Pursuing excitonic energy transfer with programmable DNA-based optical breadboards.

DNA nanotechnology has now enabled the self-assembly of almost any prescribed 3-dimensional nanoscale structure in large numbers and with high fidelity. These structures are also amenable to site-specific modification with a variety of small molecules ranging from drugs to reporter dyes. Beyond obvious application in biotechnology, such DNA structures are being pursued as programmable nanoscale optical breadboards where multiple different/identical fluorophores can be positioned with sub-nanometer resolution in a manner designed to allow them to engage in multistep excitonic energy-transfer (ET) via Förster resonance energy transfer (FRET) or other related processes. Not only is the ability to create such complex optical structures unique, more importantly, the ability to rapidly redesign and prototype almost all structural and optical analogues in a massively parallel format allows for deep insight into the underlying photophysical processes. Dynamic DNA structures further provide the unparalleled capability to reconfigure a DNA scaffold on the fly in situ and thus switch between ET pathways within a given assembly, actively change its properties, and even repeatedly toggle between two states such as on/off. Here, we review progress in developing these composite materials for potential applications that include artificial light harvesting, smart sensors, nanoactuators, optical barcoding, bioprobes, cryptography, computing, charge conversion, and theranostics to even new forms of optical data storage. Along with an introduction into the DNA scaffolding itself, the diverse fluorophores utilized in these structures, their incorporation chemistry, and the photophysical processes they are designed to exploit, we highlight the evolution of DNA architectures implemented in the pursuit of increased transfer efficiency and the key lessons about ET learned from each iteration. We also focus on recent and growing efforts to exploit DNA as a scaffold for assembling molecular dye aggregates that host delocalized excitons as a test bed for creating excitonic circuits and accessing other quantum-like optical phenomena. We conclude with an outlook on what is still required to transition these materials from a research pursuit to application specific prototypes and beyond.

Self assembling nanoparticle enzyme clusters provide access to substrate channeling in multienzymatic cascades.

Access to efficient enzymatic channeling is desired for improving all manner of designer biocatalysis. We demonstrate that enzymes constituting a multistep cascade can self-assemble with nanoparticle scaffolds into nanoclusters that access substrate channeling and improve catalytic flux by orders of magnitude. Utilizing saccharification and glycolytic enzymes with quantum dots (QDs) as a model system, nanoclustered-cascades incorporating from 4 to 10 enzymatic steps are prototyped. Along with confirming channeling using classical experiments, its efficiency is enhanced several fold more by optimizing enzymatic stoichiometry with numerical simulations, switching from spherical QDs to 2-D planar nanoplatelets, and by ordering the enzyme assembly. Detailed analyses characterize assembly formation and clarify structure-function properties. For extended cascades with unfavorable kinetics, channeled activity is maintained by splitting at a critical step, purifying end-product from the upstream sub-cascade, and feeding it as a concentrated substrate to the downstream sub-cascade. Generalized applicability is verified by extending to assemblies incorporating other hard and soft nanoparticles. Such self-assembled biocatalytic nanoclusters offer many benefits towards enabling minimalist cell-free synthetic biology.

Understanding Förster Resonance Energy Transfer in the Sheet Regime with DNA Brick-Based Dye Networks.

Controlling excitonic energy transfer at the molecular level is a key requirement for transitioning nanophotonics research to viable devices with the main inspiration coming from biological light-harvesting antennas that collect and direct light energy with near-unity efficiency using Förster resonance energy transfer (FRET). Among putative FRET processes, point-to-plane FRET between donors and acceptors arrayed in two-dimensional sheets is predicted to be particularly efficient with a theoretical 1/r4 energy transfer distance (r) dependency versus the 1/r6 dependency seen for a single donor-acceptor interaction. However, quantitative validation has been confounded by a lack of robust experimental approaches that can rigidly place dyes in the required nanoscale arrangements. To create such assemblies, we utilize a DNA brick scaffold, referred to as a DNA block, which incorporates up to five two-dimensional planes with each displaying from 1 to 12 copies of five different donor, acceptor, or intermediary relay dyes. Nanostructure characterization along with steady-state and time-resolved spectroscopic data were combined with molecular dynamics modeling and detailed numerical simulations to compare the energy transfer efficiencies observed in the experimental DNA block assemblies to theoretical expectations. Overall, we demonstrate clear signatures of sheet regime FRET, and from this we provide a better understanding of what is needed to realize the benefits of such energy transfer in artificial dye networks along with FRET-based sensing and imaging.

Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle.

Developing reliable methods of constructing cell-free multienzyme biocatalytic systems is a milestone goal of synthetic biology. It would enable overcoming the limitations of current cell-based systems, which suffer from the presence of competing pathways, toxicity, and inefficient access to extracellular reactants and removal of products. DNA nanostructures have been suggested as ideal scaffolds for assembling sequential enzymatic cascades in close enough proximity to potentially allow for exploiting of channeling effects; however, initial demonstrations have provided somewhat contradictory results toward confirming this phenomenon. In this work, a three-enzyme sequential cascade was realized by site-specifically immobilizing DNA-conjugated amylase, maltase, and glucokinase on a self-assembled DNA origami triangle. The kinetics of seven different enzyme configurations were evaluated experimentally and compared to simulations of optimized activity. A 30-fold increase in the pathway's kinetic activity was observed for enzymes assembled to the DNA. Detailed kinetic analysis suggests that this catalytic enhancement originated from increased enzyme stability and a localized DNA surface affinity or hydration layer effect and not from a directed enzyme-to-enzyme channeling mechanism. Nevertheless, the approach used to construct this pathway still shows promise toward improving other more elaborate multienzymatic cascades and could potentially allow for the custom synthesis of complex (bio)molecules that cannot be realized with conventional organic chemistry approaches.

Nanoparticle Size Influences Localized Enzymatic Enhancement-A Case Study with Phosphotriesterase.

Enhancements in enzymatic catalytic activity are frequently observed when an enzyme is displayed on a nanoparticle (NP) surface. The exact mechanisms of how this unique interfacial environment gives rise to this phenomenon are still not understood, although evidence suggests that it can help alleviate some of the enzyme's rate-limiting mechanistic steps. The physicochemical limitations that govern when this process arises are also not known including, in particular, the range of NP size and curvature that are associated with it. To investigate the latter, we undertook a case study using the enzyme phosphotriesterase (PTE) and a series of differentially sized gold NPs (AuNPs). PTE, expressed with a terminal hexahistidine sequence, was ratiometrically coordinated to a series of increasing size AuNPs (diameter ≃ 1.5, 5, 10, 20, 55, 100 nm) surface-functionalized with Ni2+-nitrilotriacetic acid ligands and its activity assayed in a comparative format versus that of equivalent amounts of free enzyme controls. PTE-AuNP samples were prepared where the total PTE concentration and NP surface density were kept fixed by varying AuNP concentration along with the converse format. Assembly to the AuNPs increased PTE kcat ca. 3-10-fold depending upon NP size, with the smaller-sized particles showing the highest increase, while enzyme efficiency only increased 2-fold. Further kinetic testing suggested that the PTE enhancement again arose from alleviating its rate limiting step of enzyme-product release and not from a change in the activation energy. Comparison of kcat and enzyme specificity with AuNP diameter revealed that enhancement was directly correlated to AuNP size and curvature with the smaller NPs showing the largest kinetic enhancements. Kinetic simulations showed that almost all of the PTE enhancement variation across AuNP sizes could be reproduced by adjusting only the rate of enzyme-product dissociation. Understanding how NP size directly affects the enhancement of an attached enzyme can provide a rational basis for designing hybrid enzyme-NP materials that specifically exploit this emergent property.

Transducing Protease Activity into DNA Output for Developing Smart Bionanosensors.

