Sequestration within peptide coacervates improves the fluorescence intensity, kinetics, and limits of detection of dye-based DNA biosensors.

Peptide-based liquid-liquid phase separated domains, or coacervates, are a biomaterial gaining new interest due to their exciting potential in fields ranging from biosensing to drug delivery. In this study, we demonstrate that coacervates provide a simple and biocompatible medium to improve nucleic acid biosensors through the sequestration of both the biosensor and target strands within the coacervate, thereby increasing their local concentration. Using the well-established polyarginine (R9) - ATP coacervate system and an energy transfer-based DNA molecular beacon we observed three key improvements: i) a greater than 20-fold reduction of the limit of detection within coacervates when compared to control buffer solutions; ii) an increase in the kinetics, equilibrium was reached more than 4-times faster in coacervates; and iii) enhancement in the dye fluorescent quantum yields within the coacervates, resulting in greater signal-to-noise. The observed benefits translate into coacervates greatly improving bioassay functionality.

Investigating the dissipation of heat and quantum information from DNA-scaffolded chromophore networks.

Scaffolded molecular networks are important building blocks in biological pigment-protein complexes, and DNA nanotechnology allows analogous systems to be designed and synthesized. System-environment interactions in these systems are responsible for important processes, such as the dissipation of heat and quantum information. This study investigates the role of nanoscale molecular parameters in tuning these vibronic system-environment dynamics. Here, genetic algorithm methods are used to obtain nanoscale parameters for a DNA-scaffolded chromophore network based on comparisons between its calculated and measured optical spectra. These parameters include the positions, orientations, and energy level characteristics within the network. This information is then used to compute the dynamics, including the vibronic population dynamics and system-environment heat currents, using the hierarchical equations of motion. The dissipation of quantum information is identified by the system's transient change in entropy, which is proportional to the heat currents according to the second law of thermodynamics. These results indicate that the dissipation of quantum information is highly dependent on the particular nanoscale characteristics of the molecular network, which is a necessary first step before gleaning the systematic optimization rules. Subsequently, the I-concurrence dynamics are calculated to understand the evolution of the vibronic system's quantum entanglement, which are found to be long-lived compared to these system-bath dissipation processes.

Smaller Gold Nanoparticles Release DNA More Efficiently During fs Laser Pulsed Optical Heating.

This work investigates the effect of plasmonic gold nanoparticle (AuNP) size on the rate of thermal release of single-stranded oligonucleotides under femtosecond (fs)-pulsed laser irradiation sources. Contrary to the theoretical predictions that larger AuNPs (50-60 nm diameter) would produce the most solution heating and fastest DNA release, it is found that smaller AuNP diameters (25 nm) lead to faster dsDNA denaturation rates. Controlling for the pulse energy fluence, AuNP concentration, DNA loading density, and the distance from the AuNP surface finds the same result. These results imply that the solution temperature increases around the AuNP during fs laser pulse optical heating may not be the only significant influence on dsDNA denaturation, suggesting that direct energy transfer from the AuNP to the DNA (phonon-phonon coupling), which is increased as AuNPs decrease in size, may play a significant role.

Multiplexed DNA and Protease Detection with Orthogonal Energy Transfer on a Single Quantum Dot Scaffolded Biosensor.

