Ken Halvorsen

Mechanoenzymatic Cleavage of the Ultralarge Vascular Protein von Willebrand Factor

Dissecting VWFs Thrombogenic Potential Von Willebrand factor (VWF) is secreted from cells in an ultralarge form (ULVWF) in response to thrombogenic stimuli. Shear forces expose a binding site for platelets, enabling formation of a hemostatic plug. The thrombogenic potential of VWF correlates with its length and is regulated by proteolytic cleavage of the A2 domain. Zhang et al. (p. 1330; see the Perspective by Gebhardt and Rief) now combine single molecule data and polymer dynamics theory to show that shear forces in the circulation are sufficient to unfold the A2 domain and allow cleavage of multimers with more than about 200 monomers. The A2 domain may thus represent the “shear bolt” of VWF, unfolding when multimers experience high forces to allow cleavage and down-regulation of thrombogenic potential. Mechanical forces regulate the length of von Willebrand factor multimers and thus regulate thrombogenic potential. Von Willebrand factor (VWF) is secreted as ultralarge multimers that are cleaved in the A2 domain by the metalloprotease ADAMTS13 to give smaller multimers. Cleaved VWF is activated by hydrodynamic forces found in arteriolar bleeding to promote hemostasis, whereas uncleaved VWF is activated at lower, physiologic shear stresses and causes thrombosis. Single-molecule experiments demonstrate that elongational forces in the range experienced by VWF in the vasculature unfold the A2 domain, and only the unfolded A2 domain is cleaved by ADAMTS13. In shear flow, tensile force on a VWF multimer increases with the square of multimer length and is highest at the middle, providing an efficient mechanism for homeostatic regulation of VWF size distribution by force-induced A2 unfolding and cleavage by ADAMTS13, as well as providing a counterbalance for VWF-mediated platelet aggregation.

Bioinspired multivalent DNA network for capture and release of cells

Capture and isolation of flowing cells and particulates from body fluids has enormous implications in diagnosis, monitoring, and drug testing, yet monovalent adhesion molecules used for this purpose result in inefficient cell capture and difficulty in retrieving the captured cells. Inspired by marine creatures that present long tentacles containing multiple adhesive domains to effectively capture flowing food particulates, we developed a platform approach to capture and isolate cells using a 3D DNA network comprising repeating adhesive aptamer domains that extend over tens of micrometers into the solution. The DNA network was synthesized from a microfluidic surface by rolling circle amplification where critical parameters, including DNA graft density, length, and sequence, could readily be tailored. Using an aptamer that binds to protein tyrosine kinase-7 (PTK7) that is overexpressed on many human cancer cells, we demonstrate that the 3D DNA network significantly enhances the capture efficiency of lymphoblast CCRF-CEM cells over monovalent aptamers and antibodies, yet maintains a high purity of the captured cells. When incorporated in a herringbone microfluidic device, the 3D DNA network not only possessed significantly higher capture efficiency than monovalent aptamers and antibodies, but also outperformed previously reported cell-capture microfluidic devices at high flow rates. This work suggests that 3D DNA networks may have broad implications for detection and isolation of cells and other bioparticles.

Physical manipulation of the Escherichia coli chromosome reveals its soft nature

Replicating bacterial chromosomes continuously demix from each other and segregate within a compact volume inside the cell called the nucleoid. Although many proteins involved in this process have been identified, the nature of the global forces that shape and segregate the chromosomes has remained unclear because of limited knowledge of the micromechanical properties of the chromosome. In this work, we demonstrate experimentally the fundamentally soft nature of the bacterial chromosome and the entropic forces that can compact it in a crowded intracellular environment. We developed a unique “micropiston” and measured the force-compression behavior of single Escherichia coli chromosomes in confinement. Our data show that forces on the order of 100 pN and free energies on the order of 105 kBT are sufficient to compress the chromosome to its in vivo size. For comparison, the pressure required to hold the chromosome at this size is a thousand-fold smaller than the surrounding turgor pressure inside the cell. Furthermore, by manipulation of molecular crowding conditions (entropic forces), we were able to observe in real time fast (approximately 10 s), abrupt, reversible, and repeatable compaction–decompaction cycles of individual chromosomes in confinement. In contrast, we observed much slower dissociation kinetics of a histone-like protein HU from the whole chromosome during its in vivo to in vitro transition. These results for the first time provide quantitative, experimental support for a physical model in which the bacterial chromosome behaves as a loaded entropic spring in vivo.

Massively Parallel Single-Molecule Manipulation Using Centrifugal Force

Precise manipulation of single molecules has already led to remarkable insights in physics, chemistry, biology, and medicine. However, widespread adoption of single-molecule techniques has been impeded by equipment cost and the laborious nature of making measurements one molecule at a time. We have solved these issues by developing an approach that enables massively parallel single-molecule force measurements using centrifugal force. This approach is realized in an instrument that we call the centrifuge force microscope in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force-field while their micro-to-nanoscopic motions are observed. We demonstrate high-throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Additionally, we verify the force accuracy of the instrument by measuring the well-established DNA overstretching transition at 66 +/- 3 pN. With significant benefits in efficiency, cost, simplicity, and versatility, single-molecule centrifugation has the potential to expand single-molecule experimentation to a wider range of researchers and experimental systems.

