Communication pubs.acs.org/cm
Chemoenzymatic Sensitization of DNA Photonic Wires Mediated through Quantum Dot Energy Transfer Relays Chris L. Dwyer,† Sebastián A. Díaz,‡ Scott A. Walper,‡ Anirban Samanta,‡ Kimihiro Susumu,§ Eunkeu Oh,§ Susan Buckhout-White,‡ and Igor L. Medintz*,‡ †
Department of Electrical and Computer Engineering, Duke University, Durham, North Carolina 27708, United States Center for Bio/Molecular Science and Engineering, Code 6900 and §Optical Sciences Division, Code 5600, U.S. Naval Research Laboratory, Washington, DC 20375, United States
‡
S Supporting Information *
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se of nanomaterials in all their diversity is driven primarily by the desire to access some new or previously unavailable property/functionality. This is either achieved by creating a new material, for example carbon nanotubes or semiconductor quantum dots (QDs), or combining diverse materials into de novo hybrids which, in concert, provide utility unavailable to either constituent when acting independently. The latter is typified by the interfacing of biological structures with inorganic nanomaterials displaying unique quantum confined effects to form the basis of a new generation of sensors and probes, smart drug delivery agents, and autonomous nanomachines among many other predicted nanodevices. Accomplishing this still requires that these almost orthogonal materials be seamlessly integrated into cohesive functional units. One of the most basic abilities that creation of such standalone nanodevices will benefit from, and can be designed around, is the capability to autonomously generate light energy, harvest that energy, and then directly exploit it by spectrally and spatially propagating it in a controlled manner.1 With this goal in mind, we integrate the bioluminescent enzyme luciferase (Luc), QDs, and peptide-modified dye-labeled DNA photonic wires into structures that undergo directed selfassembly and functionally match these core requirements by engaging in a chemoenzymatically catalyzed bioluminescent resonance energy transfer-(BRET) multistep Förster resonance energy transfer (FRET) cascade, see Figure 1 for a schematic description. From a physicochemical perspective, QDs along with Luc and DNA-based photonic wires have much to offer for the current task. QDs provide numerous unique photophysical characteristics including broad-absorption coupled to size- and composition-tunable photoluminescence (PL) spectra spanning from the visible to the near infrared (NIR), high quantum yields (QYs), and strong resistance to photo/chemical degradation.1−5 Along with amenability to multicolor detection schemes, these cumulative capabilities inherently make QDs excellent FRET donors.1−5 Use of QDs as FRET acceptors is more challenging due to their long excited-state lifetimes coupled with their broad absorption peaks, which typically results in their being better excited than a potential dye donor. Long lifetime Tb-chelates and use of chemiluminescent donors have helped address this issue.3,6,7 Nontrivial QD size and large surface-to-volume ratios also allow them to act as effective central nanoassembly platforms for display of multiple biologicals such as proteins, peptides and DNA.2,4 © XXXX American Chemical Society
Figure 1. (A) Schematic of the self-assembled energy transfer system. Luc appended with terminal (His)6 ratiometrically coordinates, i.e., control over display valency, to the QD. Dye-labeled DNA wires are formed by prehybridization and include a terminal (His)5-peptidoDNA sequence to facilitate similar QD assembly. Functionally, Coel substrate is enzymatically oxidized by Luc giving rise to excitonic energy that sensitizes the proximal QD by BRET. The QD then redirects this energy and sensitizes the proximal dye on the DNA photonic wire giving rise to a sequential FRET cascade. Assembly number of Luc and photonic wires per QD can be controlled which, along with Coel concentration, provides control over energy transfer efficiency. (B) QD surface ligand structure in thioctic acid (TA) form. (C) Electrophoretic separation of Luc−QD constructs assembled with increasing ratios of Luc/QD (0 ∼ 15).