Almost all pathogens, whether viral or bacterial, utilize key proteolytic steps in their pathogenesis. The ability to detect a pathogen's genomic material along with its proteolytic activity represents one approach to identifying the pathogen and providing initial evidence of its viability. Here, we report on a prototype biosensor design assembled around a single semiconductor quantum dot (QD) scaffold that is capable of detecting both nucleic acid sequences and proteolytic activity by using orthogonal energy transfer (ET) processes. The sensor consists of a central QD assembled via peptidyl-PNA linkers with multiple DNA sequences that encode complements to genomic sequences originating from the Ebola, Influenza, and COVID-19 viruses, which we use as surrogate targets. These are hybridized to complement strands labeled with a terbium (Tb) chelate, AlexaFluor647 (AF647), and Cy5.5 dyes, giving rise to two potential FRET cascades: the first includes Tb → QD → AF647 → Cy5.5 (→ = ET step), which is detected in a time-gated modality, and QD → AF647 → Cy5.5, which is detected from direct excitation. The labeled DNA-displaying QD construct is then further assembled with a RuII-modified peptide, which quenches QD photoluminescence by charge transfer and is recognized by a protease to yield the full biosensor. Each of the labeled DNAs and peptides can be ratiometrically assembled to the QD in a controllable manner to tune each of the ET pathways. Addition of a given target DNA displaces its labeled complement on the QD, disrupting that FRET channel, while protease addition disrupts charge transfer quenching of the central QD scaffold and boosts its photoluminescence and FRET relay capabilities. Along with characterizing the ET pathways and verifying biosensing in both individual and multiplexed formats, we also demonstrate the ability of this construct to function in molecular logic and perform Boolean operations; this highlights the construct's ability to discriminate and transduce signals between different inputs or pathogens. The potential application space for such a sensor device is discussed.

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.

Towards control of excitonic coupling in DNA-templated Cy5 aggregates: the principal role of chemical substituent hydrophobicity and steric interactions.

Understanding and controlling exciton coupling in dye aggregates has become a greater focus as potential applications such as coherent exciton devices, nanophotonics, and biosensing have been proposed. DNA nanostructure templates allow for a powerful modular approach. Using DNA Holliday junction (HJ) templates variations of dye combinations and precision dye positions can be rapidly assayed, as well as creating aggregates of dyes that could not be prepared (either due to excess or lack of solubility) through alternative means. Indodicarbocyanines (Cy5) have been studied in coupled systems due to their large transition dipole moment, which contributes to strong coupling. Cy5-R dyes were recently prepared by chemically modifying the 5,5'-substituents of indole rings, resulting in varying dye hydrophobicity/hydrophilicity, steric considerations, and electron-donating/withdrawing character. We utilized Cy5-R dyes to examine the formation and properties of 30 unique DNA templated homodimers. We find that in our system the sterics of Cy5-R dyes play the determining factor in orientation and coupling strength of dimers, with coupling strengths ranging from 50-138 meV. The hydrophobic properties of the Cy5-R modify the percentage of dimers formed, and have a secondary role in determining the packing characteristics of the dimers when sterics are equivalent. Similar to other reports, we find that positioning of the Cy5-R within the HJ template can favor particular dimer interactions, specifically oblique or H-type dimers.

Determining interchromophore effects for energy transport in molecular networks using machine-learning algorithms.

Nature uses chromophore networks, with highly optimized structural and energetic characteristics, to perform important chemical functions. Due to its modularity, predictable aggregation characteristics, and established synthetic protocols, structural DNA nanotechnology is a promising medium for arranging chromophore networks with analogous structural and energetic controls. However, this high level of control creates a greater need to know how to optimize the systems precisely. This study uses the system's modularity to produce variations of a coupled 14-Site chromophore network. It uses machine-learning algorithms and spectroscopy measurements to reveal the energy-transport roles of these Sites, paying particular attention to the cooperative and inhibitive effects they impose on each other for transport across the network. The physical significance of these patterns is contextualized, using molecular dynamics simulations and energy-transport modeling. This analysis yields insights about how energy transfers across the Donor-Relay and Relay-Acceptor interfaces, as well as the energy-transport pathways through the homogeneous Relay segment. Overall, this report establishes an approach that uses machine-learning methods to understand, in fine detail, the role that each Site plays in an optoelectronic molecular network.

Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates.

