Bioconjugate Chem. 2010, 21, 921–927
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Conjugation of Fluorescent Proteins with DNA Oligonucleotides Vidmantas Lapiene, Florian Kukolka,* Kathrin Kiko, Andreas Arndt, and Christof M. Niemeyer* Technische Universita¨t Dortmund, Fakulta¨t Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, D-44227 Dortmund, Germany. Received October 28, 2009; Revised Manuscript Received March 1, 2010
This work describes the synthesis of covalent ssDNA conjugates of six fluorescent proteins, ECFP, EGFP, E2GFP, mDsRed, Dronpa, and mCherry, which were cloned with an accessible C-terminal cystein residue to enable siteselective coupling using a heterobispecific cross-linker. The resulting conjugates revealed similar fluorescence emission intensity to the unconjugated proteins, and the functionality of the tethered oligonucleotide was proven by specific Watson-Crick base pairing to cDNA-modified gold nanoparticles. Fluorescence spectroscopy analysis indicated that the fluorescence of the FP is quenched by the gold particle, and the extent of quenching varied with the intrinsic spectroscopic properties of FP as well as with the configuration of surface attachment. Since this study demonstrates that biological fluorophores can be selectively incorporated into and optically coupled with nanoparticle-based devices, applications in DNA-based nanofabrication can be foreseen.
INTRODUCTION The investigation of the fundamental principles of photosynthesis is of great current interest. In this context, the synthesis of artificial light harvesting devices from multiple chromophoric units and the study of energy transfer processes occurring in these systems aims to mimic natural photosynthesis and to develop materials and devices with complex spectroscopic properties. Recent approaches to assemble light harvesting complexes included the use of small molecule chromophors, such as porphyrin derivatives (1), polypyridine complexes of d6 metal ions (2), perylene-based polyphenyl dendrimers (3, 4), or supramolecular assemblies of functional dyes (5). On the other hand, significant research is currently devoted to the exploitation of biomolecules for the assembly of functional devices. In this young field of science, the bioconjugation of short DNA oligonucleotides to various molecular compounds and materials has already been established as a means to fabricate nanoscaled functional devices (6). The approach is based on the extraordinary specific molecular recognition properties of short ssDNA oligonucleotides, which can be harnessed as structure-directing agents for the rational assembly of proteins, nanoparticles, and other components (7-9). In view of synthetic energy transfer systems, the naturally occurring family of fluorescent proteins (FPs) is currently attracting increasing interest (6). Starting with the initial discovery of naturally occurring Aequorea Victoria green fluorescent protein (GFP) (10), nowadays a large number of structural variants of GFP are available and they are routinely used as genetically encoded markers for cell biology and the study of biomolecular interactions by means of Fo¨rster resonance energy transfer (FRET) (11-14). Moreover, FPs are currently being exploited in materials research as chromophors for the fabrication of nanoscaled optically addressable devices. For instance, FPs have been used to decorate semiconductor nanoparticles, often termed “quantum dots” (QD), to study FRET mechanisms occurring in such light-addressable nanodevices (15-19). Also, it has recently been demonstrated that the color of natural fluorescent proteins can be controlled by photonic crystals (20).
