Conformation of von Willebrand factor in shear flow revealed with stroboscopic single-molecule imaging.

von Willebrand factor (VWF) is a multimeric blood protein that acts as a mechanical probe, responding to changes in flow to initiate platelet plug formation. Previously, our laboratory tests had shown that using single-molecule imaging that shear stress can extend surface-tethered VWF, but paradoxically, we found that the required shear stress was higher than reported for free-in-flow VWF, an observation inconsistent with basic physical principles. To resolve this inconsistency critical to VWF's molecular mechanism, we measured free-VWF extension in shear flow using pulsed laser stroboscopic imaging of single molecules. Here, laser pulses of different durations are used to capture multiple images of the same molecule within each frame, enabling accurate length measurements in the presence of motion blur. At high shear stresses, we observed a mean shift in VWF extension of <200 nm, much shorter than the multiple-micron extensions previously reported with no evidence for the predicted sharp globule-stretch conformational transition. Modeling VWF with a Brownian dynamics simulation, our results were consistent with VWF behaving as an uncollapsed polymer rather than the theorized compact ball. The muted response of free VWF to high shear rates implies that the tension experienced by free VWF in physiological shear flow is lower than indicated by previous reports and that tethering to platelets or the vessel wall is required to mechanically activate VWF adhesive function for primary hemostasis.

Mapping Single-Molecule Protein Complexes in 3D with DNA Nanoswitch Calipers.

The ability to accurately map the 3D geometry of single-molecule complexes in trace samples is a challenging goal that would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices, we call DNA nanoswitch calipers, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5 Å agreement with previously reported structures.

Resolving Molecular Heterogeneity with Single-Molecule Centrifugation.

For many classes of biomolecules, population-level heterogeneity is an essential aspect of biological function─from antibodies produced by the immune system to post-translationally modified proteins that regulate cellular processes. However, heterogeneity is difficult to fully characterize for multiple reasons: (i) single-molecule approaches are needed to avoid information lost by ensemble-level averaging, (ii) sufficient statistics must be gathered on both a per-molecule and per-population level, and (iii) a suitable analysis framework is required to make sense of a potentially limited number of intrinsically noisy measurements. Here, we introduce an approach that overcomes these difficulties by combining three techniques: a DNA nanoswitch construct to repeatedly interrogate the same molecule, a benchtop centrifuge force microscope (CFM) to obtain thousands of statistics in a highly parallel manner, and a Bayesian nonparametric (BNP) inference method to resolve separate subpopulations with distinct kinetics. We apply this approach to characterize commercially available antibodies and find that polyclonal antibody from rabbit serum is well-modeled by a mixture of three subpopulations. Our results show how combining a spatially and temporally multiplexed nanoswitch-CFM assay with BNP analysis can help resolve complex biomolecular interactions in heterogeneous samples.

Single-molecule imaging of von Willebrand factor reveals tension-dependent self-association.

von Willebrand factor (VWF) is an ultralong concatemeric protein important in hemostasis and thrombosis. VWF molecules can associate with other VWF molecules, but little is known about the mechanism. Hydrodynamic drag exerts tensile force on surface-tethered VWF that extends it and is maximal at the tether point and declines linearly to 0 at the downstream free end. Using single-molecule fluorescence microscopy, we directly visualized the kinetics of binding of free VWF in flow to surface-tethered single VWF molecules. We showed that self-association requires elongation of tethered VWF and that association increases with tension in tethered VWF, reaches half maximum at a characteristic tension of ∼10 pN, and plateaus above ∼25 pN. Association is reversible and hence noncovalent; a sharp decrease in shear flow results in rapid dissociation of bound VWF. Tethered primary VWF molecules can recruit more than their own mass of secondary VWF molecules from the flow stream. Kinetics show that instead of accelerating, the rate of accumulation decreases with time, revealing an inherently self-limiting self-association mechanism. We propose that this may occur because multiple tether points between secondary and primary VWF result in lower tension on the secondary VWF, which shields more highly tensioned primary VWF from further association. Glycoprotein Ibα (GPIbα) binding and VWF self-association occur in the same region of high tension in tethered VWF concatemers; however, the half-maximal tension required for activation of GPIbα is higher, suggesting differences in molecular mechanisms. These results have important implications for the mechanism of platelet plug formation in hemostasis and thrombosis.

DNA Nanoswitch Barcodes for Multiplexed Biomarker Profiling.

Molecular biomarkers play a key role in the clinic, aiding in diagnostics and prognostics, and in the research laboratory, contributing to our basic understanding of diseases. Detecting multiple and diverse molecular biomarkers within a single accessible assay would have great utility, providing a more comprehensive picture for clinical evaluation and research, but is a challenge with standard methods. Here, we report programmable DNA nanoswitches for multiplexed detection of up to 6 biomarkers at once with each combination of biomarkers producing a unique barcode signature among 64 possibilities. As a defining feature of our method, we show "mixed multiplexing" for simultaneous barcoded detection of different types of biomolecules, for example, DNA, RNA, antibody, and protein in a single assay. To demonstrate clinical potential, we show multiplexed detection of a prostate cancer biomarker panel in serum that includes two microRNA sequences and prostate specific antigen.

Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection.

Protein detection and quantification play critical roles in both basic research and clinical practice. Current detection platforms range from the widely used ELISA to more sophisticated, and more expensive, approaches such as digital ELISA. Despite advances, there remains a need for a method that combines the simplicity and cost-effectiveness of ELISA with the sensitivity and speed of modern approaches in a format suitable for both laboratory and rapid, point-of-care applications. Building on recent developments in DNA structural nanotechnology, we introduce the nanoswitch-linked immunosorbent assay (NLISA), a detection platform based on easily constructed DNA nanodevices that change conformation upon binding to a target protein with the results read out by gel electrophoresis. NLISA is surface-free and includes a kinetic-proofreading step for purification, enabling both enhanced sensitivity and reduced cross-reactivity. We demonstrate femtomolar-level detection of prostate-specific antigen in biological fluids, as well as reduced cross-reactivity between different serotypes of dengue and also between a single-mutation and wild-type protein. NLISA is less expensive, uses less sample volume, is more rapid, and, with no washes, includes fewer hands-on steps than ELISA, while also achieving superior sensitivity. Our approach also has the potential to enable rapid point-of-care assays, as we demonstrate by performing NLISA with an iPad/iPhone camera for imaging.

Force Spectroscopy and Beyond: Innovations and Opportunities.

Life operates at the intersection of chemistry and mechanics. Over the years, we have made remarkable progress in understanding life from a biochemical perspective and the mechanics of life at the single-molecule scale. Yet the full integration of physical and mechanical models into mainstream biology has been impeded by technical and conceptual barriers, including limitations in our ability to 1) easily measure and apply mechanical forces to biological systems, 2) scale these measurements from single-molecule characterization to more complex biomolecular systems, and 3) model and interpret biophysical data in a coherent way across length scales that span single molecules to cells to multicellular organisms. In this manuscript, through a look at historical and recent developments in force spectroscopy techniques and a discussion of a few exemplary open problems in cellular biomechanics, we aim to identify research opportunities that will help us reach our goal of a more complete and integrated understanding of the role of force and mechanics in biological systems.

DNA nanoswitches: a quantitative platform for gel-based biomolecular interaction analysis.

We introduce a nanoscale experimental platform that enables kinetic and equilibrium measurements of a wide range of molecular interactions using a gel electrophoresis readout. Programmable, self-assembled DNA nanoswitches serve both as templates for positioning molecules and as sensitive, quantitative reporters of molecular association and dissociation. We demonstrated this low-cost, versatile, 'lab-on-a-molecule' system by characterizing ten different interactions, including a complex four-body interaction with five discernible states.

Protocol for sortase-mediated construction of DNA-protein hybrids and functional nanostructures.

Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA-protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA-peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA-protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein-DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA-protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes.

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 10(5) k(B)T 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.

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.

Mapping Single-molecule Protein Complexes in 3D with DNA Nanoswitch Calipers.

The ability to accurately map the 3D geometry of single-molecule complexes in trace samples would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force-spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices we call DNA Nanoswitch Calipers, which we have previously introduced, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5 Å agreement to previously reported structures.

A non-enzymatic test for SARS-CoV-2 RNA using DNA nanoswitches.

The emergence of a highly contagious novel coronavirus in 2019 led to an unprecedented need for large scale diagnostic testing. The associated challenges including reagent shortages, cost, deployment delays, and turnaround time have all highlighted the need for an alternative suite of low-cost tests. Here, we demonstrate a diagnostic test for SARS-CoV-2 RNA that provides direct detection of viral RNA and eliminates the need for costly enzymes. We employ DNA nanoswitches that respond to segments of the viral RNA by a change in shape that is readable by gel electrophoresis. A new multi-targeting approach samples 120 different viral regions to improve the limit of detection and provide robust detection of viral variants. We apply our approach to a cohort of clinical samples, positively identifying a subset of samples with high viral loads. Since our method directly detects multiple regions of viral RNA without amplification, it eliminates the risk of amplicon contamination and renders the method less susceptible to false positives. This new tool can benefit the COVID-19 pandemic and future emerging outbreaks, providing a third option between amplification-based RNA detection and protein antigen detection. Ultimately, we believe this tool can be adapted both for low-resource onsite testing as well as for monitoring viral loads in recovering patients.

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.

Single-molecule force spectroscopy reveals the dynamic strength of the hair-cell tip-link connection.

The conversion of auditory and vestibular stimuli into electrical signals is initiated by force transmitted to a mechanotransduction channel through the tip link, a double stranded protein filament held together by two adhesion bonds in the middle. Although thought to form a relatively static structure, the dynamics of the tip-link connection has not been measured. Here, we biophysically characterize the strength of the tip-link connection at single-molecule resolution. We show that a single tip-link bond is more mechanically stable relative to classic cadherins, and our data indicate that the double stranded tip-link connection is stabilized by single strand rebinding facilitated by strong cis-dimerization domains. The measured lifetime of seconds suggests the tip-link is far more dynamic than previously thought. We also show how Ca2+ alters tip-link lifetime through elastic modulation and reveal the mechanical phenotype of a hereditary deafness mutation. Together, these data show how the tip link is likely to function during mechanical stimuli.