Coherently coupled pseudoisocyanine (PIC) dye aggregates have demonstrated the ability to delocalize electronic excitations and ultimately migrate excitons with much higher efficiency than similar designs where excitations are isolated to individual chromophores. Here, we report initial evidence of a new type of PIC aggregate, formed through heterogeneous nucleation on DNA oligonucleotides, displaying photophysical properties that differ significantly from previously reported aggregates. This new aggregate, which we call the super aggregate (SA) due to the need for elevated dye excess to form it, is clearly differentiated from previously reported aggregates by spectroscopic and biophysical characterization. In emission spectra, the SA exhibits peak narrowing and, in some cases, significant quantum yield variation, indicative of stronger coupling in cyanine dyes. The SA was further characterized with circular dichroism and atomic force microscopy observing unique features depending on the DNA substrate. Then by integrating an AlexaFluorTM647 (AF) dye as an energy transfer acceptor into the system, we observed mixed energy transfer characteristics using the different DNA. For example, SA formed with a rigid DNA double crossover tile (DX-tile) substrate resulted in AF emission sensitization. While SA formed with more flexible non-DX-tile DNA (i.e. duplex and single strand DNA) resulted in AF emission quenching. These combined characterizations strongly imply that DNA-based PIC aggregate properties can be controlled through simple modifications to the DNA substrate's sequence and geometry. Ultimately, we aim to inform rational design principles for future device prototyping. For example, one key conclusion of the study is that the high absorbance cross-section and efficient energy transfer observed with rigid substrates made for better photonic antennae, compared to flexible DNA substrates.

Quantum Dot-Based Molecular Beacons for Quantitative Detection of Nucleic Acids with CRISPR/Cas(N) Nucleases.

Strategies utilizing the CRISPR/Cas nucleases Cas13 and Cas12 have shown great promise in the development of highly sensitive and rapid diagnostic assays for the detection of pathogenic nucleic acids. The most common approaches utilizing fluorophore-quencher molecular beacons require strand amplification strategies or highly sensitive optical setups to overcome the limitations of the readout. Here, we demonstrate a flexible strategy for assembling highly luminescent and colorimetric quantum dot-nucleic acid hairpin (QD-HP) molecular beacons for use in CRISPR/Cas diagnostics. This strategy utilizes a chimeric peptide-peptide nucleic acid (peptide-PNA) to conjugate fluorescently labeled DNA or RNA hairpins to ZnS-coated QDs. QDs are particularly promising alternatives for molecular beacons due to their greater brightness, strong UV absorbance with large emission offset, exceptional photostability, and potential for multiplexing due to their sharp emission peaks. Using Förster resonance energy transfer (FRET), we have developed ratiometric reporters capable of pM target detection (without nucleotide amplification) for both target DNA and RNA, and we further demonstrated their capabilities for multiplexing and camera-phone detection. The flexibility of this system is imparted by the dual functionality of the QD as both a FRET donor and a central nanoscaffold for arranging nucleic acids and fluorescent acceptors on its surface. This method also provides a generalized approach that could be applied for use in other CRISPR/Cas nuclease systems.

Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors.

Cell-free synthetic biology has emerged as a valuable tool for the development of rapid, portable biosensors that can be readily transported in the freeze-dried form to the point of need eliminating cold chain requirements. One of the challenges associated with cell-free sensors is the ability to simultaneously detect multiple analytes within a single reaction due to the availability of a limited set of fluorescent and colorimetric reporters. To potentially provide multiplexing capabilities to cell-free biosensors, we designed a modular semiconductor quantum dot (QD)-based reporter platform that is plugged in downstream of the transcription-translation functionality in the cell-free reaction and which converts enzymatic activity in the reaction into distinct optical signals. We demonstrate proof of concept by converting restriction enzyme activity, utilized as our prototypical sensing output, into optical changes across several distinct spectral output channels that all use a common excitation wavelength. These hybrid Förster resonance energy transfer (FRET)-based QD peptide PNA-DNA-Dye reporters (QD-PDDs) are completely self-assembled and consist of differentially emissive QD donors paired to a dye-acceptor displayed on a unique DNA encoding a given enzyme's cleavage site. Three QD-based PDDs, independently activated by the enzymes BamHI, EcoRI, and NcoI, were prototyped in mixed enzyme assays where all three demonstrated the ability to convert enzymatic activity into fluorescent output. Simultaneous monitoring of each of the three paired QD-donor dye-acceptor spectral channels in cell-free biosensing reactions supplemented with added linear genes encoding each enzyme confirmed robust multiplexing capabilities for at least two enzymes when co-expressed. The modular QD-PDDs are easily adapted to respond to other restriction enzymes or even proteases if desired.

