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


Y Jiang, T Feldman, JAM Bakx, D Yang, WP Wong. Lab Chip (2020). PDF

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... Read more ›

The Dynamic Strength of the Hair-Cell Tip Link Reveals Mechanisms of Hearing and Deafness


EM Mulhall, A Ward, D Yang, MA Koussa, DP Corey^, WP Wong^. bioRxiv (2019). PDF

Our senses of hearing and balance rely on the extraordinarily sensitive molecular machinery of the inner ear to convert deflections as small as the width of a single carbon atom into electrical signals that the brain can process. In humans and other vertebrates, transduction is mediated by hair cells, where tension on tip links conveys force to mechanosensitive ion channels. Each tip link comprises two helical filaments of atypical cadherins bound at their N-termini through two unique adhesion bonds. Tip links must be strong enough to maintain a connection to the mechanotransduction channel under the dynamic forces exerted by sound or head movement—yet might also act as mechanical circuit breakers, releasing under extreme conditions to preserve the delicate structures within the hair cell. Previous studies have argued that this connection.... Read more ›

Electrostatic Steering Enables Flow-Activated Von Willebrand Factor to Bind Platelet Glycoprotein, Revealed by Single-Molecule Stretching and Imaging

Electrostatic Steering Enables Flow-Activated Von Willebrand Factor to Bind Platelet Glycoprotein, Revealed by Single-Molecule Stretching and Imaging

Y Jiang*, H Fu*, T Springer^, WP Wong^. J. Mol. Biol. (2019). PDF

Von Willebrand factor (VWF), a large multimeric blood protein, senses changes in shear stress during bleeding and responds by binding platelets to plug ruptures in the vessel wall. Molecular mechanisms underlying this dynamic process are difficult to uncover using standard approaches due to the challenge of applying mechanical forces while monitoring structure and activity. By combining single-molecule fluorescence imaging with high-pressure, rapidly switching microfluidics, we reveal the key role of electrostatic steering in accelerating the binding between flow-activated VWF and GPIbα, and in rapidly immobilizing platelets under flow. We measure the elongation and tension-dependent activation of individual VWF multimers under a range of ionic strengths and pH levels, and find that the association rate is enhanced by 4 orders of magnitude by... Read more ›

Force Spectroscopy and Beyond: Innovations and Opportunities

Force Spectroscopy and Beyond: Innovations and Opportunities

Nathwani^, WM Shih, WP Wong^. Biophys. J. (2018). PDF

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... Read more ›

Repurposing a benchtop centrifuge for high-throughput single-molecule force spectroscopy

Repurposing a Benchtop Centrifuge for High-Throughput Single-Molecule Force Spectroscopy

D Yang, WP Wong. Methods in Molecular Biology: Single Molecule Analysis: Methods and Protocols, 2nd Edition 1665, EJG Peterman, ed., Springer, 353-366 (2018). PDF

We present high-throughput single-molecule manipulation using a benchtop centrifuge, overcoming limitations common in other single-molecule approaches such as high cost, low throughput, technical difficulty, and strict infrastructure requirements. An inexpensive and compact Centrifuge Force Microscope (CFM) adapted to a commercial centrifuge enables use by nonspecialists, and integration with DNA nanoswitches facilitates both reliable measurements and repeated molecular interrogation. Here, we provide detailed protocols for constructing the CFM, creating DNA nanoswitch samples, and carrying out single-molecule force measurements. Read more ›

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

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

CH Hansen, D Yang, MA Koussa, WP Wong. PNAS (2017). PDF

Basic research and medical diagnostics rely on the ability to detect and quantify specific proteins in biological fluids. While numerous current detection techniques exist, these are often limited by trade-offs between ease of use, sensitivity, and cost. Here, we present the nanoswitch-linked immunosorbent assay (NLISA), an accessible, sensitive, and low-cost detection platform that is based upon nanoscale devices that change confirmation upon binding a target protein. NLISA is surface-free and includes a kinetic-proofreading purification step, enabling both enhanced sensitivity and the ability to accurately distinguish between similar proteins from different strains of the same virus or that differ by only a single mutation. Our method is also readily transferable to point-of-care devices due to an easy readout and few hands-on steps. Read more ›

Flow-induced elongation of von Willebrand factor precedes tension-dependent activation

Flow-induced elongation of von Willebrand factor precedes tension-dependent activation

H Fu*, Y Jiang*, D Yang, F Scheiflinger, WP Wong^, TA Springer^. Nat. Commun. 8, 324 (2017). PDF

Von Willebrand factor, an ultralarge concatemeric blood protein, must bind to platelet GPIbα during bleeding to mediate hemostasis, but not in the normal circulation to avoid thrombosis. Von Willebrand factor is proposed to be mechanically activated by flow, but the mechanism remains unclear. Using microfluidics with single-molecule imaging, we simultaneously monitored reversible Von Willebrand factor extension and binding to GPIbα under flow. We show that Von Willebrand factor is activated through a two-step conformational transition: first, elongation from compact to linear form, and subsequently, a tension-dependent local transition to a state with high affinity for GPIbα. High-affinity sites develop only in upstream regions of VWF where tension exceeds ~21 pN and depend upon electrostatic interactions... Read more ›

