI-TOF mass spectrometry indicated the expected molecular weights with slight deviations of less than 2%. The 1:1 stoichiometric ratio was additionally confirmed, on one hand, by quantifying the absorbances of the Atto647-cA24 conjugates at both 260 and 649 nm and the independent calculation of conjugate concentrations. On the other hand, the Atto647-cA24 conjugates were titrated in the course of the assembly of the FRET system with the complementary CysEYFP-A24. These titrations showed a maximum in the quenching of the EYFP fluorescence as well as in the FRET-based Atto647 emission at the stoichiometric ratio of 1:1, thus confirming the 1:1 stoichiometry of both conjugates (data not shown). Assembly of FRET Systems. The four different supramolecular FRET systems with differing numbers of base pair separations (FS0bp-24bp, Scheme 2) were assembled by hybridization of CysEYFP-A24 with the Atto647-labeled oligomers, such that the variation in donor and acceptor distance should be reflected in different FRET efficiencies. To this end, the CysEYFP-A24 conjugate and Atto-oligomers were mixed in TETBS at a final concentration of 0.5 µM. For higher hybridization efficiencies, the samples were heated up to 50 °C for 5 min, slowly cooled to room temperature, and then stored at 4 °C until used in PLE measurements. As controls, the single fluorophore DNA conjugates were hybridized with complementary unlabeled DNA, and an EYFP-DNA conjugate (CysEYFP-cA24) containing the same DNA sequence as the Atto647-DNA conjugates was incubated with the Attooligomer species. 2D-PLE Measurements. For measuring the PLE spectra, the sample was illuminated using a 150 W xenon lamp spectrally filtered by a 1 m monochromator. The collected luminescence was spectrally dispersed in a 0.25 m spectrometer and recorded with an optical multichannel analyzer, yielding spectra over the full visible wavelength range. These spectra were then normalized to the photon flux at the excitation wavelength, which was monitored by a photodiode. The excitation wavelength was scanned over 240 nm with a step size of 1 nm. All signals were corrected with respect to the spectral sensitivity of both the excitation monitoring and the fluorescence detection systems.
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Scheme 2
We present the PLE signals of the individual samples as twodimensional, color-coded plots of the emission intensity, spectrally dispersed along the x-axis, while the y-axis represents the different excitation wavelengths. The diagonal line, stemming from scattered excitation light, indicates the resonance case of excitation energy being equal to the detection energy. 2D-PLE of the Fluorescent Protein EYFP. In order to establish the major constituent of our FRET system, we initially investigated the internal energy relaxation pathway of the EYFP. An ideal donor system, in general, should emit at a single, homogeneously broadened ground-state energy with intensities proportional to the density and oscillator strength of the absorbing states of the corresponding donor absorption spectrum. The photoluminescence (PL) line width of the emitting state should not be influenced by an internal fine structure of the optical ground state, which would cause inhomogeneous broadening by a superposition of several optically allowed and dark states. Figure 1 shows the measured 2D-PLE intensity map of solely the EYFP-A24 donor conjugate. The excitation wavelength was tuned from short excitation wavelengths/high energies (460 nm/2.694 eV) toward longer wavelengths/lower
energies (535 nm/2.316 eV) in steps of 1 nm while recording for each excitation the corresponding emission spectrum within a detection range between 500 and 620 nm. As shown in Figure 1 (left), the EYFP emission has a maximum at 528 nm with an asymmetric line shape showing a long spectral tail toward longer wavelengths, reaching the zero level around 650 nm. The signal on the upper left side of the diagonal line might indicate the presence of excited vibrational degrees of freedom in the sample, that contribute to the complete energy balance for radiative recombination. This observation is not affected by DNA, since it is observed in samples consisting of the fluorophores only. To analyze the PL line shape as a function of excitation energy, we normalized the full emission spectra with respect to the number of exciting photons derived from the absorption strength at the corresponding wavelength measured before in the same experimental setup. The result is shown in Figure 1 (right). It confirms that the shape of the emission spectrum is independent of the excitation energy, thereby ruling out a fine structure that may interfere with an energy transfer signature in the 2D-PLE plot of the combined donor-acceptor system.
Figure 1. Left: 2D-PLE of the EYFP donor in color-coded (linear) intensities covering a range from 0 (blue) to 50 000 (red) counts. Right: 2D-PLE of the EYFP donor normalized with respect to the number of absorbing states showing an excitation-independent emission line shape and emission maximum. The color-coded (linear) intensity covers a range from 0 (blue) to 1 (red). The intensity which is recorded as the diagonal signal arises from scattered excitation light.