Tunable Electronic Structure via DNA-Templated Heteroaggregates of Two Distinct Cyanine Dyes.

Molecular excitons are useful for applications in light harvesting, organic optoelectronics, and nanoscale computing. Electronic energy transfer (EET) is a process central to the function of devices based on molecular excitons. Achieving EET with a high quantum efficiency is a common obstacle to excitonic devices, often owing to the lack of donor and acceptor molecules that exhibit favorable spectral overlap. EET quantum efficiencies may be substantially improved through the use of heteroaggregates-aggregates of chemically distinct dyes-rather than individual dyes as energy relay units. However, controlling the assembly of heteroaggregates remains a significant challenge. Here, we use DNA Holliday junctions to assemble homo- and heterotetramer aggregates of the prototypical cyanine dyes Cy5 and Cy5.5. In addition to permitting control over the number of dyes within an aggregate, DNA-templated assembly confers control over aggregate composition, i.e., the ratio of constituent Cy5 and Cy5.5 dyes. By varying the ratio of Cy5 and Cy5.5, we show that the most intense absorption feature of the resulting tetramer can be shifted in energy over a range of almost 200 meV (1600 cm-1). All tetramers pack in the form of H-aggregates and exhibit quenched emission and drastically reduced excited-state lifetimes compared to the monomeric dyes. We apply a purely electronic exciton theory model to describe the observed progression of the absorption spectra. This model agrees with both the measured data and a more sophisticated vibronic model of the absorption and circular dichroism spectra, indicating that Cy5 and Cy5.5 heteroaggregates are largely described by molecular exciton theory. Finally, we extend the purely electronic exciton model to describe an idealized J-aggregate based on Förster resonance energy transfer (FRET) and discuss the potential advantages of such a device over traditional FRET relays.

Polyhistidine-Tag-Enabled Conjugation of Quantum Dots and Enzymes to DNA Nanostructures.

DNA nanostructures self-assemble into almost any arbitrary architecture, and when combined with their capability to precisely position and orient dyes, nanoparticles, and biological moieties, the technology reaches its potential. We present a simple yet multifaceted conjugation strategy based on metal coordination by a multi-histidine peptide tag (Histag). The versatility of the Histag as a means to conjugate to DNA nanostructures is shown by using Histags to capture semiconductor quantum dots (QDs) with numerical and positional precision onto a DNA origami breadboard. Additionally, Histag-expressing enzymes, such as the bioluminescent luciferase, can also be captured to the DNA origami breadboard with similar precision. DNA nanostructure conjugation of the QDs or luciferase is confirmed through imaging and/or energy transfer to organic dyes integrated into the DNA nanostructure.

Determining the Cytosolic Stability of Small DNA Nanostructures In Cellula.

DNA nanostructures have proven potential in biomedicine. However, their intracellular interactions─especially cytosolic stability─remain mostly unknown and attempts to discern this are confounded by the complexities of endocytic uptake and entrapment. Here, we bypass the endocytic uptake and evaluate the DNA structural stability directly in live cells. Commonly used DNA structures─crosshairs and a tetrahedron─were labeled with a multistep Förster resonance energy transfer dye cascade and microinjected into the cytosol of transformed and primary cells. Energy transfer loss, as monitored by fluorescence microscopy, reported the structure's direct time-resolved breakdown in cellula. The results showed rapid degradation of the DNA crosshair within 20 min, while the tetrahedron remained consistently intact for at least 1 h postinjection. Nuclease assays in conjunction with a current understanding of the tetrahedron's torsional rigidity confirmed its higher stability. Such studies can inform design parameters for future DNA nanostructures where programmable degradation rates may be required.

Observation of fractional spin textures in a Heusler material.

