Sithara S. Wijeratne

Single molecule force measurements of perlecan/HSPG2: A key component of the osteocyte pericellular matrix.

Perlecan/HSPG2, a large, monomeric heparan sulfate proteoglycan (HSPG), is a key component of the lacunar canalicular system (LCS) of cortical bone, where it is part of the mechanosensing pericellular matrix (PCM) surrounding the osteocytic processes and serves as a tethering element that connects the osteocyte cell body to the bone matrix. Within the pericellular space surrounding the osteocyte cell body, perlecan can experience physiological fluid flow drag force and in that capacity function as a sensor to relay external stimuli to the osteocyte cell membrane. We previously showed that a reduction in perlecan secretion alters the PCM fiber composition and interferes with bones response to a mechanical loading in vivo. To test our hypothesis that perlecan core protein can sustain tensile forces without unfolding under physiological loading conditions, atomic force microscopy (AFM) was used to capture images of perlecan monomers at nanoscale resolution and to perform single molecule force measurement (SMFMs). We found that the core protein of purified full-length human perlecan is of suitable size to span the pericellular space of the LCS, with a measured end-to-end length of 170±20 nm and a diameter of 2-4 nm. Force pulling revealed a strong protein core that can withstand over 100 pN of tension well over the drag forces that are estimated to be exerted on the individual osteocyte tethers. Data fitting with an extensible worm-like chain model showed that the perlecan protein core has a mean elastic constant of 890 pN and a corresponding Youngs modulus of 71 MPa. We conclude that perlecan has physical properties that would allow it to act as a strong but elastic tether in the LCS.

Melting Transitions of DNA-Capped Gold Nanoparticle Assemblies

The recognition capabilities of DNA are remarkable in their specificity. This makes DNA highly suitable as the basis for the creation of self-assembling nanostructures. Understanding the effects of various parameters such as nanoparticle size and DNA sequences provides important information about the phase behavior of DNA-based nanotechnology and DNA-capped colloidal systems. Quantifying the interactions of surface-bound DNA hybridization and their effects on colloidal gold phase transition may help in the development of these DNA-capped nanoparticles into a practical tool for DNA detection and biosensing.

DNA under Force: Mechanics, Electrostatics, and Hydration

Quantifying the basic intra- and inter-molecular forces of DNA has helped us to better understand and further predict the behavior of DNA. Single molecule technique elucidates the mechanics of DNA under applied external forces, sometimes under extreme forces. On the other hand, ensemble studies of DNA molecular force allow us to extend our understanding of DNA molecules under other forces such as electrostatic and hydration forces. Using a variety of techniques, we can have a comprehensive understanding of DNA molecular forces, which is crucial in unraveling the complex DNA functions in living cells as well as in designing a system that utilizes the unique properties of DNA in nanotechnology.

Multiscale mechanobiology: mechanics at the molecular, cellular, and tissue levels

Mechanical force is present in all aspects of living systems. It affects the conformation of molecules, the shape of cells, and the morphology of tissues. All of these are crucial in architecture-dependent biological functions. Nanoscience of advanced materials has provided knowledge and techniques that can be used to understand how mechanical force is involved in biological systems, as well as to open new avenues to tailor-made bio-mimetic materials with desirable properties.In this article, we describe models and show examples of how force is involved in molecular functioning, cell shape patterning, and tissue morphology.

Helicity Distributions of Single-Walled Carbon Nanotubes and Its Implication on the Growth Mechanism

Single-walled nanotubes (SWNT) have attracted significant attention because of the substance’s superior crystal quality, high thermal conductivity and current carrying capacity, thus emerging as an attractive material for nanoelectrics. To optimize the selection of SWNT structures in large-scale synthesis, an understanding of their growth mechanism is necessary. We report studies of the helicity distributions of SWNT using electron nanodiffraction. The overall statistical distribution of helicity has peaks at 0° and 30°. The peak evident at 0° was found to be a sharp local maximum, while the peak at 30° was broader. We also found that the helicity distribution varies from region to region of micrometer size. This observation indicates that local environment affects nanotube growth, resulting in different structural distributions.

Reconstructing multiple free energy pathways of DNA stretching from single molecule experiments.