In these examples, the decoration of the inorganic materials with FPs was achieved by adsorption and electrostatic interactions (21), thus allowing only limited control of the assembly process. To extend controlability of supramolecular FP assembly and enable rational design of FP-based FRET systems, we have previously established the synthesis of a covalent ssDNA conjugate, using the enhanced yellow fluorescent protein (EYFP) (22). The use of this conjugate indeed enabled the DNA-based assembly of FRET systems, in which the spatial proximity of the chromophors can be controlled by the helicity of the dsDNA carrier backbone (23, 24). Moreover, the ssDNA-EYFP conjugate was used to explore FRET processes in threechromophore QD assemblages (17) and to investigate particleprotein interactions, taking advantage of EYFP fluorescence emission quenching by gold nanoparticles (25). Very recently, a ssDNA conjugate of the photoswitchable FP Dronpa, one of the conjugates developed in the course of the work reported here, was used in a single-molecule FRET study (26). In this full paper, we describe the synthesis of six novel DNA oligonucleotide-FP conjugates, based on mutant variants of the native FPs (Figure 1). In particular, the fluorescent proteins ECFP, a cyan mutant of jellyfish Aequorea Victoria GFP (27), EGFP (28), E2GFP (29), mDsRed, a monomeric variant of tetrameric DsRed (30, 31), available from Clontech, photoswitchable Dronpa (32), and mCherry (33) were cloned with an N-terminal hexahistidine sequence and a C-terminal cystein residue. The engineered cystein residue was positioned at the C-terminal region of the FPs because known crystal structures suggest that this region is easily accessible for chemical conjugation. Indeed, all six proteins could be derivatized with maleimide-activated DNA oligomers to yield the desired ssDNA-FP conjugates. These hybrid molecules were characterized and then used for the investigation of fluorescence emission quenching as a result of their specific hybridization with ssDNAmodified gold nanoparticles (DNA-AuNP), bearing the complementary oligonucleotides. Determination of hybridization kinetics and quenching efficiency revealed slight but distinct differences between the six DNA-FP conjugates, as a consequence of the optical and steric properties of the individual FPs.
EXPERIMENTAL PROCEDURES * Telefax: Int. + 49 (0)231/755 7082, E-mail: christof.niemeyer@ tu-dortmund.de.
Molecular cloning and overexpression of the recombinant FPs was carried out similar to previously described methods (22, 34).
10.1021/bc900471q 2010 American Chemical Society Published on Web 04/30/2010
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Figure 1. Schematic representation of the cross-linking of alkylamino-modified DNA with mutant CysFP by initial maleimide activation of the DNA cA24 and subsequent reaction with the genetically engineered cystein residue of the FP. Table 1. Spectroscopic Properties and Yields Obtained from Overexpression and Covalent ssDNA-Conjugation of the Various Fluorescent Proteins Described in This Study entry
protein
λExc [nm]
λEM [nm]
vector
expression yield [µg/mL]
conjugation yield [%]a
1 2 3 4 5 6
ECFP-Cys EGFP-Cys Dronpa-Cys E2GFP-Cys mDsRed-Cys mCherry-Cys
434 488 503 514 558 587
476 508 518 528 583 615
pQE30 pQE30 pQE30 pQE30 pQE30 pEXP17-N-mCherry-Cys
76 156 258 71 215 105
33 22 28 36 18 15
a
Calculated with respect to the amount of ssDNA oligonucleotide.
Synthesis of ssDNA-FP conjugates was achieved from 5′alkylamino-modified DNA using the heterobispecific crosslinker sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane1-carboxylate (sSMCC) (22). Preparation of 23 nm diameter ssDNA-modified gold nanoparticles and hybridization with ssDNA-FP conjugates was carried out as described before (25). Full details on cloning and expression of FPs, experimental protocols for conjugate synthesis, and binding studies with DNA-AuNP are available in the Supporting Information.