Recently a zoology of non-collinear chiral spin textures has been discovered, most of which, such as skyrmions and antiskyrmions, have integer topological charges. Here we report the experimental real-space observation of the formation and stability of fractional antiskyrmions and fractional elliptical skyrmions in a Heusler material. These fractional objects appear, over a wide range of temperature and magnetic field, at the edges of a sample, whose interior is occupied by an array of nano-objects with integer topological charges, in agreement with our simulations. We explore the evolution of these objects in the presence of magnetic fields and show their interconversion to objects with integer topological charges. This means the topological charge can be varied continuously. These fractional spin textures are not just another type of skyrmion, but are essentially a new state of matter that emerges and lives only at the boundary of a magnetic system. The coexistence of both integer and fractionally charged spin textures in the same material makes the Heusler family of compounds unique for the manipulation of the real-space topology of spin textures and thus an exciting platform for spintronic and magnonic applications.

Tuning between Quenching and Energy Transfer in DNA-Templated Heterodimer Aggregates.

Molecular excitons, which propagate spatially via electronic energy transfer, are central to numerous applications including light harvesting, organic optoelectronics, and nanoscale computing; they may also benefit applications such as photothermal therapy and photoacoustic imaging through the local generation of heat via rapid excited-state quenching. Here we show how to tune between energy transfer and quenching for heterodimers of the same pair of cyanine dyes by altering their spatial configuration on a DNA template. We assemble "transverse" and "adjacent" heterodimers of Cy5 and Cy5.5 using DNA Holliday junctions. We find that the transverse heterodimers exhibit optical properties consistent with excitonically interacting dyes and fluorescence quenching, while the adjacent heterodimers exhibit optical properties consistent with nonexcitonically interacting dyes and disproportionately large Cy5.5 emission, suggestive of energy transfer between dyes. We use transient absorption spectroscopy to show that quenching in the transverse heterodimer occurs via rapid nonradiative decay to the ground state (∼31 ps) and that in the adjacent heterodimer rapid energy transfer from Cy5 to Cy5.5 (∼420 fs) is followed by Cy5.5 excited-state relaxation (∼700 ps). Accessing such drastically different photophysics, which may be tuned on demand for different target applications, highlights the utility of DNA as a template for dye aggregation.

Rapid DNA origami nanostructure detection and classification using the YOLOv5 deep convolutional neural network.

The intra-image identification of DNA structures is essential to rapid prototyping and quality control of self-assembled DNA origami scaffold systems. We postulate that the YOLO modern object detection platform commonly used for facial recognition can be applied to rapidly scour atomic force microscope (AFM) images for identifying correctly formed DNA nanostructures with high fidelity. To make this approach widely available, we use open-source software and provide a straightforward procedure for designing a tailored, intelligent identification platform which can easily be repurposed to fit arbitrary structural geometries beyond AFM images of DNA structures. Here, we describe methods to acquire and generate the necessary components to create this robust system. Beginning with DNA structure design, we detail AFM imaging, data point annotation, data augmentation, model training, and inference. To demonstrate the adaptability of this system, we assembled two distinct DNA origami architectures (triangles and breadboards) for detection in raw AFM images. Using the images acquired of each structure, we trained two separate single class object identification models unique to each architecture. By applying these models in sequence, we correctly identified 3470 structures from a total population of 3617 using images that sometimes included a third DNA origami structure as well as other impurities. Analysis was completed in under 20 s with results yielding an F1 score of 0.96 using our approach.

Laser-Controlled Real- and Reciprocal-Space Topology in Multiferroic Insulators.

Magnetic materials in which it is possible to control the topology of their magnetic order in real space or the topology of their magnetic excitations in reciprocal space are highly sought after as platforms for alternative data storage and computing architectures. Here we show that multiferroic insulators, owing to their magnetoelectric coupling, offer a natural and advantageous way to address these two different topologies using laser fields. We demonstrate that via a delicate balance between the energy injection from a high-frequency laser and dissipation, single skyrmions-archetypical topological magnetic textures-can be set into motion with a velocity and propagation direction that can be tuned by the laser field amplitude and polarization, respectively. Moreover, we uncover an ultrafast Floquet magnonic topological phase transition in a laser-driven skyrmion crystal and we propose a new diagnostic tool to reveal it using the magnonic thermal Hall conductivity.