DNA can process information through sequence-based reorganization but cannot typically receive input information from most biological processes and translate that into DNA compatible language. Coupling DNA to a substrate responsive to biological events can address this limitation. A two-component sensor incorporating a chimeric peptide-DNA substrate is evaluated here as a protease-to-DNA signal convertor which transduces protease activity through DNA gates that discriminate between different input proteases. Acceptor dye-labeled peptide-DNAs are assembled onto semiconductor quantum dot (QD) donors as the input gate. Addition of trypsin or chymotrypsin cleaves their cognate peptide sequence altering the efficiency of Förster resonance energy transfer (FRET) with the QD and frees a DNA output which interacts with a tetrahedral output gate. Downstream output gate rearrangement results in FRET sensitization of a new acceptor dye. Following characterization of component assembly and optimization of individual steps, sensor ability to discriminate between the two proteases is confirmed along with effects from joint interactions where potential for cross-talk is highest. Processing multiple bits of information for a sensing outcome provides more confidence than relying on a single change especially for the discrimination between different targets. Coupling other substrates to DNA that respond similarly could help target other types of enzymes.

Enhancing Coupled Enzymatic Activity by Colocalization on Nanoparticle Surfaces: Kinetic Evidence for Directed Channeling of Intermediates.

Multistep enzymatic cascades are becoming more prevalent in industrial settings as engineers strive to synthesize complex products and pharmaceuticals in economical, environmentally friendly ways. Previous work has shown that immobilizing enzymes on nanoparticles can enhance their activity significantly due to localized interfacial effects, and this enhancement remains in place even when that enzyme's activity is coupled to another enzyme that is still freely diffusing. Here, we investigate the effects of displaying two enzymes with coupled catalytic activity directly on the same nanoparticle surface. For this, the well-characterized enzymes pyruvate kinase (PykA) and lactate dehydrogenase (LDH) were utilized as a model system; they jointly convert phosphoenolpyruvate to lactate in two sequential steps as part of downstream glycolysis. The enzymes were expressed with terminal polyhistidine tags to facilitate their conjugation to semiconductor quantum dots (QDs) which were used here as prototypical nanoparticles. Characterization of enzyme coassembly to two different sized QDs showed a propensity to cross-link into nanoclusters consisting of primarily dimers and some trimers. Individual and joint enzyme activity in this format was extensively investigated in direct comparison to control samples lacking the QD scaffolds. We found that QD association enhances LDH activity by >50-fold and its total turnover by at least 41-fold, and that this high activation appears to be largely due to stabilization of its quarternary structure. When both enzymes are simultaneously bound to the QD surfaces, their colocalization leads to >100-fold improvements in the overall rates of coupled activity. Experimental results in conjunction with detailed kinetic simulations provide evidence that this significant improvement in coupled activity is partially attributable to a combination of enhanced enzymatic activity and stabilization of LDH. More importantly, experiments aimed at disrupting channeled processes and further kinetic modeling suggest that the bulk of the performance enhancement arises from intermediary "channeling" between the QD-colocalized enzymes. A full understanding of the underlying processes that give rise to such enhancements from coupled enzymatic activity on nanoparticle scaffolds can provide design criteria for improved biocatalytic applications.

Pursuing the Promise of Enzymatic Enhancement with Nanoparticle Assemblies.

The growing emphasis on green chemistry, renewable resources, synthetic biology, regio-/stereospecific chemical transformations, and nanotechnology for providing new biological products and therapeutics is reinvigorating research into enzymatic catalysis. Although the promise is profound, many complex issues remain to be addressed before this effort will have a significant impact. Prime among these is to combat the degradation of enzymes frequently seen in ex vivo formats following immobilization to stabilize the enzymes for long-term application and to find ways of enhancing their activity. One promising avenue for progress on these issues is via nanoparticle (NP) display, which has been found in a number of cases to enhance enzyme activity while also improving long-term stability. In this feature article, we discuss the phenomenon of enhanced enzymatic activity at NP interfaces with an emphasis on our own work in this area. Important factors such as NP surface chemistry, bioconjugation approaches, and assay formats are first discussed because they can critically affect the observed enhancement. Examples are given of improved performance for enzymes such as phosphotriesterase, alkaline phosphatase, trypsin, horseradish peroxidase, and ß-galactosidase and in configurations with either the enzyme or the substrate attached to the NP. The putative mechanisms that give rise to the performance boost are discussed along with how detailed kinetic modeling can contribute to their understanding. Given the importance of biosensing, we also highlight how this configuration is already making a significant contribution to NP-based enzymatic sensors. Finally, a perspective is provided on how this field may develop and how NP-based enzymatic enhancement can be extended to coupled systems and multienzyme cascades.