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Figure 2. 2D-PLE signals of the different FRET systems FS0bp, FS10bp, FS17bp, and FS24bp with the corresponding donor-acceptor distances. The signature at the upper right corner of each graph arises from the Atto647 acceptor, and the signature at the lower left corner from the EYFP donor. The off-diagonal signature at the acceptor emission energy (lower right corner) shows the emission due to transfer. A distance-dependent quenching of the donor emission is accompanied by an enhanced acceptor PL.
RESULTS AND DISCUSSION 2D-PLE of the Donor-Acceptor System EYFP-Atto647. In the four different supramolecular FRET systems (FS0bp-24bp; for a schematic representation of the structures, see Scheme 2), the donor and acceptor distance was increased stepwise from 0 over 10, 17, to 24 base pairs (bp), by varying the binding site of the Atto dye on the DNA in the corresponding Atto647DNA conjugates. As will be discussed below, these variations in the number of base pairs leads to donor-acceptor distances of 4.17, 5.38, 6.39, and 8.37 nm, respectively. The 2D-PLE spectra of these four different supramolecular FRET systems are shown in Figure 2. In case of a too large donor-acceptor distance, no transfer was expected, and the 2D-PLE signals of donor and acceptor should appear as two independent features, namely, the above-discussed 2D-PLE of the control EYFPA24 in addition to a similar spectral feature of the acceptor Atto647. Indeed, such a pattern was clearly observed for FS24bp, with a donor-acceptor separation of 8.37 nm. If the donoracceptor pair is separated by a distance that allows for significant FRET, a third feature should be visible in the PLE plot. It appears as PL intensity at the spectral position of the acceptor’s preferential recombination wavelength, while exciting the system at the wavelength of maximum donor absorption (see the 2DPLE plot for FS0bp; Figure 2). The strength of that off-diagonal signal is considered to be the fingerprint of energy transfer. For a quantitative analysis, the transfer efficiency is extracted from the luminescence intensity F of both emitting species, analyzing either the intensity at the peak positions or the volume under the curve down to 50% of the maximum. The transfer efficiency E is then extracted as E ) [1 + (FDηA/FAηD)]-1. For the FRET systems shown in Figure 2, we obtained values for E of 0.79, 0.53, 0.26, and 0.074 for FS0bp, FS10bp, FS17bp, and FS24bp, respectively. More obvious information about the energy transfer pathways and the absolute transfer efficiency can be obtained by
transforming the PLE signals into a loss/gain statistic by subtracting the intensities of the control samples, i.e., the hybridized samples of the individual fluorophores CysEYFPA24 + bcA24 and Atto647-cA24 + bA24 (see Figure 3). Figure 3 shows the spectral window of the differential 2DPLE around the donor absorption. The violet color code indicates fluorescence quenching, while the red color code corresponds to absolute gain. With increasing donor-acceptor distance, the donor emission becomes less quenched while the acceptor gain decreases until the transfer is completely hindered by a too large donor-acceptor separation. In case the EYFP was quenched via another competitive channel without significant transfer to the Atto647 acceptor, we would expect a strong negative (violet) signal at the donor emission in the differential 2D-PLE plot but no (or weak) positive (red) signal at the acceptor energy. Since such a feature and also a significant gain at another spectral position was not observed, we again concluded that the energy of the EYFP donor is directly transferred to the Atto647 acceptor in the FRET systems FS0bp, FS10bp, and FS17bp. FRET Efficiencies and Determination of the DonorAcceptor Distance. To estimate the distance between the donor and the acceptor, one has to take into account the helical nature of double-stranded DNA. Under the conditions used in our experiments, we expect the DNA to adopt the regular conformation of the right-handed B-DNA helix. In this conformation, the DNA has a diameter of 2 nm, the increase in height per base pair is 0.34 nm, and ten base pairs comprise one entire helical turn. To confirm the assumption that the DNA in the FRET systems is indeed in the B-form conformation, we measured CD spectra of FS0bp and a sample containing an equimolar mixture of unlabeled A24 and cA24 (Figure 5). Both curves display strong negative and positive cotton effects around 250 and 280 nm, respectively. This pattern is characteristic for B-form DNA and is in agreement with previously reported similar systems (16). Since the persistence length of duplex
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Figure 3. Differential 2D-PLE spectra obtained by subtracting the PLE signals of the control samples (isolated, hybridized donor and acceptor) from the PLE of the FRET systems. The linear color scale shows quenching (violet), enhancement (red), and constancy (green) of the emission intensities on a scale corresponding to -50% to +50% for the top row and -10% to +10% for the bottom row.
and acceptor pair. According to Fo¨rster theory, the Fo¨rster radius R0 can be calculated by
R06 ) 8.79‚10-5‚[QDκ2n-4J]
Figure 4. Measured donor emission spectrum (black) and acceptor absorbance (red). The blue curve shows the resulting overlap integral kernel yielding J ) 2.09‚1015 nm4.