Mechanistic Understanding of DNA Denaturation in Nanoscale Thermal Gradients Created by Femtosecond Excitation of Gold Nanoparticles.

There is significant interest in developing photothermal systems that can precisely control the structure and function of biomolecules through local temperature modulation. One specific application is the denaturation of double-stranded (ds) DNA through femtosecond (fs) laser pulse optical heating of gold nanoparticles (AuNPs); however, the mechanism of DNA melting in these systems is not fully understood. Here, we utilize 55 nm AuNPs with surface-tethered dsDNA, which are locally heated using fs laser pulses to induce DNA melting. By varying the dsDNA distance from the AuNP surface and the laser pulse energy fluence, this system is used to study how the nanosecond duration temperature increase and the steep temperature gradient around the AuNP affect dsDNA dehybridization. Through modifying the distance between the dsDNA and AuNP surface by 3.8 nm in total and the pulse energy fluence from 7.1 to 14.1 J/m2, the dehybridization rates ranged from 0.002 to 0.05 DNA per pulse, and the total amount of DNA released into solution was controlled over a range of 26-93% in only 100 s of irradiation. By shifting the dsDNA position as little as ∼1.1 nm, the average dsDNA dehybridization rate is altered up to 30 ± 2%, providing a high level of control over DNA melting and release. By comparing the theoretical temperature around the dsDNA to the experimentally derived temperature, we find that maximum or peak temperatures have a greater influence on the dehybridization rate when the dsDNA is closer to the AuNP surface and when lower laser pulse fluences are used. Furthermore, molecular dynamics simulations mimicking the photothermal heat pulse around a AuNP provide mechanistic insight into the stochastic nature of dehybridization and demonstrate increased base pair separation near the AuNP surface during laser pulse heating when compared to steady-state heating. Understanding how biological materials respond to the short-lived and non-uniform temperature increases innate to fs laser pulse optical heating of AuNPs is critical to improving the functionality and precision of this technique so that it may be implemented into more complex biological systems.

Understanding Self-Assembled Pseudoisocyanine Dye Aggregates in DNA Nanostructures and Their Exciton Relay Transfer Capabilities.

Progress has been made using B-form DNA duplex strands to template chromophores in ordered molecular aggregates known as J-aggregates. These aggregates can exhibit strong electronic coupling, extended coherent lifetimes, and long-range exciton delocalization under appropriate conditions. Certain cyanine dyes such as pseudoisocyanine (PIC) dye have shown a proclivity to form aggregates in specific DNA sequences. In particular, DX-tiles containing nonalternating poly(dA)-poly(dT) dinucleotide tracks (AT-tracks), which template noncovalent PIC dye aggregates, have been demonstrated to exhibit interesting emergent photonic properties. These DNA-based aggregates are referred to as J-bits for their similarity to J-aggregates. Here, we assemble multifluorophore DX-tile scaffolds which template J-bits into both contiguous and noncontiguous linear arrays. Our goal is to understand the relay capability of noncontiguous J-bit arrays and probe the effects that orientation and position have on the energy transfer between them. We find that linearly contiguous J-bits can relay excitons from an initial AlexaFluor 405 donor to a terminal AlexaFluor 647 acceptor across a distance of up to 16.3 nm. We observed a maximum increase in energy transfer of 41% in the shortest scaffold and an 11% increase in energy transfer across the maximum distance. However, in nonlinear arrays, exciton transfer is not detectable, even when off-axis J-bit-to-J-bit transfer distances were <2 nm. These results, in conjunction with the previous work on PIC-DNA systems, suggest that PIC-DNA-based systems may currently be limited to simple 1-D designs, which prevent isolating J-bits for enhanced energy-transfer characteristics until further understanding and improvements to the system can be made.