Research Overview

NANOENGINEERING FUNCTIONAL SINGLE-MOLECULE TOOLS WITH DNA SELF-ASSEMBLY

Sat, 10 Mar 2012 20:31:02 -0500

New methods in DNA self-assembly, such as DNA origami, are enabling the precise engineering of nanoscale structures with unique properties. We are using these powerful methods to create new useful tools for studying biomolecular interactions.

For example, we have created a nanoscale mechanical switch that is able to change states under force to report the interactions between molecules of interest. When combined with modern methods in single-molecule manipulation, this becomes a powerful, accessible, and reliable system for measuring molecular kinetics and conformations at the single-molecule level.

This switch is fabricated using DNA self-assembly, taking advantage of the specific and reliable pairing between complementary bases to localize structures with sub-nanometer accuracy using standard test tube chemistry. By mixing a long piece of single-stranded DNA with a carefully designed soup of short DNA oligomers, a looped single-molecule linker with an integrated receptor–ligand pair is self-assembled. Significantly, this approach is easy and accessible, requiring minimal time and equipment.

This functional nanoscale structure enables new and more reliable studies of biomolecular structure and function at the single-molecule level. Experiments show that this force-switchable molecule improves single molecule experiments by providing a tunable molecular “signature” to filter out spurious data. Furthermore, the force-switching behavior of the linker is reversible, enabling repeated measurement of binding and unbinding of a single pair of molecules, paving the way for measurements of on-rates and population heterogeneity. When combined with our recently developed centrifuge force microscope (CFM), these molecular constructs will enable reliable, high-throughput studies of biomolecular interactions, enabling new insights into how force affects single-molecule mechanics and dynamics, including enzymatic activity, binding of receptor-ligand pairs, and drug interactions.

Single-molecule centrifugation: a new approach for massively-parallel single-molecule manipulation

Mon, 14 Jun 2010 16:55:07 -0400

Precise manipulation of single molecules is leading to remarkable insights in physics, chemistry, biology and medicine. However, widespread adoption of single-molecule techniques is impeded by equipment cost and the laborious nature of making measurements one molecule at a time. We are developing a new approach to solve these issues: massively parallel single-molecule force measurements using centrifugal force. This approach is realized in a novel instrument that we call the Centrifuge Force Microscope (CFM), 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 have demonstrated 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 have verified 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.

Mechanoenzymatic cleavage of the ultralarge vascular protein von willebrand factor

Fri, 5 Jun 2009 02:00:33 -0400

We have been studying the vascular protein von Willebrand factor (VWF) and the regulation of blood clotting in collaboration with Timothy A. Springer’s group. The A2 domain of VWF is the key to this largely mechanical molecular feedback loop. Using optical tweezers, we have characterized the unfolding and refolding kinetics of the A2 domain, and have shown that force acts as an “cofactor”, enabling the cleavage of A2 by the ADAMTS13 enzyme. Furthermore, the hydrodynamic tensile forces encountered by VWF in the circulation are sufficient to enable cleavage, which in turn down regulates the body’s hemostatic potential. Here is the abstract of work that has recently been published in Science Magazine [link]:

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, 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.

Beyond the frame rate: measuring high-frequency fluctuations with motion blur

Fri, 29 May 2009 03:00:43 -0400

Motion blur results from the finite exposure time (or shutter speed) of video cameras and other detection and acquisition systems. When properly understood, this effect is not a liability but instead provides valuable dynamical information. Below, we present two methods we have developed for using motion blur: one to calibrate force probes such as optical traps, and the second to measure the power spectral density (PSD) of any signal above the Nyquist frequency of an acquisition system.

Spectrin unfolding/refolding kinetics from dynamic force spectroscopy

Fri, 30 Jan 2009 04:00:30 -0500

We have been studying the kinetics of protein folding and unfolding for spectrin in collaboration with Evan Evans’ group. Spectrin repeats are found in numerous structural proteins that are subject to mechanical stresses in the cell, most notably in the spectrin filaments of the red blood cell (RBC) membrane cytoskeleton. This scaffolding is largely responsible for the extraordinary softness and resilience of the red blood cell; the unfolding/refolding of spectrin molecules may play a key role in these unusual material properties.

We have examined the dynamical behavior of the spectrin repeat as it unfolds and refolds under force. Probing single poly-spectrin molecules with ascending and descending ramps of force, we demonstrated an extension of dynamic force spectroscopy (DFS) applicable to bi-directional chemical transitions involving both rupture and formation of molecular bonds between/within molecules.

3D high-resolution, feedback-stabilized optical tweezers

Mon, 26 Jan 2009 05:00:05 -0500

Single-molecule techniques enable complex biomolecular interactions to be studied in a detailed way. In particular, molecular transitions can be observed directly rather than inferred from ensemble averages, and systems can be studied out of equilibrium and under force. Such investigations require the use of high-precision ultrasensitive force probes with nanometer length and piconewton force resolution.

We have developed a unique dual-mode optical tweezers system designed to explore both forward and reverse biomolecular transitions (e.g bond rupture &formation, protein unfolding & refolding). Our system incorporates a high-resolution 3D particle tracking technique based on reflection-interference imaging, as well as feedback systems which result in longtime stability of 1-2 nm. The system is described in more detail below.

In the two complimentary modes of operation, a functionalized glass probe bead is held by an optical trap near a reactive substrate, allowing single molecular tethers to bridge the two surfaces. The nanoscale motion of the probe bead reports molecular transitions such as bond rupture/formation and protein unfolding/refolding. The optical trap acts as a tunable spring, allowing precise and controllable application of force to the probe bead, and thus the molecular tether.