Eukaryotic genomic DNA is repeatedly wrapped into nucleosome spools: the basic building block of chromatin. This organization regulates the physical accessibility of the genome to gene transcription, replication, and repair regulatory factors. Chromatin compaction is controlled by multivalent weak interactions, resulting in a complicated conformational landscape that remains challenging to characterize. This work reports a method for characterizing chromatin compaction, Free Energy Spectroscopy (FES), which is based on DNA nanotechnology and transmission electron microscopy. This method experimentally determines the chromatin compaction free energy landscape in terms of end-to-end distance and nucleosome stacking interactions. By deconvolving the free energy landscapes of partially and fully compact tetranucleosomes, FES revealed three separate mechanisms by which linker histones reshape the compaction energetics to condense chromatin. This study establishes FES as a method with the potential to help answer a broad range of mechanistic questions about genome and epigenome function.

Ion gradients play a vital role in cellular signaling, mechanobiology, and organ-level homeostasis. Despite their importance, accurately mapping these spatial gradients at biologically relevant length scales remains a challenge due to the limited tunability and spatial resolution of conventional fluorescent sensors. Here, we present a DNA origami-based sensor (NanoDyn) with tunable sensitivity that enables the detection of Na+-ion gradients across micron to millimeter scales. The sensor design leverages programmable DNA base-pairing interactions to control both the detection range and sensitivity of the sensor. Using fluorescence spectroscopy, we show that NanoDyn can exhibit programmable sensing ranges spanning ∼100-1675 mM Na+. To validate the ability to quantify ion gradients and investigate their spatial resolution, we use a custom microfluidic gradient generator, showing that NanoDyn can resolve changes in ion gradients across multiple scales and over distances as little as ∼6 μm, which, here, is limited by the resolution of the microfluidic device. By highlighting the potential of DNA nanodevices as multiscale, tunable ion-gradient sensors, together with their biocompatibility, high temporal resolution, and potential for multiplexed functionalization, this work expands on the role that DNA nanodevices can play in spatial sensing to study ion-mediated processes in microenvironments. Overall, this work advances DNA nanotechnology as a versatile foundation for biosensing with capabilities to probe ion-mediated signaling in health and disease.

Acute myeloid leukemia (AML) is the most common and lethal leukemia in adults. AML consists of many genetic subtypes, which limits broad applicability of targeted therapy. We discovered that the hematopoiesis-restricted tetraspanin CD37 is expressed on the majority of primary AML blasts and thus may represent a common therapeutic target for AML regardless of subtype. We demonstrate that the internalization properties of CD37 are distinct in AML blasts when compared with normal blood cells, and that CD37 rapidly accumulates inside AML blasts via dynamin-dependent endocytosis. Our work revealed that the clinically relevant anti-CD37 antibody-drug conjugate (ADC) Debio 1562 (alpha CD37DM1) is highly cytotoxic to AML blasts, but not normal hematopoietic stem cells. We found that alpha CD37-DM1 improved clinical outcomes and overall survival in multiple in vivo models of AML. Together, these data demonstrate that targeting CD37 with an ADC such as alpha CD37DM1 is a feasible and promising therapeutic option for the treatment of AML.