Time-Gated FRET and DNA-Based Photonic Molecular Logic Gates: AND, OR, NAND, and NOR.

Molecular logic devices (MLDs) constructed from DNA are promising for applications in bioanalysis, computing, and other applications requiring Boolean logic. These MLDs accept oligonucleotide inputs and generate fluorescence output through changes in structure. Although fluorescent dyes are most common in MLD designs, nontraditional luminescent materials with unique optical properties can potentially enhance MLD capabilities. In this context, luminescent lanthanide complexes (LLCs) have been largely overlooked. Here, we demonstrate a set of high-contrast DNA photonic logic gates based on toehold-mediated strand displacement and time-gated FRET. The gates include NAND, NOR, OR, and AND designs that accept two unlabeled target oligonucleotide sequences as inputs. Bright "true" output states utilize time-gated, FRET-sensitized emission from an Alexa Fluor 546 (A546) dye acceptor paired with a luminescent terbium cryptate (Tb) donor. Dark "false" output states are generated through either displacement of the A546, or through competitive and sequential quenching of the Tb or A546 by a dark quencher. Time-gated FRET and the long luminescence lifetime and spectrally narrow emission lines of the Tb donor enable 4-10-fold contrast between Boolean outputs, ≤10% signal variation for a common output, multicolor implementation of two logic gates in parallel, and effective performance in buffer and serum. These metrics exceed those reported for many other logic gate designs with only fluorescent dyes and with other non-LLC materials. Preliminary three-input AND and NAND gates are also demonstrated. The powerful combination of an LLC FRET donor with DNA-based logic gates is anticipated to have many future applications in bioanalysis.

Evaluating Dye-Labeled DNA Dendrimers for Potential Applications in Molecular Biosensing.

DNA nanostructures provide a reliable and predictable scaffold for precisely positioning fluorescent dyes to form energy transfer cascades. Furthermore, these structures and their attendant dye networks can be dynamically manipulated by biochemical inputs, with the changes reflected in the spectral response. However, the complexity of DNA structures that have undergone such types of manipulation for direct biosensing applications is quite limited. Here, we investigate four different modification strategies to effect such dynamic manipulations using a DNA dendrimer scaffold as a testbed, and with applications to biosensing in mind. The dendrimer has a 2:1 branching ratio that organizes the dyes into a FRET-based antenna in which excitonic energy generated on multiple initial Cy3 dyes displayed at the periphery is then transferred inward through Cy3.5 and/or Cy5 relay dyes to a Cy5.5 final acceptor at the focus. Advantages of this design included good transfer efficiency, large spectral separation between the initial donor and final acceptor emissions for signal transduction, and an inherent tolerance to defects. Of the approaches to structural rearrangement, the first two mechanisms we consider employed either toehold-mediated strand displacement or strand replacement and their impact was mainly via direct transfer efficiency, while the other two were more global in their effect using either a belting mechanism or an 8-arm star nanostructure to compress the nanostructure and thereby modulate its spectral response through an enhancement in parallelism. The performance of these mechanisms, their ability to reset, and how they might be utilized in biosensing applications are discussed.

Enhancing coupled enzymatic activity by conjugating one enzyme to a nanoparticle.