The Renilla reniformis Luc (EC # 1.13.12.5) utilized was mutationally optimized by Rao to improve performance as described.5 Luc oxidizes a variety of substrates to produce light with Coelenterazine H (Coel) being the preferred substrate utilized here, see Supporting Information (SI) for structure and reaction scheme. Luc catalysis of Coel produces a relatively short-lived electronically excited state and broad blue emission centered at 475 nm with a QY approaching ∼0.40,8 making it useful for sensitizing QDs emitting at ≥500 nm. Rao also pioneered Luc utility as a chemoenzymatic QD sensitizer demonstrating multicolor QD imaging in vivo along with active sensors.5 Maye focused on how QDs and semiconductor Received: July 25, 2015 Revised: September 1, 2015
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DOI: 10.1021/acs.chemmater.5b02870 Chem. Mater. XXXX, XXX, XXX−XXX
Communication
Chemistry of Materials nanorods could be sensitized by a similar enzyme and demonstrated that such chemocatalysis could drive a multistep BRET−FRET cascade through a QD intermediary to sensitize a terminal fluorescent protein.9 We demonstrated a BRET− FRET1−FRET2 construct using Luc to sensitize red-emitting QDs which, in turn, sensitized a FRET cascade through dye acceptors concentrically arranged around the nanocrystal.10 Energy transfer efficiency was modulated by the ratio of Luc or acceptor(s) arrayed per QD. Here, we define DNA photonic wires as a DNA scaffolding displaying multiple chromophores that interact with each other through energy transfer.1,11 Given their inherent ability to arrange individual chromophores with high density, close proximity, and 3-D structural complexity, such DNA architectures often serve as a nanoscale synthetic optical table for studying energy harvesting and FRET systems.1,11 Different fluorescent materials have already been incorporated into photonic wires including QDs, fluorescent proteins, longlifetime metal cryptates, and a plethora of organic dyes; the unique photophysical properties of these materials often permit fine-tuning of the energy transfer within the wires.1,11−13 Although many bioconjugation chemistries are available for engineering the current constructs, we again utilize directed self-assembly driven by polyhistidine (Hisn) metal-affinity coordination to the QD’s ZnS surface due to the ratiometric control which it affords over the final construct along with its simple implementation.4 To increase potential QD surface display capacity, the QDs were functionalized with dihydrolipoic acid (reduced form of TA)-modified nitrilotriacetic acid ligands (structure Figure 1B, synthesis SI), which provided an additional Hisn binding site at the terminus of each short ligand (length < 1.8 nm).14 Luc is expressed with a C-terminal hexahistidine (His6) facilitating purification over Ni2+−NTA resin and ratiometric QD assembly as verified by QD electrophoretic mobility shifts in an agarose gel (Figure 1C). Luc’s C-terminus provides some flexibility and movement when QD attached although we did not observe any separation differences between Luc attachment to the ZnS surface or the NTA-ligand terminus through comparative testing with other ligands (data not shown). Photonic wires were similarly attached to the QD by appending the DNA sequence located closest to the nanocrystal surface with a (His)5C-peptide. A 3′amine DNA modification was linked to the peptidyl cysteinethiol using the heterobifunctional cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, see SI). Luc and DNA valencey per QD was controlled through the molar average of each mixed with QD and constructs utilized directly without further purification.4 We estimate that the 540 nm emitting CdSe/CdZnS/ZnS core−shell−shell QDs15 (diameter ∼4.6 ± 0.4 nm, TEM in SI) can display ∼15 Luc (Mw ∼ 36 kDa, 5.2 × 7.6 × 8.9 nm, PDB entry 2PSH) or ∼40 linear DNA wires without significant steric hindrance. Constructs are described as Luc n −QD−DNA n with n designating the average number of Luc/DNA wires assembled per QD. Photophysically, Luc−QD−DNA constructs function via a BRET−FRET4 cascade. Initially, Coel is enzymatically oxidized by Luc to form an excited state that sensitizes the proximal QD. The QD then sensitizes the closest dye on the DNA wire giving rise to a directed FRET cascade through the remaining sequentially arrayed dyes, see Figure 1. This requires that Coel, QD, and dyes all display the necessary spectral overlap as shown in Figure 2 and SI Table 2. Dyes were chosen based on
Figure 2. Spectroscopic properties of the Luc−QD−DNA relay system. (A) Extinction coefficients and (B) emission spectra, normalized to component emitter QY. (C) Spectral overlap for nearest neighbor donor−acceptor pairs.