Deoxyribonucleic acid (DNA)-based nanomaterials can template growth of nanostructured films on their surfaces, generating complex morphologies. However, previous work has not explored the application of this approach to DNA nanostructures capable of large shape transformations. This study investigated the application of in situ reduction chemistries to dynamic DNA origami materials. Extending beyond past work using gold or silica, iron oxide nanostructures were grown on a variety of DNA origami geometries at different iron:DNA origami molar ratios (i.e., 25,000 to 1,000,000). Growth was visualized using transmission electron microscopy (TEM) with energy dispersive X-ray spectroscopy (EDS), which indicated the presence of electron dense iron oxide. Structures generally retained their geometric form factors, with some modifications observed in TEM. Structures formed at the highest ratios (i.e., 500,000 to 1,000,000) aggregated, providing an upper limit for this method. DNA origami nanostructures were programmed with single-stranded DNA (ssDNA) overhangs for binding complementary ssDNA-modified cargoes, inducing structural transformations, and for hierarchical assembly. Overhang functionality in coated structures was assessed by gold nanoparticle (AuNP) binding, actuation of two different DNA origami nanostructures, and polymerization into nanotube bundles. These findings indicate that the in situ reduction technique can be applied to dynamic DNA origami structures, retaining their capacity for large shape changes, and that overhangs presented by those structures retain functionality. This approach enables dynamic transformation of individual inorganic nanostructure shapes and assembly of units into larger, arrayed materials.

DNA nanotechnology enables the precise construction of intricate nanoscale structures. Over the past two decades, significant progress has been made in incorporating dynamic functionalities into these nanostructures. Concurrently, innovative strategies have emerged for their self-assembly and surface patterning into larger, more complex architectures. This review explores the convergence of these two key capabilities-reconfigurability and hierarchical assembly-to engineer DNA origami superstructures with intrinsic dynamic behavior. We begin by outlining foundational strategies in dynamic design, hierarchical assembly, and surface placement, then review recent progress in leveraging these strategies to construct dynamic superstructures with emergent behaviors. The article concludes with a roadmap of major challenges and opportunities shaping the future of this rapidly evolving field.

Mechanical forces play key roles in biological processes such as cell migration and sensory perception. In recent years, molecular force sensors have been developed as tools for in situ force measurements. Here, we use all-atom steered molecular dynamics simulations to predict and study the relationship between design parameters and mechanical properties for three types of molecular force sensors commonly used in cellular biological research: two peptide and one DNA based. The peptide-based sensors consist of a pair of fluorescent proteins that can undergo Förster resonance energy transfer, linked by spider silk (GPGGA)n or synthetic (GGSGGS)n disordered regions. The DNA-based sensor consists of two fluorophore-labeled strands of DNA that can be unzipped or sheared upon force application with a Förster resonance energy transfer signal as readout of dissociation. We simulated nine sensors, three of each kind. After equilibration, flexible peptide linkers of three different lengths were stretched by applying forces to their N- and C-terminal Cα atoms in opposite directions. Similarly, we equilibrated a DNA-based sensor and pulled on the phosphate atom of the terminal guanine of one strand and a selected phosphate atom on the other strand for pulling in the opposite direction. These simulations were performed at constant velocity (0.01-10 nm/ns) and constant force (10-500 pN) for all versions of the sensors. Our results show how the force response of these sensors depends on their length, sequence, configuration, and loading rate. Mechanistic insights gained from simulations analyses indicate that interpretation of experimental results should consider the influence of transient formation of secondary structure in peptide-based sensors and of overstretching in DNA-based sensors. These predictions can guide optimal fluorophore choice and facilitate the rational design of new sensors for use in protein, DNA, hybrid systems, and molecular devices.

Structural DNA nanotechnology, a research field in which scientists use DNA as the primary material to make designer nanostructures, has experienced rapid growth in the past few decades. The continuous development of the field has produced a rich repository of impressive, complex nanostructures for applications in materials science, biological research, and therapeutics. The unprecedented programmability of DNA nanostructures, particularly DNA origami, combined with the biocompatibility and rich functionality of DNA molecules make them attractive candidates for building nanocarriers for cellular delivery. While the initial research toward this direction focused on the delivery of small molecule drugs and short nucleic acids, emerging efforts in the last two years have expanded to gene delivery by leveraging the capacity of DNA origami to fold gene sequences into compact structures amenable for cell delivery. Here, we review this exciting research direction and provide our perspective on the challenges and opportunities in this field.