Single-molecule mechanical fingerprinting with DNA nanoswitch calipers.

Decoding the identity of biomolecules from trace samples is a longstanding goal in the field of biotechnology. Advances in DNA analysis have substantially affected clinical practice and basic research, but corresponding developments for proteins face challenges due to their relative complexity and our inability to amplify them. Despite progress in methods such as mass spectrometry and mass cytometry, single-molecule protein identification remains a highly challenging objective. Towards this end, we combine DNA nanotechnology with single-molecule force spectroscopy to create a mechanically reconfigurable DNA nanoswitch caliper capable of measuring multiple coordinates on single biomolecules with atomic resolution. Using optical tweezers, we demonstrate absolute distance measurements with ångström-level precision for both DNA and peptides, and using multiplexed magnetic tweezers, we demonstrate quantification of relative abundance in mixed samples. Measuring distances between DNA-labelled residues, we perform single-molecule fingerprinting of synthetic and natural peptides, and show discrimination, within a heterogeneous population, between different posttranslational modifications. DNA nanoswitch calipers are a powerful and accessible tool for characterizing distances within nanoscale complexes that will enable new applications in fields such as single-molecule proteomics.

Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection.

MicroRNAs are short, non-coding RNA sequences involved in gene expression regulation. Quantification of miRNAs in biological fluids involves time consuming and laborious methods such as Northern blotting or PCR-based techniques. Molecular beacons (MB) are an attractive means for rapid detection of miRNAs, although the need for sophisticated readout methods limits their use in research and clinical settings. Here, we introduce a novel method based on delayed electrophoretic mobility, as a quantitative means for detection of miRNAs-MB hybridization. Upon hybridization with the target miRNAs, MB form a fluorescent duplex with reduced electrophoretic mobility, thus bypassing the need for additional staining. In addition to emission of light, the location of the fluorescent band on the gel acts as an orthogonal validation of the target identity, further confirming the specificity of binding. The limit of detection of this approach is approximately 100 pM, depending on the MB sequence. The method is sensitive enough to detect specific red blood cell miRNAs molecules in total RNA, with single nucleotide specificity. Altogether, we describe a rapid and affordable method that offers sensitive detection of single-stranded small DNA and RNA sequences.

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.

Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping.

Single-molecule force spectroscopy has brought many new insights into nanoscale biology, from the functioning of molecular motors to the mechanical response of soft materials within the cell. To expand the single-molecule toolbox, we have developed a surface-free force spectroscopy assay based on a high-speed hydrodynamic trap capable of applying extremely high tensions for long periods of time. High-speed single-molecule trapping is enabled by a rigid and gas-impermeable microfluidic chip, rapidly and inexpensively fabricated out of glass, double-sided tape and UV-curable adhesive. Our approach does not require difficult covalent attachment chemistries, and enables simultaneous force application and single-molecule fluorescence. Using this approach, we have induced a highly extended state with twice the contour length of B-DNA in regions of partially intercalated double-stranded (dsDNA) by applying forces up to 250 pN. This highly extended state resembles the hyperstretched state of dsDNA, which was initially discovered as a structure fully intercalated by dyes under high tension. It has been hypothesized that hyperstretched DNA could also be induced without the aid of intercalators if high-enough forces were applied, which matches our observation. Combining force application with single-molecule fluorescence imaging is critical for distinguishing hyperstretched DNA from single-stranded DNA that can result from peeling. High-speed hydrodynamic trapping is a powerful yet accessible force spectroscopy method that enables the mechanics of biomolecules to be probed in previously difficult to access regimes.

Dual functions of Aire CARD multimerization in the transcriptional regulation of T cell tolerance.

Aggregate-like biomolecular assemblies are emerging as new conformational states with functionality. Aire, a transcription factor essential for central T cell tolerance, forms large aggregate-like assemblies visualized as nuclear foci. Here we demonstrate that Aire utilizes its caspase activation recruitment domain (CARD) to form filamentous homo-multimers in vitro, and this assembly mediates foci formation and transcriptional activity. However, CARD-mediated multimerization also makes Aire susceptible to interaction with promyelocytic leukemia protein (PML) bodies, sites of many nuclear processes including protein quality control of nuclear aggregates. Several loss-of-function Aire mutants, including those causing autoimmune polyendocrine syndrome type-1, form foci with increased PML body association. Directing Aire to PML bodies impairs the transcriptional activity of Aire, while dispersing PML bodies with a viral antagonist restores this activity. Our study thus reveals a new regulatory role of PML bodies in Aire function, and highlights the interplay between nuclear aggregate-like assemblies and PML-mediated protein quality control.