previous utilization in similar roles.10,12,13 DNA sequences were designed such that when hybridized together, dyes would be sequentially displayed in the order Cy3−Cy3.5−Alexa647− Cy5.5 proceeding from the QD surface, see SI Table 1. Wire length is estimated at ∼14 nm with ∼3.6 nm spacing between dyes corresponding to a range of ∼0.5−0.7 × R0 (Förster distance) for each sequential donor−acceptor pair.16 Estimated theoretical efficiencies are ≥90% for each sequential FRET step in the wire.3,4 Initial experiments focused on optimizing Luc−Coel catalytic sensitization of the 540 nm QDs. Increasing molar ratios of Luc to QD were assembled and spectra were collected following addition of 50-fold excess Coel/Luc with a 1 min sample equilibration as described unless noted,10 representative data in Figure 3A. Similar to other multidonor-single acceptor FRET systems, increasing the ratio of Luc−Coel donors/QD increases the probability that excitonic energy is transferred to the QD but not the underlying efficiency (assuming homogeneous separation) as only one excited-state Coel can couple to the QD dipole at one time.3,4 As Coel emission overlaps the QD’s absorption minima, sensitized QD emission is not as intense as noted before,10 and to compensate we utilize a reciprocal BRET ratio (ILuc/IQD).5,8−10 An average ratio of 7.7 ± 1.3 was derived across all the samples as shown in Figure 3B. 6 Luc/QD was utilized in subsequent experiments; this provides a value close to average while still leaving considerable QD surface available for displaying multiple DNA wires. Although Luc/QD assembly follows a Poisson distribution, the small changes in Figure 3B reflect that this has minimal impact on final energy transfer efficiency. Further empirical experiments were implemented with Luc6−QD and an increasing ratio of DNA wires displaying only the first Cy3 acceptor dye to optimize QD−Cy3 FRET1, see SI. Comparing the BRET-driven sensitization of Cy3 versus that of FRET by direct QD donor excitation (480 nm excitation corresponding to Luc) demonstrated that ∼8 DNA, and hence 8 Cy3, were optimal, manifesting high FRET efficiency (E ∼ 75 ± 5%, ∼ 7.3 ± 0.4 nm donor−acceptor separation distance, SI) while remaining within the linear response portion; this was B
DOI: 10.1021/acs.chemmater.5b02870 Chem. Mater. XXXX, XXX, XXX−XXX
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Chemistry of Materials
step) but less than the >20% achieved by a Pacific Blue → Cy3 dye-only system incorporating YO-PRO-1 intercalating dye as a homoFRET relay with more putative FRET steps.17 For the Luc contribution, we hypothesize that the 6 Luc/QD average leads to >95% of the QDs being BRET-sensitized during an assay. Having established a baseline view for Luc6−QD−DNA8 construct efficiency, some of the internal photophysical processes were next probed. The ratio of fully assembled wires/QD was modified from 2 to 10, see Figure 4A,B. An
Figure 3. Lucn−QD−DNAn spectral characterization. (A) Normalized emission spectra of Lucn−QD as the number of Luc/QD increased. Inset: deconvoluted Coel and QD components. (B) Luc to QD intensity ratio as a function of Lucn/QD. Data in B is derived from fluorescence spectra in part A. (C) Normalized spectra of Luc6−QD− DNA8 constructs as the displayed acceptor dyes are extended along the DNA from Cy3 up to Cy5.5 (→ = BRET/FRET step). (D) Deconvoluted Luc6−QD−DNA8 component spectra.