RESULTS AND DISCUSSION To facilitate site-selective conjugation with DNA oligonucleotides, we followed the bioconjugation approach previously explored for the coupling of the fluorescent protein EYFP with alkylamino-modified DNA (22). This method is based on the cloning and heterologous expression of a mutant FP, containing an N-terminal hexahistidine sequence and a C-terminal cystein residue. While the His6-tag allows for convenient purification of the recombinant FP, the accessible C-terminal Cys residue enabled conjugation with thiol-reactive DNA oligonucleotides. Genetic engineering of the cystein residue into the FP genes was achieved by PCR techniques. Detailed information on the cloning of the expression vectors is given in the Supporting Information. Transformation of E. coli BL21 and overexpression of the recombinant FPs was followed by Ni-NTA affinity chromatography. Expression yields obtained ranged 70-260 µg/ mL cell culture (Table 1). Gel electrophoretic analysis of the
recombinant FPs revealed that the proteins were sufficiently pure (>85%) to enable chemical coupling with DNA oligomers. Site-selective chemical coupling of the mutant FPs with DNA oligomers was achieved by using the heterobispecific crosslinker sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane1-carboxylate (sSMCC), which contains a thiol-reactive maleimide functionality in addition to an amino-reactive NHS group (Figure 1). To this end, commercially available alkylaminomodified oligonucleotide cA24 was reacted with an excess of sSMCC to install a maleimide group at the 5′-end of the oligomer. Subsequent to purification by gel filtration chromatography, the oligomer was allowed to react with the recombinant FP, and crude reaction products were purified by ultrafiltration and anion-exchange chromatography (Figure S1, in the Supporting Information). Isolated yields, determined by UV/vis spectroscopy after alkaline denaturation (35-37), ranging 15-36% (Table 1), thus indicating that the synthetic protocol allows ready production of the desired DNA-FP conjugates. Gel electrophoretic analysis of the conjugates (Figure 2) showed that the products essentially contained a single molecular species, with only slight impurities of the unconjugated protein. Denaturing SDS-gel electrophoresis indicated the expected increase in molecular weight of ∼8 kDa due to the attachement of one oligonucleotide per FP molecule (Figure 2A). This result was also obtained for Dronpa, which contains an additional Cys residue at position 113. We assume that the exclusive generation of the monoconjugated cA24-Dronpa is due to our optimized
Conjugation of FPs with DNA Oligonucleotides
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Figure 2. Gel electrophoretic analysis of recombinant FPs and their respective ssDNA conjugates. (A) 14% denaturing SDS polyacrylamide gel, stained with Coomassie: lane M, SDS Broadrange Marker (Bio-Rad); lane 1, EGFP; 2, cA24-EGFP; 3, E2GFP; 4, cA24-E2GFP; 5, ECFP; 6, cA24-ECFP; 7, mDsRed; 8, cA24-mDsRed; 9, Dronpa; 10, cA24-Dronpa; 11, mCherry; 12, cA24-mCherry. (B) 14% nondenaturing polyacrylamide gel after DNA staining with SybrGold: lane M, O’RangeRuler 50bp DNA ladder (Fermentas); lanes 1-12, same samples as in (A). Note that, although SybrGold dye is usually selective for nucleic acid staining, some FPs are visible as weak bands (e.g., lanes 1, 5, 7). This phenomenon has previously been observed (22), and it results from the native FP fluorescence at the filter settings used for SybrGold imaging (λExc ) 365 nm, λEm ) 520 nm, see also Figure S7 in the Supporting Information).
Figure 3. Fluorescence emission spectra of the DNA-FP conjugates cA24-ECFP (cyan), cA24-EGFP (brown), cA24-Dronpa (purple), cA24E2GFP green), cA24-mDsRed (red), and cA24-mCherry (pink). For comparison, the absorbance spectrum of AuNP is shown as the black curve.