Free energy landscapes provide information on the dynamics of proteins and nucleic acid folding. It has been demonstrated that such landscapes can be reconstructed from single molecule force measurement data using Jarzynskis equality, which requires only stretching data. However, when the process is reversible, the Crooks fluctuation theorem combines both stretch and relaxation force data for the analysis and can offer more rapid convergence of free energy estimates of different states. Here we demonstrate that, similar to Jarzynskis equality, the Crooks fluctuation theorem can be used to reconstruct the full free energy landscapes. In addition, when the free energy landscapes exhibit multiple folding pathways, one can use Jarzynskis equality to reconstruct individual free energy pathways if the experimental data show distinct work distributions. We applied the method to reconstruct the overstretching transition of poly(dA) to demonstrate that the nonequilibrium work theorem combined with single molecule force measurements provides a clear picture of the free energy landscapes.

Detecting the Biopolymer Behavior of Graphene Nanoribbons in Aqueous Solution.

Graphene nanoribbons (GNR), can be prepared in bulk quantities for large-area applications by reducing the product from the lengthwise oxidative unzipping of multiwalled carbon nanotubes (MWNT). Recently, the biomaterials application of GNR has been explored, for example, in the pore to be used for DNA sequencing. Therefore, understanding the polymer behavior of GNR in solution is essential in predicting GNR interaction with biomaterials. Here, we report experimental studies of the solution-based mechanical properties of GNR and their parent products, graphene oxide nanoribbons (GONR). We used atomic force microscopy (AFM) to study their mechanical properties in solution and showed that GNR and GONR have similar force-extension behavior as in biopolymers such as proteins and DNA. The rigidity increases with reducing chemical functionalities. The similarities in rigidity and tunability between nanoribbons and biomolecules might enable the design and fabrication of GNR-biomimetic interfaces.

Single-molecule force measurements of the polymerizing dimeric subunit of von Willebrand factor.

Von Willebrand factor (VWF) multimers are large adhesive proteins that are essential to the initiation of hemostatic plugs at sites of vascular injury. The binding of VWF multimers to platelets, as well as VWF proteolysis, is regulated by shear stresses that alter VWF multimeric conformation. We used single molecule manipulation with atomic force microscopy (AFM) to investigate the effect of high fluid shear stress on soluble dimeric and multimeric forms of VWF. VWF dimers are the smallest unit that polymerizes to construct large VWF multimers. The resistance to mechanical unfolding with or without exposure to shear stress was used to evaluate VWF conformational forms. Our data indicate that, unlike recombinant VWF multimers (RVWF), recombinant dimeric VWF (RDVWF) unfolding force is not altered by high shear stress (100dynes/cm^{2} for 3 min at 37^{∘}C). We conclude that under the shear conditions used (100dynes/cm^{2} for 3 min at 37^{∘}C), VWF dimers do not self-associate into a conformation analogous to that attained by sheared large VWF multimers.

Principles Involved in Interpreting Single-Molecule Force Measurement of Biomolecules

Single-molecule manipulation techniques provide a unique tool for a close-up investigation of the complex biological properties and interactions. During the force measurement, a single molecule is pulled while its force response is monitored. However, quantifying these non-equilibrium data and using them to understand the structure-function relationship of biological systems have been challenging. We describe the mechanics of nanoscale biomolecules and the use of these force measurements for the free energy reconstruction using the recently derived non-equilibrium work theorem, i.e., Jarzynski’s equality. We also compare the results with those from other phenomenological approaches. Finally, mechanical characterization of systems such as overstretching transitions of DNA are presented, and the implications and challenges of these single-molecule force studies are discussed.

PRINCIPLES OF SINGLE-MOLECULE MANIPULATION AND ITS APPLICATION IN BIOLOGICAL PHYSICS

Recent advances in nanoscale manipulation and piconewton force detection provide a unique tool for studying the mechanical and thermodynamic properties of biological molecules and complexes at the single-molecule level. Detailed equilibrium and dynamics information on proteins and DNA have been revealed by single-molecule manipulation and force detection techniques. The atomic force microscope (AFM) and optical tweezers have been widely used to quantify the intra- and inter-molecular interactions of many complex biomolecular systems. In this article, we describe the background, analysis, and applications of these novel techniques. Experimental procedures that can serve as a guide for setting up a single-molecule manipulation system using the AFM are also presented.