Imaging biomolecular interactions by fast three-dimensional tracking of laser-confined carrier particles

The quantitative study of the near-equilibrium structural behavior of individual biomolecules requires high-resolution experimental approaches with longtime stability. We present a new technique to explore the dynamics of weak intramolecular interactions. It is based on the analysis of the 3D Brownian fluctuations of a laser-confined glass bead that is tethered to a flat surface by the biomolecule of interest. A continuous autofocusing mechanism allows us to maintain or adjust the height of the optical trap with nanometer accuracy over long periods of time. The resulting remarkably stable trapping potential adds a well-defined femto-to-piconewton force bias to the energy landscape of molecular configurations. A combination of optical interferometry and advanced pattern-tracking algorithms provides the 3D bead positions with nanometer spatial and >120 Hz temporal resolution. The analysis of accumulated 3D positions has allowed us not only to identify small single biomolecules but also to characterize their nanomechanical behavior, for example, the force-extension relations of short oligonucleotides and the unfolding/refolding transitions of small protein tethers.

Nanoengineering a single-molecule mechanical switch using DNA self-assembly

The ability to manipulate and observe single biological molecules has led to both fundamental scientific discoveries and new methods in nanoscale engineering. A common challenge in many single-molecule experiments is reliably linking molecules to surfaces, and identifying their interactions. We have met this challenge by nanoengineering a novel DNA-based linker that behaves as a force-activated switch, providing a molecular signature that can eliminate errant data arising from non-specific and multiple interactions. By integrating a receptor and ligand into a single piece of DNA using DNA self-assembly, a single tether can be positively identified by force-extension behavior, and receptor-ligand unbinding easily identified by a sudden increase in tether length. Additionally, under proper conditions the exact same pair of molecules can be repeatedly bound and unbound. Our approach is simple, versatile and modular, and can be easily implemented using standard commercial reagents and laboratory equipment. In addition to improving the reliability and accuracy of force measurements, this single-molecule mechanical switch paves the way for high-throughput serial measurements, single-molecule on-rate studies, and investigations of population heterogeneity.

Beyond the Fold: Emerging Biological Applications of DNA Origami

The use of DNA as a material for nanoscale construction has blossomed in the past decade. This is largely attributable to the DNA origami technique, which has enabled construction of nanostructures ranging from simple two‐dimensional sheets to complex three‐dimensional objects with defined curves and edges. These structures are amenable to site‐specific functionalization with nanometer precision, and have been shown to exhibit cellular biocompatibility and permeability. The DNA origami technique has already found widespread use in a variety of emerging biological applications such as biosensing, enzyme cascades, biomolecular analysis, biomimetics, and drug delivery. We highlight a few of these applications and comments on the prospects for this rapidly expanding field of research.

Multiplexed single-molecule force spectroscopy using a centrifuge

We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise.

Complex Thermodynamic Behavior of Single-Stranded Nucleic Acid Adsorption to Graphene Surfaces

In just over a decade since its discovery, research on graphene has exploded due to a number of potential applications in electronics, materials, and medicine. In its water-soluble form of graphene oxide, the material has shown promise as a biosensor due to its preferential absorption of single-stranded polynucleotides and fluorescence quenching properties. The rational design of these biosensors, however, requires an improved understanding of the binding thermodynamics and ultimately a predictive model of sequence-specific binding. Toward these goals, here we directly measured the binding of nucleosides and oligonucleotides to graphene oxide nanoparticles using isothermal titration calorimetry and used the results to develop molecular models of graphene-nucleic acid interactions. We found individual nucleosides binding KD values lie in the submillimolar range with binding order of rG < rA < rC < dT < rU, while 5mer and 15mer oligonucleotides had markedly higher binding affinities in the range of micromolar and submicromolar KD values, respectively. The molecular models developed here are calibrated to quantitatively reproduce the above-mentioned experimental results. For oligonucleotides, our model predicts complex binding features such as double-stacked bases and a decrease in the fraction of graphene stacked bases with increasing oligonucleotide length until plateauing beyond ∼10-15 nucleotides. These experimental and computational results set the platform for informed design of graphene-based biosensors, further increasing their potential and application.

Binary DNA Nanostructures for Data Encryption

We present a simple and secure system for encrypting and decrypting information using DNA self-assembly. Binary data is encoded in the geometry of DNA nanostructures with two distinct conformations. Removing or leaving out a single component reduces these structures to an encrypted solution of ssDNA, whereas adding back this missing “decryption key” causes the spontaneous formation of the message through self-assembly, enabling rapid read out via gel electrophoresis. Applications include authentication, secure messaging, and barcoding.