Figure 5. CD spectra of FRET system FS0bp (solid curve) and an equimolar mixture of unlabeled A24 and cA24 (dotted line).
DNA is about 50 nm (31), the 24 bp dsDNA system with a nominal length of 8.16 nm studied here can be regarded as a rigid-rod linear system. Donor and acceptor positions were therefore represented as vectors in a cylindrical coordinate system (32) with the symmetry axis of the DNA as the z-axis and the DNA-donor link defining the direction of the x-axis (see schematic representation in Figure 6). The absolute value of the donor and acceptor connecting vector then represents the distance. To calculate the values RD and RA for the distances between the z-axis and the donor and acceptor, respectively, one first needs to estimate the Fo¨rster distance for the donor
yielding R0 in Å with J in M-1 cm-1(nm) (4). QD is the quantum efficiency of the donor, κ2 ) 2/3 of the orientational factor, and n ) 1.33 is the refractive index of the medium. J is the spectral overlap integral, which can be derived from the experimentally obtained absorption and emission spectra. The Fo¨rster radius R0 is the distance at which the energy transfer efficiency is 50%. For the calculation of J, the measured donor emission spectrum was normalized to an area of 1 on a wavelength scale (see Figure 4). It was multiplied by the measured acceptor’s extinction, taking into account the optical path length within the cuvette as well as the concentration. After multiplication with the fourth power of the wavelength, the result was numerically integrated to obtain J ) ∫fDon(λ)Acc(λ)λ4 dλ. Using the experimentally determined J, we find R0 ) 5.38 nm as a first estimate for the Fo¨rster radius. Correlation of the cylindrical model with the data measured for the FRET efficiencies (Figure 2) and the calculated R0 allows one to calculate the values of RD ) 2.8 nm and RA ) 1.38 nm and to translate the number of base pairs between donor and acceptor into metric values of 4.17, 5.38, 6.39, and 8.37 nm for the FS0bp-24bp. To calculate the transfer efficiencies from the maximal emission intensities of donor and acceptor upon donor excitation FD and FA, respectively, we took quantum efficiencies of 20% for the Atto647 (according to information of the manufacturer) and 62% for the EYFP (33) into account via E ) [1 + (FDηA/FAηD)]-1. The independence of the PL line shape from the excitation wavelength ensures equal results when using peak intensities or volumes under the 2D-PLE intensity plots (not shown). Figure 6 illustrates the derived change in donor-acceptor distance when increasing the number of base pairs (bp). The closest distance is found for a distance of approximately 5 bp. In this FRET system, the donor would just sit on top of the acceptor, having the same angular coordinates. The minimum distance for 0 bp separation is determined by the diameter of the DNA cylinder and the approximate length of the linking molecules, as well as the distance from the EYFP chromophore inside of the protein structure to the surface. The measured transfer efficiencies in dependence of the donor-acceptor distance is displayed in Figure 7. The data points are in very good agreement with a Fo¨rster-type curve for a distance-
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Figure 6. Cylindrical model of the FRET systems and translation of base pair separation into metric distances. Left: Scheme of the different arrangements of donor and acceptor in the four FRET systems depending on the attachment site of the Atto dye on the DNA. Right: The donoracceptor distance as a function of the number of base pair separation between the two fluorophores. The vectors on top sketch the angular orientation.
PROTEOMICS project by the Ministry of Innovation and Research of the state Northrhine Westfalia.
LITERATURE CITED
Figure 7. Transfer efficiencies deduced from PL intensities as a function of donor-acceptor distance (black dots) and expected transfer efficiencies calculated from Fo¨rster theory (red curve).
dependent energy transfer, proving that DNA-fluorescent protein based supramolecular FRET systems can be rationally designed for generating energy transfer systems with predictable optical properties.
CONCLUSION We reported here on four supramolecular FRET systems which were obtained by hybridizing the CysEYFP-DNA conjugates with the complementary Atto647-cDNA conjugates. The donor-acceptor distance was altered in a controlled way by variation of the number of base pairs separating the donor EYFP and the acceptor dye Atto647. The interchromophore distances were calculated within a cylinder model of the DNA helix yielding 4.17, 5.38, 6.38, and 8.37 nm for the used separations of 0, 10, 17, and 24 base pairs, respectively. For these donor-acceptor distances, the energy transfer was evidenced in 2D-PLE spectra which clearly showed the loss/gain statistics. The calculated values for the FRET efficiencies and the Fo¨rster radius are in good agreement with the experimentally obtained values. We anticipate that the molecular construction approach reported here should allow one to rationally assemble more complex, nanoscaled optical systems by taking advantage of the incredibly diverse physicochemical properties of fluorescent proteins.
ACKNOWLEDGMENT This work was supported by Deutsche Forschungsgemeinschaft (DFG, grants Ni-399/6-1/6-2) and by funding of the
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