Mechanical loading of the intervertebral disc induces biophysical cues at the cellular level that regulate cellular biology and tissue maintenance [1–3]. Of these cues, fluid shear, hydrostatic pressure, and osmotic swelling are key to tissue function, and as such are of importance to measure. The goal of this study was to develop and characterize the response of DNA origami biosensors to these biophysical cues. Tunable DNA origami sensors were created with 2 barrels connected by 6 radially oriented fluctuating connectors.

To date, studies on the thermodynamic and kinetic processes that underlie biological function and nanomachine actuation in biological- and biology-inspired molecular constructs have primarily focused on photothermal heating of ensemble systems, highlighting the need for probes that are localized within the molecular construct and capable of resolving single-molecule response. Here we present an experimental demonstration of wavelength-selective, localized heating at the single-molecule level using the surface plasmon resonance of a 15 nm gold nanoparticle (AuNP). Our approach is compatible with force-spectroscopy measurements and can be applied to studies of the single-molecule thermodynamic properties of DNA origami nanomachines as well as biomolecular complexes. We further demonstrate wavelength selectivity and establish the temperature dependence of the reaction coordinate for base-pair disruption in the shear-rupture geometry, demonstrating the utility and flexibility of this approach for both fundamental studies of local (nanometer-scale) temperature gradients and rapid and multiplexed nanomachine actuation.

Abstract Gene therapy is a cornerstone of modern medicine's quest for precision treatment to cure human disease including cancer. The current approach to gene delivery technology employs viral vector delivery systems. While effective, these vectors present significant challenges including immunogenicity, safety concerns due to off-target non-tissue specific delivery, payload limitations, and significant cost ultimately diluting their therapeutic potential. Thus, novel approaches to gene delivery are necessary. One such approach to address these critical limitations involves DNA Nanobots, a DNA-based nanotechnology that allows for a tissue-/target-cell specific gene delivery system. Utilizing DNA origami molecular assembly techniques, our DNA Nanobots offer a customizable and highly precise delivery platform, free from viral vector constraints. This innovative approach has the potential to transform the landscape of gene therapy by providing a safer, more efficient, and targeted alternative. Key technical advancements with our DNA Nanobots Gene Delivery Platform include: •CRISPR Cas-9 Integration: By incorporating CRISPR Cas-9 functional proteins, our nanobots were shown to enhance gene editing precision in primary human T cells, broadening therapeutic applications across various genetic conditions. •Targeted mRNA Delivery: Our nanobots revealed promising results in the targeted delivery of mRNA, vital for vaccine development and personalized treatments. •Efficient antisense/siRNA Delivery: Prior studies have overcome traditional challenges including endosomal escape and cytoplasmic entry, demonstrating effective siRNA delivery critical for gene silencing therapeutic development. In summary, DNA Nanobots mark a paradigm shift in gene therapy. Their ability to deliver various nucleic acids with high specificity and efficiency addresses many of the current methodological limitations especially off-target safety concerns and high cost. This advancement not only enhances the therapeutic index of existing treatments but also opens avenues for novel therapeutic strategies, potentially reshaping the future of molecular medicine and gene therapy. Citation Format: Melika Shahhoessini, Jeffrey R. Spitzner, Christopher R. Lucas, Carlos E. Castro, Patrick D. Halley. Exploring the potential of targeted DNA Nanobot delivery systems for gene delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7238.

Comprehending cellular interactions within the extracellular matrix (ECM) is crucial for unraveling the intricacies of cell fate and functions, with far-reaching implications for diseases like metabolic disorders and cancer. These interactions are influenced by the physical and biochemical properties of the surrounding ECM, which constantly remodels through crosstalk between its constituents, maintaining microenvironmental homeostasis. Consequently, investigating intercellular interactions within a 3D tissue model and between ECM components becomes pivotal for elucidating the underlying mechanisms driving disease progression.