Enzymes have long been a prime research target for the commercial production of commodity and specialty chemicals, design of sensing devices, and the development of therapeutics and new chemical processes. Industrial applications for enzymes can potentially be enhanced by enzyme immobilization which often allows for increased enzyme stability, facile product purification, and minimized substrate diffusion times in multienzymatic cascades, but this is usually at the cost of a significant decrease in catalytic rates. Recently, enzyme immobilization has been advanced by the discovery that nanoparticle surfaces are frequently able to enhance the activity of the bound enzyme. Here we extend this observation to a multienzymatic coupled system using semiconductor quantum dots (QDs) as a model nanoparticle material and the prototypical enzyme pair of glucose oxidase (GOX) and horseradish peroxidase (HRP). We first demonstrate that HRP binding to QDs has a significant beneficial effect on enzymatic activity, producing a >2-fold improvement in kcat. We argue that this enhancement is due to affinity of the QD surface for the substrate. Furthermore, we demonstrate that when the ratio of GOX to HRP is adjusted to allow HRP to be the rate-limiting step of the pathway, the QD-induced rate enhancement of HRP can be maintained in a multi-enzyme cascade. Kinetic analysis shows that the underlying processes can be simulated numerically and provide insight into the governing mechanisms. The potential of nanoparticle-based catalytic enhancement is then discussed in the context of multienzyme cascades and synthetic biology.

Fluorescence and Energy Transfer in Dye-Labeled DNA Crystals.

DNA crystals make it possible to organize guest molecules into specific periodic 3D patterns at the nanoscale, and thereby to create novel macroscopic objects with potentially useful functionality. Here, we describe the fluorescence and energy transfer properties of DNA crystals that are self-assembled from DNA tensegrity triangles with covalently attached Cy3 and Cy5 dyes. When compared to reference DNA strands in solution, the fluorescence measurements indicate that the dyes in the crystal experience a more homogeneous environment, resulting in a 2-fold increase in Cy3 quantum yield and single-exponential Cy3 fluorescence decays. Energy transfer in a network of coupled Cy3 and Cy5 dyes in the DNA crystal is demonstrated experimentally. Numerical simulation finds the experiments to be consistent with a Förster model of the dyes in the periodic crystalline environment, and particularly if the transition dipoles are assumed random in orientation but static on the time scale of the excitation decay.

Time-Resolved Nucleic Acid Hybridization Beacons Utilizing Unimolecular and Toehold-Mediated Strand Displacement Designs.

Nucleic acid hybridization probes are sought after for numerous assay and imaging applications. These probes are often limited by the properties of fluorescent dyes, prompting the development of new probes where dyes are paired with novel or nontraditional luminescent materials. Luminescent terbium complexes are an example of such a material, and these complexes offer several unique spectroscopic advantages. Here, we demonstrate two nonstem-loop designs for light-up nucleic acid hybridization beacons that utilize time-resolved Förster resonance energy transfer (TR-FRET) between a luminescent Lumi4-Tb cryptate (Tb) donor and a fluorescent reporter dye, where time-resolved emission from the dye provides an analytical signal. Both designs are based on probe oligonucleotides that are labeled at their opposite termini with Tb and a fluorescent reporter dye. In one design, a probe is partially blocked with a quencher dye-labeled oligonucleotide, and target hybridization is signaled through toehold-mediated strand displacement and loss of a competitive FRET pathway. In the other design, the intrinsic folding properties of an unblocked probe are utilized in combination with a temporal mechanism for signaling target hybridization. This temporal mechanism is based on a recently elucidated "sweet spot" for TR-FRET measurements and exploits distance control over FRET efficiencies to shift the Tb lifetime within or outside the time-gated detection window for measurements. Both the blocked and unblocked beacons offer nanomolar (femtomole) detection limits, response times on the order of minutes, multiplexing through the use of different reporter dyes, and detection in complex matrices such as serum and blood. The blocked beacons offer better mismatch selectivity, whereas the unblocked beacons are simpler in design. The temporal mechanism of signaling utilized with the unblocked beacons also plays a significant role with the blocked beacons and represents a new and effective strategy for developing FRET probes for bioassays.

Understanding How Nanoparticle Attachment Enhances Phosphotriesterase Kinetic Efficiency.