reaction conditions (i.e., excess of protein and short reaction time) in addition to careful purification by gradient anionexchange chromatography. Analysis under nondenaturing conditions (Figure 2B) showed that the electrophoretic mobility of the DNA-FP conjugates was increased, as compared to the individual native FPs, due to the appended negatively charged oligonucleotide. The DNA-FP conjugates were analyzed by fluorescence spectroscopy (Figure 3), their fluorescence emission and UV/ vis absorbance spectra were compared to those of the native FPs (Figures S2, S3, in the Supporting Information), and quantum yields were determined (Table S3, in the Supporting Information). The spectroscopic properties of all DNA-FP conjugates were basically identical to those of the unconjugated FPs, thus confirming that (i) all FPs under investigation here were fully maturated and (ii) conjugation with ssDNA did not significantly affected the chromophore of the native FP. With the desired DNA-FP conjugates in hand, we investigated their nucleic acid hybridization with gold nanoparticles (AuNPs), containing the complementary ssDNA oligomer A24. It should be noted that the generation of protein-functionalized gold nanoparticles is of great current interest, due to their applications in biosensing and diagnostics (8, 9). While protein-AuNPs are long known as versatile probes in immunohistochemistry and related techniques (7), mixed hybrid nanoparticles containing both nucleic acid fragments and proteins have only recently been established (25, 38-42), because they offer additional functionality due to the presence of both classes of biomolecular recognition elements. We had previously demonstrated that such hybrid particles can be readily
prepared by specific DNA hybridization using DNA-AuNPs and appropriate ssDNA-protein conjugates bearing the complementary sequence (25, 38, 41, 43), and here we applied this method to investigate whether the various ssDNA-FP conjugates are functional with respect to both the FP fluorescence and their specific Watson-Crick hybridization capability. Similar to that reported for small-molecule organic fluorescent dyes (44-50), the FP fluorescence should be quenched upon binding to the AuNP, and it was interesting to see whether and how quenching would be influenced by the optical properties of the various FPs under investigation here. To this end, binding of the various covalent DNA-FP conjugates was investigated, using three different kinds of DNA-coated 23 nm AuNPs (Figure 4a). 5′A24-AuNP contained the complementary oligomer A24 bound via its 5′-end, while 3′A24-AuNP contained the A24 sequence attached via its 3′-end. Due to the directionality of the DNA double helical spacer, the distance between the gold surface and the FP is greater in the case of 5′A24-AuNP than for 3′A24-AuNP. Moreover, the noncomplementary oligonucleotide AuNP-dT124b was used as control. Gel electrophoretic analysis was used to initially confirm specificity of binding (Figure S4, in the Supporting Information). In the case of DNA-AuNPs bearing complementary oligomers, a decrease in electrophoretic mobility of the nanoparticle bands indicated formation of the respective FP-AuNPs, while incubation with dT124b-AuNP only revealed very small shifts of the particle bands (lanes 11-15, in Supporting Information Figure S4; see also Figure S6). This confirmed that the binding of all ssDNA-FP conjugates to the DNA-AuNPs primarily occurred due to specific Watson-Crick base-pairing of the oligonucleotides. We then investigated whether and to what extent the binding of the various ssDNA-FP conjugates to the DNA-AuNP leads to the quenching of FP fluorescence. In an initial set of experiments, solutions of the DNA-FP conjugate (1.2 pmol in TETBS buffer, pH 7.3, containing 150 mM NaCl) were mixed with solutions of the various DNA-AuNP (36 fmol in TETBS buffer containing 150 mM NaCl, corresponding to 32 mol equiv of DNA-FP to DNA-AuNP) and incubated for 6 h at room temperature. While the entire data is represented in Supporting Information Figure S4, it is examplified here for cA24-EGFP and cA24-mCherry (Figure 4b) that end-point determination of the fluorescence showed marked differences between the samples. The fluorescence of the pure ssDNA-FP conjugates (blue curves) is significantly decreased upon addition of noncomplementary dT124b-AuNP, and this effect occurrs due to strong absorption and scattering caused by the AuNPs. The extent of decrease differed clearly for the various FPs under investigation, which is a logical consequence of the different wavelenghts used for FP excitation. The absorption spectrum of AuNP (Figure 3) indicates that the particle’s broad plasmon
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Figure 4. AuNP-induced quenching of FP fluoresence. (A) Schematic representations of DNA-directed binding of ssDNA-FP conjugates to DNA-AuNPs and the DNA-AuNPs used for immobilization of cA24-FP conjugates to DNA-AuNPs and the DNA-AuNPs used for immobilization of cA24-FP conjugates. (B) End point determination of the fluorescence measured for cA24-EGFP and cA24-mCherry before (blue curves) and after incubation with 5′A24-AuNP (red), 3′A24-AuNP (green), or dT124b-AuNP (black). (C,D) Variation of coupling ratio FP/AuNP; fluorescence values of the ssDNA-FP conjugates after binding to 5′A24-AuNP (C) or 3′A24-AuNP (D) were normalized with respect to the fluorescence of analogous samples after incubation with noncomplementary dT124b-AuNP.