Many experimental and computational efforts have sought to understand DNA origami folding, but the time and length scales of this process pose significant challenges. Here, we present a mesoscopic model that uses a switchable force field to capture the behavior of single- and double-stranded DNA motifs and transitions between them, allowing us to simulate the folding of DNA origami up to several kilobases in size. Brownian dynamics simulations of small structures reveal a hierarchical folding process involving zipping into a partially folded precursor followed by crystallization into the final structure. We elucidate the effects of various design choices on folding order and kinetics. Larger structures are found to exhibit heterogeneous staple incorporation kinetics and frequent trapping in metastable states, as opposed to more accessible structures which exhibit first-order kinetics and virtually defect-free folding. This model opens an avenue to better understand and design DNA nanostructures for improved yield and folding performance. The self-assembly process of DNA nanostructures is still not well understood, especially for DNA origami. Here, the authors present a mesoscopic model that uses a switchable force field to capture the mechanical behavior of single- and double-stranded DNA motifs and transition between them, allowing access to the long assembly timescales of DNA origami up to several kilobases in size.

DNA origami nanostructures (DOs) are promising tools for applications including drug delivery, biosensing, detecting biomolecules, and probing chromatin substructures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing, visualizing, and controlling biomolecular processes within live cells. We present an approach to deliver DOs into live-cell nuclei. We show that these DOs do not undergo detectable structural degradation in cell culture media or cell extracts for 24 hours. To deliver DOs into the nuclei of human U2OS cells, we conjugated 30-nanometer DO nanorods with an antibody raised against a nuclear factor, specifically the largest subunit of RNA polymerase II (Pol II). We find that DOs remain structurally intact in cells for 24 hours, including inside the nucleus. We demonstrate that electroporated anti–Pol II antibody–conjugated DOs are piggybacked into nuclei and exhibit subdiffusive motion inside the nucleus. Our results establish interfacing DOs with a nuclear factor as an effective method to deliver nanodevices into live-cell nuclei.

DNA origami nanotechnology has great potential in multiple fields including biomedical, biophysical, and nanofabrication applications. However, current production pipelines lead to single-use devices incorporating a small fraction of initial reactants, resulting in a wasteful manufacturing process. Here, we introduce two complementary approaches to overcome these limitations by recycling the strand components of DNA origami nanostructures (DONs). We demonstrate reprogramming entire DONs into new devices, reusing scaffold strands. We validate this approach by reprogramming DONs with complex geometries into each other, using their distinct geometries to verify successful scaffold recycling. We reprogram one DON into a dynamic structure and show both pristine and recycled structures display similar properties. Second, we demonstrate the recovery of excess staple strands postassembly and fold DONs with these recycled strands, showing these structures exhibit the expected geometry and dynamic properties. Finally, we demonstrate the combination of both approaches, successfully fabricating DONs solely from recycled DNA components.

Scaffolded DNA origami relies on the programmed interaction between many different single-stranded DNAs (ssDNA). While short oligonucleotides can be readily produced through solid-phase synthesis, production of the scaffold strand is mostly limited to a few available sources. Even though there has been significant effort to expand the available sources for long ssDNAs, sources of ssDNA are still significantly limited compared to widely available sources (e.g. plasmids) of double-stranded DNA (dsDNA).