As a specific example of the enhancement of enzymatic activity that can be induced by nanoparticles, we investigate the hydrolysis of the organophosphate paraoxon by phosphotriesterase (PTE) when the latter is displayed on semiconductor quantum dots (QDs). PTE conjugation to QDs underwent extensive characterization including structural simulations, electrophoretic mobility shift assays, and dynamic light scattering to confirm orientational and ratiometric control over enzyme display which appears to be necessary for enhancement. PTE hydrolytic activity was then examined when attached to ca. 4 and 9 nm diameter QDs in comparison to controls of freely diffusing enzyme alone. The results confirm that the activity of the QD conjugates significantly exceeded that of freely diffusing PTE in both initial rate (∼4-fold) and enzymatic efficiency (∼2-fold). To probe kinetic acceleration, various modified assays including those with increased temperature, presence of a competitive inhibitor, and increased viscosity were undertaken to measure the activation energy and dissociation rates. Cumulatively, the data indicate that the higher activity is due to an acceleration in enzyme-product dissociation that is presumably driven by the markedly different microenvironment of the PTE-QD bioconjugate's hydration layer. This report highlights how a specific change in an enzymatic mechanism can be both identified and directly linked to its enhanced activity when displayed on a nanoparticle. Moreover, the generality of the mechanism suggests that it could well be responsible for other examples of nanoparticle-enhanced catalysis.

Assembling programmable FRET-based photonic networks using designer DNA scaffolds.

DNA demonstrates a remarkable capacity for creating designer nanostructures and devices. A growing number of these structures utilize Förster resonance energy transfer (FRET) as part of the device's functionality, readout or characterization, and, as device sophistication increases so do the concomitant FRET requirements. Here we create multi-dye FRET cascades and assess how well DNA can marshal organic dyes into nanoantennae that focus excitonic energy. We evaluate 36 increasingly complex designs including linear, bifurcated, Holliday junction, 8-arm star and dendrimers involving up to five different dyes engaging in four-consecutive FRET steps, while systematically varying fluorophore spacing by Förster distance (R0). Decreasing R0 while augmenting cross-sectional collection area with multiple donors significantly increases terminal exciton delivery efficiency within dendrimers compared with the first linear constructs. Förster modelling confirms that best results are obtained when there are multiple interacting FRET pathways rather than independent channels by which excitons travel from initial donor(s) to final acceptor.

Extending FRET cascades on linear DNA photonic wires.

Photonic wires were constructed by sequentially arranging up to 7 fluorophores along a concatenated DNA scaffold. This yielded nanostructures displaying from one- to six-energy transfer steps where end-to-end efficiency reflected the multiple underlying photophysical processes and the ability of long-range interactions to compensate for localized non-ideal dye behaviour.

Complex logic functions implemented with quantum dot bionanophotonic circuits.

We combine quantum dots (QDs) with long-lifetime terbium complexes (Tb), a near-IR Alexa Fluor dye (A647), and self-assembling peptides to demonstrate combinatorial and sequential bionanophotonic logic devices that function by time-gated Förster resonance energy transfer (FRET). Upon excitation, the Tb-QD-A647 FRET-complex produces time-dependent photoluminescent signatures from multi-FRET pathways enabled by the capacitor-like behavior of the Tb. The unique photoluminescent signatures are manipulated by ratiometrically varying dye/Tb inputs and collection time. Fluorescent output is converted into Boolean logic states to create complex arithmetic circuits including the half-adder/half-subtractor, 2:1 multiplexer/1:2 demultiplexer, and a 3-digit, 16-combination keypad lock.

Biophotonic logic devices based on quantum dots and temporally-staggered Förster energy transfer relays.

Integrating photonic inputs/outputs into unimolecular logic devices can provide significantly increased functional complexity and the ability to expand the repertoire of available operations. Here, we build upon a system previously utilized for biosensing to assemble and prototype several increasingly sophisticated biophotonic logic devices that function based upon multistep Förster resonance energy transfer (FRET) relays. The core system combines a central semiconductor quantum dot (QD) nanoplatform with a long-lifetime Tb complex FRET donor and a near-IR organic fluorophore acceptor; the latter acts as two unique inputs for the QD-based device. The Tb complex allows for a form of temporal memory by providing unique access to a time-delayed modality as an alternate output which significantly increases the inherent computing options. Altering the device by controlling the configuration parameters with biologically based self-assembly provides input control while monitoring changes in emission output of all participants, in both a spectral and temporal-dependent manner, gives rise to two input, single output Boolean Logic operations including OR, AND, INHIBIT, XOR, NOR, NAND, along with the possibility of gate transitions. Incorporation of an enzymatic cleavage step provides for a set-reset function that can be implemented repeatedly with the same building blocks and is demonstrated with single input, single output YES and NOT gates. Potential applications for these devices are discussed in the context of their constituent parts and the richness of available signal.