utilized in subsequent experiments unless noted. The increasing Cy3 dye surrounding a single QD donor proportionally increases the acceptor absorption cross section and allows increased tuning of FRET efficiency.3,4 Luc6−QD−DNA8 constructs were monitored as dye acceptor display on the wire evolved from just Cy3 to the full complement of 4 organic dyes. As seen in Figure 3C, despite the intense, broad Coel signal centered at 480 nm, peaks corresponding to each of the dye maxima appear in the composite spectrum as they are incorporated (QD-540 nm, Cy3-558 nm, Cy3.5-603 nm, Alexa647-663 nm, and Cy5.5-699 nm). Figure 3D presents the deconvoluted emission components for the QD and 4 dyes in the fully assembled construct. Efficiencies for the latter FRET transfer steps were good, though below the theoretical ≥90% values. For example, Figure 3C,D yields FRET efficiencies of 87, 83, 60, and 63% starting from the QD to Cy3 step and ending with the Alexa647 to Cy5.5 step, respectively. Clearly, chemically derived excitonic energy originating from Luc−Coel catalysis can be propagated through the QD by a BRET step and then to the terminal Cy5.5 dye by a 4-step sequential FRET cascade. End-to-end energy transfer efficiency (Eee) through the cascade was estimated with16,17 Eee = 100 × [(IAD − IA )/ΦA ]/(ID/ΦD)
Figure 4. Modifying Coel concentration and other variables in the Lucn−QD−DNAn system. (A) Emission of Luc−QD−DNAn as the number of wires/QD was modified. Inset: dye emission spectra. (B) Normalized emission of dyes (normalized to Alexa647 with 2 DNA/ QD) as the number of wires/QD increased. Solid lines, QD excitation by BRET; dashed lines, direct excitation of QD at 400 nm (FRET). (C) Normalized emission of the total photon counts (not deconvolved) from the Luc−Coel and QD as Coel concentration increased with linear fits. Note, slope of the Luc line is ∼2× slope of QD line. (D) Fluorescence emission of individual dyes within the Luc6−QD−DNA8 construct as Coel concentration increased.
optimal ratio of 8 wires/QD was observed coinciding with the results of the Cy3 acceptor-only format above (Figure 3,B) and confirming that i-DNA wire output efficiency was directly dependent on the efficiency of the first QD-dye FRET step; and ii-increasing wire number around the QD increases output efficiency by providing more viable acceptor cross section and transfer pathways and thus a higher probability of the exciton reaching the terminus.13 There is, however, a limit to accessing such increases as a ratio of 10 DNA/QD decreased dye output. As seen in Figure 4B, the decrease was independent of whether the original excitation occurred through a BRET or FRET mechanism. We hypothesize that this higher ratio brought about either steric constraints in the full construct, allowed some of the dyes to physically interact by homoFRET leading to competitive ineffectual FRET pathways, or created localized dark quencher duplexes or similar structures. Effects of modifying relative Coel concentration were also probed. Examining the simplified Luc6−QD system over a range of 10−100 Coel/Luc (Figure 4C) revealed that as the amount of substrate increased the overall emission of the system increased proportionally, yet BRET efficiency remained unchanged as Luc emission outpaced QD emission by almost 2-to-1 (of undeconvolved signal). This may again reflect that only one
(1)