absorption has its maximum at about 540 nm. This wavelength almost coincides with the excitation/emission wavelenghts of mDsRed (558/583 nm). In contrast, plasmon absorption is significantly less effective at the excitation/emission wavelenghts of ECFP (434/476 nm), EGFP (488/508 nm), or mCherry (587/615 nm). Consequently, the amount of scattering induced by addition of dT124b-AuNP, and thus immediate decrease in FP fluorescence, is much higher for mDsRed than for ECFP, EGFP, or mCherry. Normalized values of ssDNA-FP conjugate’s fluorescence, measured in the presence of dT124b-AuNP, 5′A24-AuNP, or 3′A24-AuNP, are summarized in Table 2. To account for the aforementioned wavelength-dependent differences in scattering, fluorescence of the ssDNA-FP conjugate in the presence 5′A24AuNP or 3′A24-AuNP was normalized with respect to the value measured in the presence of noncomplementary dT124b-AuNP, set as 100%. It is clearly evident from the data of Table 2 as well as from the original data (Figure 4b and Supporting Information Figure S5) that addition of the complementary AuNPs led to a significantly higher quenching of the fluorescence of the FP, and this effect was markedly different for particles 5′A24-AuNP and 3′A24-AuNP (red and green curves, in Figure 4b). This result is in aggreement with the expectation that Watson-Crick hybridization with 5′A24-AuNP should lead to the formation of a 24 base-pair dsDNA spacer of approximately 8.5 nm in length between the protein and the gold
Table 2. Numerical Values of ssDNA-FP Conjugate Fluorescence, Measured in the Presence of Gold Nanoparticlesa entry
conjugate
dT124b-AuNPb
5′A24-AuNPc
3′A24-AuNPc
1 2 3 4 5 6
cA24-ECFP cA24-EGFP cA24-Dronpa cA24-E2GFP cA24-mDsRed cA24-mCherry
70 72 61 55 42 82
61 58 56 63 62 76
52 46 45 50 53 60
a End point determination after 6 h of incubation with 32 mol equiv of AuNP. b Normalized values [%] with respect to the fluorescence of pure cA24-FP in the absence of AuNP (100%). c Normalized values [%] with respect to the fluorescence of cA24-FP in the presence of noncomplementary dT124b-AuNP set as 100%.
