Search for: All records

Abstract 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. 

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. 

Abstract 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. 

Recent advances in structural DNA nanotechnology have been facilitated by design tools that continue to push the limits of structural complexity while simplifying an often-tedious design process. We recently introduced the software MagicDNA, which enables design of complex 3D DNA assemblies with many components; however, the design of structures with free-form features like vertices or curvature still required iterative design guided by simulation feedback and user intuition. Here, we present an updated design tool, MagicDNA 2.0, that automates the design of free-form 3D geometries, leveraging design models informed by coarse-grained molecular dynamics simulations. Our GUI-based, stepwise design approach integrates a high level of automation with versatile control over assembly and subcomponent design parameters. We experimentally validated this approach by fabricating a range of DNA origami assemblies with complex free-form geometries, including a 3D Nozzle, G-clef, and Hilbert and Trifolium curves, confirming excellent agreement between design input, simulation, and structure formation. 

In this work, we describe the development of a computational model for arrays of rotary DNA origami elements which can self-organize on a large scale and explore the interesting morphologies and order–disorder transition behavior of these systems. 

Abstract Transcription factors (TF) require access to target sites within nucleosomes to initiate transcription. The target site position within the nucleosome significantly influences TF occupancy, but how is not quantitatively understood. Using ensemble and single-molecule fluorescence measurements, we investigated the targeting and occupancy of the transcription factor, Gal4, at different positions within the nucleosome. We observe a dramatic decrease in TF occupancy to sites extending past 30 base pairs (bp) into the nucleosome which cannot be explained by changes in the TF dissociation rate or binding site orientation. Instead, the nucleosome unwrapping free energy landscape is the primary determinant of Gal4 occupancy by reducing the Gal4 binding rate. The unwrapping free energy landscape defines two distinct regions of accessibility and kinetics with a boundary at 30 bp into the nucleosome where the inner region is over 100-fold less accessible. The Gal4 binding rate in the inner region no longer depends on its concentration because it is limited by the nucleosome unwrapping rate, while the frequency of nucleosome rewrapping decreases because Gal4 exchanges multiple times before the nucleosome rewraps. Our findings highlight the importance of the nucleosome unwrapping free energy landscape on TF occupancy and dynamics that ultimately influences transcription initiation. 

Chromatin, a dynamic protein-DNA complex that regulates eukaryotic genome accessibility and essential functions, is composed of nucleosomes connected by linker DNA with each nucleosome consisting of DNA wrapped around an octamer of histones H2A, H2B, H3 and H4. Magic angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy can yield unique insights into histone structure and dynamics in condensed nucleosomes and nucleosome arrays representative of chromatin at physiological concentrations. Recently we used J-coupling-based solid-state NMR methods to investigate with residue-specific resolution the conformational dynamics of histone H3 N-terminal tails in 16-mer nucleosome arrays containing 15, 30 or 60 bp DNA linkers. Here, we probe the H3 core domain in the 16-mer arrays as a function of DNA linker lengthviadipolar coupling-based¹H-detected solid-state NMR techniques. Specifically, we established nearly complete assignments of backbone chemical shifts for H3 core residues in arrays with 15–60 bp DNA linkers reconstituted with²H,¹³C,¹⁵N-labeled H3. Overall, these chemical shifts were similar irrespective of the DNA linker length indicating no major changes in H3 core conformation. Notably, however, multiple residues at the H3-nucleosomal DNA interface in arrays with 15 bp DNA linkers exhibited relatively pronounced differences in chemical shifts and line broadening compared to arrays with 30 and 60 bp linkers. These findings are consistent with increased heterogeneity in nucleosome packing and structural strain within arrays containing short DNA linkers that likely leads to side-chains of these interfacial residues experiencing alternate conformations or shifts in their rotamer populations relative to arrays with the longer DNA linkers. 

Abstract Single molecule force spectroscopy is a powerful approach to probe the structure, conformational changes, and kinetic properties of biological and synthetic macromolecules. However, common approaches to apply forces to biomolecules require expensive and cumbersome equipment and relatively large probes such as beads or cantilevers, which limits their use for many environments and makes integrating with other methods challenging. Furthermore, existing methods have key limitations such as an inability to apply compressive forces on single molecules. We report a nanoscale DNA force spectrometer (nDFS), which is based on a DNA origami hinge with tunable mechanical and dynamic properties. The angular free energy landscape of the nDFS can be engineered across a wide range through substitution of less than 5% of the strand components. We further incorporate a removable strut that enables reversible toggling of the nDFS between open and closed states to allow for actuated application of tensile and compressive forces. We demonstrate the ability to apply compressive forces by inducing a large bend in a 249bp DNA molecule, and tensile forces by inducing DNA unwrapping of a nucleosome sample. These results establish a versatile tool for force spectroscopy and robust methods for designing nanoscale mechanical devices with tunable force application.