where IAD, IA, and ID are the emission intensities of acceptor in the presence of donor, acceptor alone, and donor alone respectively, whereas ΦA and ΦD are the respective QY’s. Because of the complication of ascertaining Luc−Coel emission ID, we consider only the FRET components of the system, making the directly excited QD emission without dyes present ID. For Figure 3D, we estimate an Eee value of 9 ± 1% which, remarkably, appears unchanged regardless of whether the QD is directly excited or sensitized by Luc−Coel (SI Table S3). For the QD−Cy5.5 system, the 9% efficiency achieved here is comparable to that we previously described for an optimized QD → Cy3 → Cy3.5 → Cy5 → Cy5.5 system14 (→ = FRET C
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Semiconductor Quantum Dots in Biological Applications. Curr. Opin. Biotechnol. 2015, 34, 30−40. (3) FRET − Förster Resonance Energy Transfer From Theory to Applications; Medintz, I. L., Hildebrandt, N., Eds.; Wiley-VCH: Weinheim, Germany, 2013. (4) Blanco-Canosa, J.; Wu, M.; Susumu, K.; Petryayeva, E.; Jennings, T. L.; Dawson, P. E.; Algar, W. R.; Medintz, I. L. Recent Progress in the Bioconjugation of Quantum Dots. Coord. Chem. Rev. 2014, 263− 264, 101−137. (5) So, M. K.; Xu, C. J.; Loening, A. M.; Gambhir, S. S.; Rao, J. H. Self-Illuminating Quantum Dot Conjugates For In Vivo Imaging. Nat. Biotechnol. 2006, 24, 339−343. (6) Charbonnière, L. J.; Hildebrandt, N. Lanthanide Complexes and Quantum Dots: A Bright Wedding for Resonance Energy Transfer. Eur. J. Inorg. Chem. 2008, 2008, 3241−3251. (7) Huang, X.; Li, L.; Qian, H.; Dong, C.; Ren, J. A Resonance Energy Transfer between Chemiluminescent Donors and Luminescent Quantum-Dots as Acceptors (CRET). Angew. Chem., Int. Ed. 2006, 45, 5140−5143. (8) Ando, Y.; Niwa, K.; Yamada, N.; Enomoto, T.; Irie, T.; Kubota, H.; Ohmiya, Y.; Akiyama, H. Firefly Bioluminescence Quantum Yield and Colour Change by pH-Sensitive Green Emission. Nat. Photonics 2008, 2, 44−47. (9) Alam, R.; Zylstra, J.; Fontaine, D. M.; Branchini, B.; Maye, M. M. Novel Multistep BRET-FRET Energy Transfer using Nanoconjugates of Firefly Proteins, Quantum Dots, and Red Fluorescent Proteins. Nanoscale 2013, 5, 5303−5306. (10) Samanta, A.; Walper, S. A.; Susumu, K.; Dwyer, C. L.; Medintz, I. L. An Enzymatically-Sensitized Sequential and Concentric Energy Transfer Relay Self-Assembled Around Semiconductor Quantum Dots. Nanoscale 2015, 7, 7603−7614. (11) Albinsson, B.; Hannestad, J. K.; Borjesson, K. Functionalized DNA Nanostructures for Light Harvesting and Charge Separation. Coord. Chem. Rev. 2012, 256, 2399−2413. (12) Massey, M.; Ancona, M. G.; Medintz, I. L.; Algar, W. R. TimeGated DNA Photonic Wires with Förster Resonance Energy Transfer Cascades Initiated by a Luminescence Terbium Donor. ACS Photonics 2015, 2, 639−652. (13) Buckhout-White, S.; Spillmann, C. M.; Algar, W. R.; Khachatrian, A.; Melinger, J. S.; Goldman, E. R.; Ancona, M. G.; Medintz, I. L. Assembling Programmable FRET-Based Photonic Networks Using Designer DNA scaffolds. Nat. Commun. 2014, 5, 5615. (14) Susumu, K.; Oh, E.; Delehanty, J. B.; Blanco-Canosa, J. B.; Johnson, B.; Jain, V.; Hervey, W. J.; Algar, W. R.; Boeneman, K.; Dawson, P.; Medintz, I. L. Multifunctional Compact Zwitterionic Ligands for Preparing Robust Biocompatible Semiconductor Quantum Dots and Gold Nanoparticles. J. Am. Chem. Soc. 2011, 133, 9480− 9496 (13). (15) Susumu, K.; Oh, E.; Delehanty, J. B.; Pinaud, F.; Gemmill, K. B.; Walper, S.; Breger, J.; Schroeder, M. J.; Stewart, M. H.; Jain, V.; Whitaker, C. M.; Huston, A. L.; Medintz, I. L. A New Family of Pyridine-Appended Multidentate Polymers as Hydrophilic Ligands for Preparing Stable Biocompatible Quantum Dots. Chem. Mater. 2014, 26, 5327−5344. (16) Spillmann, C. M.; Ancona, M. G.; Buckhout-White, S.; Algar, W. R.; Stewart, M. H.; Susumu, K.; Huston, A. L.; Goldman, E. R.; Medintz, I. L. Achieving Effective Terminal Exciton Delivery in Quantum Dot Antenna-Sensitized Multistep DNA Photonic Wires. ACS Nano 2013, 7, 7101−7118. (17) Hannestad, J. K.; Sandin, P.; Albinsson, B. Self-Assembled DNA Photonic Wire for Long-Range Energy Transfer. J. Am. Chem. Soc. 2008, 130, 15889−15895. (18) Buckhout-White, S.; Claussen, J. C.; Melinger, J. S.; Dunningham, Z.; Ancona, M. G.; Goldman, E. R.; Medintz, I. L. A Triangular Three-Dye DNA Switch Capable of Reconfigurable Molecular Logic. RSC Adv. 2014, 4, 48860−48871. (19) Johnson, B. J.; Algar, W. R.; Malanoski, A. P.; Ancona, M. G.; Medintz, I. L. Understanding Enzymatic Acceleration at Nanoparticle