Small but mighty: Nanoparticles probe cellular signaling pathways

Small but Mighty: Nanoparticles Probe Cellular Signaling Pathways

D Yang, WP Wong. Dev. Cell 37 (5), 397-398 (2016). PDF

Mechanical forces play key roles in regulating cellular pathways but are challenging to study using standard biological approaches. In a recent issue of Cell, Seo et al. (2016) present a platform for in vivo single-molecule manipulation, using magnetoplasmonic nanoparticles capable of imaging, localizing, and force-loading receptor proteins at high spatiotemporal resolution. Read more ›

Multiplexed single-molecule force spectroscopy using a centrifuge

Multiplexed single-molecule force spectroscopy using a centrifuge

D Yang, A Ward, K Halvorsen, WP Wong. Nat. Commun. 7, 11026 (2016). PDF

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... Read more ›


Featured as a research highlight in Nature Methods:

  • “Putting a spin on high-throughput force spectroscopy,” Nat. Methods 13, 396 (2016).

DNA Nanoswitches: A quantitative platform for gel-based biomolecular interaction analysis

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

MA Koussa*, K Halvorsen*, A Ward, WP Wong. Nat. Methods 12, 123-126 (2015). PDF, Protocol, Videos

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. Read more ›

New class of biological detectors for WIMPs 

New class of biological detectors for WIMPs

AK Drukier, Ch Cantor, M Chonofsky, GM Church, RL Fagaly, K Freese, A Lopez, T Sano, C Savage, WP Wong. Int. J. Mod. Phys. 29 (19), 1443007 (2014). PDF  

Weakly Interacting Massive Particles (WIMPs) may constitute a large fraction of the matter in the Universe. There are excess events in the data of DAMA/LIBRA, CoGeNT, CRESST-II, and recently CDMS-Si, which could be consistent with WIMP masses of approximately 10 GeV/c2. However, for MDM > 10 GeV/c2 null results of the CDMS- Ge, XENON, and LUX detectors may be in tension with the potential detections for certain dark matter scenarios and assuming a certain light response. We propose the use of a new class of biological dark matter (DM) detectors to further examine this light dark matter hypothesis, taking advantage of new signatures with low atomic number targets... Read more ›

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

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

MA Koussa, M Sotomayor, WP Wong. Methods 67 (2), 134-141 (2014). PDF

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. Read more ›

A bioinspired multivalent DNA network for capture and release of cells

Bioinspired multivalent DNA network for capture and release of cells

W Zhao, CH Cui, S Bose, D Guo, C Shen, WP Wong, K Halvorsen, OC Farokhzad, GSL Teo, JA Phillips, DM Dorfman, R Karnik, JM Karp. PNAS 109, 19626-19631 (2012). PDF    

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... Read more ›

Physical manipulation of the Escherichia coli chromosome reveals its soft nature

Physical manipulation of the Escherichia coli chromosome reveals its soft nature

J Pelletier, K Halvorsen, BY Ha, R Paparcone, SJ Sandler, CL Woldringh, WP Wong, S Jun. PNAS 109, E2649-E2656 (2012). PDF 

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... Read more ›


Featured as a research highlight in Nature Methods and PNAS:

  • “Soft bacterial chromosomes,” Nat. Methods 9, 1047 (2012). PDF

  • “Under pressure, bacterial chromosomes mimic soft springs,” PNAS 109 (2012). PDF

Binary DNA Nanostructures for Data Encryption

Binary DNA Nanostructures for Data Encryption

K Halvorsen, WP Wong. PLoS ONE 7 (9), e44212 (2012). PDF

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. Read more ›

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

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

K Halvorsen, D. Schaak, WP Wong. Nanotechnology 22 (49), 494005 (2011). PDF

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... Read more ›

Massively parallel single-molecule manipulation using centrifugal force

Massively Parallel Single-Molecule Manipulation Using Centrifugal Force

K Halvorsen, WP Wong. Biophys. J. 98 (11), L53-L55 (2010). PDF

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... Read More ›


Featured as a research highlight in Nature and Nature Methods:

  • “Biophysics: Molecular carnival ride,” Nature 465, 669 (2010). PDF

  • “High-throughput single-molecule force spectroscopy,” Nat. Methods 7, 489 (2010). PDF

High-precision microsphere sorting using velocity sedimentation 

D Cheng, K Halvorsen, WP Wong. Reviews of Scientific Instruments 81, 026106 (2010). PDF

Monodisperse populations of microspheres are desirable for a variety of research and industrial applications, but many desirable sizes and materials can be difficult to synthesize and have limited commercial availability. We present an effective, straightforward, and low cost method for sorting polydisperse microspheres into many separate monodisperse samples. The approach is to use velocity sedimentation through a density gradient in a long vertical column, followed by carefully targeted extraction. We demonstrate this technique by reducing the coefficient of variation of melamine microspheres from 13% to 1%–4% and glass microspheres from 35% to 3%–8%. This simple and inexpensive method can be used to sort microspheres of many sizes and materials, and is easily scalable, opening the possibility of cheap, monodisperse microspheres. Read more ›