surface, while the hybridization with 3′A24-AuNP should localize the protein in closer proximity to the particle’s surface. The difference in quenching efficiency observed for the two different configurations, however, is relatively small. This suggests that binding and quenching aspects are involved. The fundamental mechanisms of the quenching of organic fluorophoresbymetalnanoparticlesarestillunderinvestigation(44-50), and one important factor concerns the knowledge of real distances between the chromophore and the metal surface. The AuNP-bound DNA should be able to adopt a number of different conformations, such as being fully stretched and
Conjugation of FPs with DNA Oligonucleotides
pointing perpendicular from the surface, adopting a random coil shape, or possibly being tilted and interacting with the AuNP surface (51). Hence, the aforementioned nominal difference in distance of about 8.5 nm is most likely affected by the presence of various conformational states. Moreover, slight changes in protein loading should also induce different distances and/or different dynamics and, thus, might affect the quenching efficiency. To further analyze the system, we also studied the influence of surface coverage of AuNP with ssDNA-FP conjugate on the quenching efficiency. To this end, particles 5′A24-AuNP (Figure 4c) and 3′A24-AuNP (Figure 4d) were incubated with various molar equivalents of ssDNA-FP conjugate, and fluorescence emission was determined after 15 h. The fluorescence emission values measured were normalized to that of similar samples incubated with noncomplementary dT124b-AuNP. It is evident that the nature of FP did not markedly influence the stoichiometry of binding. In all cases, the fluorescence decreased for the binding of up to about 32 mol equiv of ssDNA-FP conjugate, and a further increase led to an increase in fluorescence, due to increasing amounts of free conjugate in the solution. It should be noted that the about 32 mol equiv of ssDNA-FP conjugate, which hybridizes to the 25 nm AuNP, correlates with an approximately 18% hybridization efficiency of the in total about 175 oligonucleotide molecules bound at the AuNP (see Supporting Information). This result is in good agreement with earlier studiesconcerningthehybridizationefficiencyofDNA-AuNPs(52,53). As expected, the extent of quenching differed significantly with the configuration of binding (Figure 4c vs d), and interestingly, the quenching efficiency was different for the various FPs studied (see also Table 2). In particular, strongest quenching was observed for binding of EGFP- and Dronpaconjugate to either 5′A24-AuNP or 3′A24-AuNP, and this maximum was slightly more pronounced in the case of 3′A24AuNP. The relative quenching efficiency observed for the six ssDNA-FP conjugates is in good aggreement with what one would expect from the overlap of FP fluorescence emission and AuNP plasmon absorption (Figure 3). It was also analyzed whether the two different configurations, realized in the binding to either 5′A24-AuNP or 3′A24AuNP, affect the kinetics of protein immobilization (Supporting Information Figure S5). Time-dependent fluorescence measurements showed that the ssDNA-FP conjugates rapidly hybridize with 5′A24-AuNP, and the reaction was completed within less than 4 h. The curves also suggest that the binding of ssDNA-FP to 3′A24-AuNP was slightly slower than binding to 5′A24-AuNP. This observation is in agreement with the proposed configuration, since binding to 3′A24AuNP requires the bulky FP to penetrate, at least partially, the dense layer of DNA oligomers attached to the particle’s surface. In conclusion, we have shown that fluorescent proteins can be effectively conjugated with DNA oligonucleotides, using a combination of genetic engineering and heterobispecific cross-linking chemistry. The resulting ssDNA-FP conjugates revealed similar spectroscopic properties than the unconjugated FPs. Moreover, the functionality of the tethered oligonucleotide was proven by specific Watson-Crick base pairing to cDNA-modified gold nanoparticles. Fluorescence spectroscopy analysis indicated that the FP fluorescence is quenched by the gold particle and the extent of quenching varied with the intrinsic spectroscopic properties of FP as well as with the configuration of surface attachement. Thus, this study demonstrates that these biological fluorophores can be incorporated into and optically coupled with nanoparticle-based devices using DNA-based assembly schemes. Given the enormous power of structural DNA nanotechnology and nanofabrication, we therefore
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anticipate that further elaboration on covalent DNA-FP conjugates might open up ways toward nanostructured materials with programmable functionalities for a broad range of applications in nanobiotechnology.
ACKNOWLEDGMENT This work was supported in part by Deutsche Forschungsgemeinschaft (DFG, grants Ni-399/6-1/6-2) Max-Planck Research School in Chemical Biology (IMPRS-CB), Dortmund, and the project SMD in the course of FP7-NMP-2008SMALL-2, founded by European Comission. We thank Martina Reibner for preparation of DNA-conjugated gold nanoparticles. We are also grateful to the reviewer for very important comments on the manuscript. Supporting Information Available: Full details on experimental protocols for molecular cloning of FPs, conjugate and nanoparticle synthesis, hybridization assays, characterization of ssDNA-FP conjugates, and spectroscopic analysis of their binding to DNA-AuNP. This material is available free of charge via the Internet at http://pubs.acs.org.
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