excited-state Coel can sensitize the central QD at any one time given dipole−dipole coupling constraints.3,4 Emission from each of the DNA dye components also increased as the amount of substrate increased, see Figure 4D. This data revealed that Cy3 and Cy3.5 emission increased much faster than the other components. Comparing excesses of 64× to 16× Coel/Luc, QD emission increases ∼30-fold whereas Cy3/Cy3.5 emission increases ∼50-fold. This likely arises from the increasing number of excited-state Coel which can directly excite the dyes: Cy3 and Cy3.5 have significant spectral overlap with Coel emission (SI Table 2). Although this represents just an initial foray into coupling enzymatic BRET with QD−DNA photonic wire FRET cascades, the energy transfer observed in the construct is promising. As long as the QD is sufficiently excited by BRET or direct excitation, downstream energy transfer efficiency appears comparable showing the QD to be the key intermediary. Further improvements may be realized by optimizing QD and dye choice to enhance Förster coupling. DNA wires displaying multiple copies of donor−acceptor fluorophores may help improve Eee by providing multiple-overlapping FRET pathways for exciton travel.13 O2 supplementation may also help as it is required for Coel catalysis and can be limiting due to its low solubility in aqueous buffers.5,8−10 It is also unclear if Luc demonstrates the catalytic improvements noted for other enzyme systems when displayed at a nanoparticle interface.19 Experiments are planned to understand the primary causes of energy loss in the cascade. Potential applications, even for this rudimentary first generation system, may include providing activation and chemically controlled access to FRET-based DNA−dye Boolean logic systems.18 Overall, this confirms that inorganic nanoparticles can be combined with enzymes, DNA, peptides, and dyes using simple chemistry to give rise to de novo designer integrated nanoconstructs capable of self-generating, harvesting, and directing their own light energy.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.5b02870. DNA sequences, ligand synthesis, experimental details and supporting data/results (PDF).
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AUTHOR INFORMATION
Corresponding Author
*I. L. Medintz. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS CLD acknowledges an NRL Summer Faculty Fellowship and the National Security Science and Engineering Faculty Fellowship # N00014-15-1-0032. IM acknowledges the NRL Nanosciences Institute, and DTRA JSTO MIPR # B112582M.
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REFERENCES
(1) Spillmann, C. M.; Medintz, I. L. Use of Biomolecular Scaffolds for Assembling Multistep Light Harvesting and Energy Transfer Devices. J. Photochem. Photobiol., C 2015, 23, 1−24. (2) Massey, M.; Wu, M.; Conroy, E. M.; Algar, W. R. Mind Your P’s and Q’s: The Coming of Age of Semiconducting Polymer Dots and D
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Chemistry of Materials Interfaces: Approaches and Challenges. Nano Today 2014, 9, 102− 131.
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