Achieving effective terminal exciton delivery in quantum dot antenna-sensitized multistep DNA photonic wires.

Assembling DNA-based photonic wires around semiconductor quantum dots (QDs) creates optically active hybrid architectures that exploit the unique properties of both components. DNA hybridization allows positioning of multiple, carefully arranged fluorophores that can engage in sequential energy transfer steps while the QDs provide a superior energy harvesting antenna capacity that drives a Förster resonance energy transfer (FRET) cascade through the structures. Although the first generation of these composites demonstrated four-sequential energy transfer steps across a distance >150 Å, the exciton transfer efficiency reaching the final, terminal dye was estimated to be only ~0.7% with no concomitant sensitized emission observed. Had the terminal Cy7 dye utilized in that construct provided a sensitized emission, we estimate that this would have equated to an overall end-to-end ET efficiency of ≤ 0.1%. In this report, we demonstrate that overall energy flow through a second generation hybrid architecture can be significantly improved by reengineering four key aspects of the composite structure: (1) making the initial DNA modification chemistry smaller and more facile to implement, (2) optimizing donor-acceptor dye pairings, (3) varying donor-acceptor dye spacing as a function of the Förster distance R0, and (4) increasing the number of DNA wires displayed around each central QD donor. These cumulative changes lead to a 2 orders of magnitude improvement in the exciton transfer efficiency to the final terminal dye in comparison to the first-generation construct. The overall end-to-end efficiency through the optimized, five-fluorophore/four-step cascaded energy transfer system now approaches 10%. The results are analyzed using Förster theory with various sources of randomness accounted for by averaging over ensembles of modeled constructs. Fits to the spectra suggest near-ideal behavior when the photonic wires have two sequential acceptor dyes (Cy3 and Cy3.5) and exciton transfer efficiencies approaching 100% are seen when the dye spacings are 0.5 × R0. However, as additional dyes are included in each wire, strong nonidealities appear that are suspected to arise predominantly from the poor photophysical performance of the last two acceptor dyes (Cy5 and Cy5.5). The results are discussed in the context of improving exciton transfer efficiency along photonic wires and the contributions these architectures can make to understanding multistep FRET processes.

Assembly of a concentric Förster resonance energy transfer relay on a quantum dot scaffold: characterization and application to multiplexed protease sensing.

Semiconductor nanocrystals, or quantum dots (QDs), are one of the most widely utilized nanomaterials for biological applications. Their cumulative physicochemical and optical properties are both unique among nanomaterials and highly advantageous. In particular, Förster resonance energy transfer (FRET) has been widely utilized as a spectroscopic tool with QDs, whether for characterizing QD bioconjugates as a "molecular ruler" or for modulating QD luminescence "on" and "off" in biosensing configurations. Here, we investigate the assembly and utility of a new "concentric" FRET relay that comprises a central QD conjugated with multiple copies of two different peptides, each labeled with one of two fluorescent dyes, Alexa Fluor 555 (A555) or Alexa Fluor 647 (A647). Energy transfer occurs from the QD to the A555 (FRET(1)) then to the A647 (FRET(2)) and, to a lesser extent, directly from the QD to the A647 (FRET(3)). We show that such an arrangement can provide insight into the interfacial distribution of peptides assembled to the QD and can further be utilized for sensing proteolytic activity. In the latter, progress curves for digestion of the assembled peptides by two prototypical proteases, trypsin and chymotrypsin, were measured from the relative QD, A555 and A647 PL contributions, and used to extract Michaelis-Menten kinetic parameters. We further show that the concentric FRET relay, as a single nanoparticle vector, can track the tryptic activation of a proenzyme, chymotrypsinogen, to active chymotrypsin. The concentric FRET relay is thus a potentially powerful tool for the characterization of QD bioconjugates and multiplexed sensing of coupled biological activity.