Abstract Acute Myeloid Leukemia (AML) treatment faces considerable challenges, in particular the delivery of therapeutics with sufficient potency and specificity. Traditional Antibody-Drug Conjugates (ADCs) offer a strategic approach to cancer therapy by combining the targeting capabilities of monoclonal antibodies with the cell-killing effect of cytotoxic drugs. However, the limitations in drug-to-antibody ratio (DAR) and the challenges in controlling the release kinetics have often hindered their therapeutic window suggesting novel approaches are necessary. Our approach integrates DNA nanobot technology to facilitate targeted delivery of anthracycline agents directly to CD37-expressing leukemic cells. CD37, a tetraspanin superfamily antigen, has shown promise as a target for AML due to its selective expression on immune cells and favorable internalization properties. By harnessing DNA origami assembly techniques, we developed a customizable platform capable of accommodating a significantly higher payload of chemotherapeutic agents than traditional ADCs. This increased payload potential, paired with the precision targeting of CD37, presents a potent therapeutic strategy with the possibility of reduced systemic toxicity. Here we present preclinical evidence demonstrating targeted delivery of our DNA nanobot drug delivery device and anthracycline payload to CD37+ AML target cells in vitro. In addition, we show targeted efficacy in vitro and in vivo of our anthracycline-loaded DNA nanobot-enabled delivery system, which significantly outperformed free anthracycline in AML target cells. Collectively, our findings suggest an improvement in the therapeutic index of anthracycline and antimitotic agents as well as the ability to combine multiple drug pathways in a single delivery device. The implications of this work extend beyond AML, offering a versatile platform that could revolutionize ADC chemistry and targeted drug delivery. We propose that our DNA nanobot delivery system signifies a pivotal advancement in the field of targeted cancer therapy, with the potential to overcome longstanding barriers in the treatment of AML and potentially other malignancies. Citation Format: Nicholas Vantangoli, Patrick D. Halley, Jeffrey R. Spitzner, John C. Byrd, Karilyn T. Larkin, Carlos E. Castro, Christopher R. Lucas. Enhanced payload delivery for acute myeloid leukemia treatment through CD37-targeting DNA nanobots [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 479.

While there has been much effort to miniaturize manufacturing methods of robotics to micron and millimeter scale, there are few approaches to integrate sensing, communication, and actuation at the nanometer to micron scale. In an effort to create new DNA-based nanoscale robotic materials, we have created a DNA origami structure designed to communicate signals over a distance of several microns, and to do so faster by several orders of magnitude than hybridization based dynamic DNA structures. Signal transduction assemblies can be triggered specifically at one end by an external stimulus, and we aim to communicate a signal through a sequence of conformational changes of dynamic modules within a filament assembly.

Biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces, but they are not optimized for studies in constrained environments as they require attachment to micron-scale probes such as beads or cantilevers. Nanomechanical devices are a promising alternative, but this requires versatile designs that can be tuned to respond to a wide range of forces. We investigate the properties of a nanoscale force sensitive DNA origami device which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, has a binary (open or closed) response to an applied force by undergoing a reversible structural transition. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). The DNA oligonucleotide design parameters also strongly influence the efficiency of resetting the initial state, with higher stability devices (≳10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show the opening force is tunable in real time by adding a single DNA oligonucleotide. These results establish the potential of the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.

ABSTRACT DNA origami is a rapidly emerging nanotechnology that enables researchers to create nanostructures with unprecedented geometric precision that have tremendous potential to advance a variety of fields, including molecular sensing, robotics, and nanomedicine. Hence, many students could benefit from exposure to basic knowledge of DNA origami nanotechnology. However, due to the complexity of design, cost of materials, and cost of equipment, experiments with DNA origami have been limited mainly to research institutions in graduate-level laboratories with significant prior expertise and well-equipped laboratories. This work focuses on overcoming critical barriers to translating DNA origami methods to educational laboratory settings. In particular, we present a streamlined protocol for fabrication and analysis of DNA origami nanostructures that can be carried out within a 2-h laboratory course using low-cost equipment, much of which is readily available in educational laboratories and science classrooms. We focus this educational experiment module on a DNA origami nanorod structure that was previously developed for drug delivery applications. In addition to fabricating nanostructures, we demonstrate a protocol for students to analyze structures via gel electrophoresis using classroom-ready gel equipment. These results establish a basis to expose students to DNA origami nanotechnology and can enable or reinforce valuable learning milestones in fields such as biomaterials, biological engineering, and nanomedicine. Furthermore, introducing students to DNA nanotechnology and related fields can also have the potential to increase interest and future involvement by young students.