Mechanoenzymatic cleavage of the ultralarge vascular protein, von Willebrand Factor

Mechanoenzymatic cleavage of the ultralarge vascular protein, von Willebrand Factor

X Zhang*, K Halvorsen*, CZ Zh, WP Wong^, TA Springer^. Science 324, 1330-1334 (2009). PDF

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... Read more ›


Featured as a research highlight in Science and Nature Medicine:

  • “Force signaling in Biology,” J Christof, M Gebhardt, M Rief. Science 324, 1278-1280 (2009).

  • “Vascular disorders: Biological shear bolt,” Nature Medicine 7 (15), 738-739 (2009).

A new approach to analysis of single molecule force measurements 

A New Approach to Analysis of Single-Molecule Force Measurements

E Evans, K Halvorsen, K Kinoshita, WP Wong. Handbook of Single Molecule Biophysics, P. Hinterdorfer, ed., Springer, 571-589 (2009). PDF

Using sample data taken from tests of ligand–receptor unbinding and protein unfolding/refolding, we show that populations of “single molecule” events, arranged into statistical arrays expressing the numbers of bonds or initial conformers remaining as a function of time and cumulated into histograms of transitions over fixed time increments, provide the bases for a model-independent assay of the kinetic rates of transition throughout the course of an experiment. Most important, this assay for kinetic rates can be employed with any deterministic mode of force spectroscopy, whether the pulling force increases or decreases with time. Read more ›

Beyond the frame rate: Measuring high-frequency fluctuations with light intensity modulation 

Beyond the frame rate: measuring high-frequency fluctuations with light-intensity modulation

WP Wong*^, K Halvorsen*. Optics Letters 34, 277-279 (2009). PDF

Power-spectral-density measurements of any sampled signal are typically restricted by both acquisition rate and frequency response limitations of instruments, which can be particularly prohibitive for video-based measurements. We have developed a new method called intensity modulation spectral analysis that circumvents these limitations, dramatically extending the effective detection bandwidth. We demonstrate this by video tracking an optically trapped microsphere while oscillating an LED illumination source. This approach allows us to quantify fluctuations of the microsphere at frequencies over 10 times higher than the Nyquist frequency, mimicking a significantly higher frame rate. Read more ›

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

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

V Heinrich*^, WP Wong*, K Halvorsen, E Evans. Langmuir 24, 1194-1203 (2008). PDF

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... Read more ›

The effect of integration time on fluctuation measurements: Calibrating an optical trap in the presence of motion blur


WP Wong*^, K Halvorsen*. Optics Express 14, 12517-12531 (2006). PDF

Dynamical instrument limitations, such as finite detection bandwidth, do not simply add statistical errors to fluctuation measurements, but can create significant systematic biases that affect the measurement of steady-state properties. Such effects must be considered when calibrating ultra-sensitive force probes by analyzing the observed Brownian fluctuations. In this article, we present a novel method for extracting the true spring constant and diffusion coefficient of a harmonically confined Brownian particle that extends the standard equipartition and power spectrum techniques to account for video-image motion blur. These results are confirmed both numerically with a Brownian dynamics simulation, and experimentally with laser optical tweezers. Read more ›

Rare returns on lost effort

Rare returns on lost effort

WP Wong, E Evans, Nature 437, 198-199 (2005). PDF

How does the size of a system affect its thermodynamic irreversibility? A deft experiment that observes the unfolding and refolding of a single molecule of RNA provides insights into the question at a small scale. Read more ›

Exploring reaction pathways of single-molecule interactions through the manipulation and tracking of a potential-confined microsphere in three dimensions

wong_mrs_proceedings_2003-3 (dragged).jp

WP Wong, V Heinrich, E Evans. Mat. Res. Soc. Symp. Proc. 790, P5.1.1-P5.1.12 (2004). PDF

We present a novel method to quantify subtle features of weak chemical transitions by analyzing the 3D Brownian fluctuations of a functionalized microsphere held near a reactive substrate. A weak optical-trapping potential is used to confine motion of the bead to a nanoscale domain, and to apply a controlled bias field to the interaction. Stochastic interruptions in the monitored bead dynamics report formation and release of single molecular bonds. In addition, variations in the motion of a bead linked to the substrate via a biomolecule (a protein or nucleic acid) signal conformational changes in the molecule, such as the folding/unfolding of protein domains or the unzipping of DNA. Read more ›

Exploring single-molecule interactions through 3D optical trapping and tracking: from thermal noise to protein refolding

WP Wong. Ph. D Thesis (advisors: D.R. Nelson and E. Evans), Department of Physics, Harvard University, USA (2006). PDF

Modelling a Tethered Polymer Loop: Shape and thickness compressibility

WP Wong. Master’s Thesis, The University of British Columbia, Canada (2001). PDF