Nolan C. Harris

Experimental free energy surface reconstruction from single-molecule force spectroscopy using Jarzynski's equality.

We used the atomic force microscope to manipulate and unfold individual molecules of the titin I27 domain and reconstructed its free energy surface using Jarzynskis equality. The free energy surface for both stretching and unfolding was reconstructed using an exact formula that relates the nonequilibrium work fluctuations to the molecular free energy. In addition, the unfolding free energy barrier, i.e., the activation energy, was directly obtained from experimental data for the first time. This Letter demonstrates that Jarzynskis equality can be used to analyze nonequilibrium single-molecule experiments, and to obtain the free energy surfaces for molecular systems, including interactions for which only nonequilibrium work can be measured.

Defects Can Increase the Melting Temperature of DNA-Nanoparticle Assemblies

DNA-gold nanoparticle assemblies have shown promise as an alternative technology to DNA microarrays for DNA detection and RNA profiling. Understanding the effect of DNA sequences on the melting temperature of the system is central to developing reliable detection technology. We studied the effects of DNA base-pairing defects, such as mismatches and deletions, on the melting temperature of DNA-nanoparticle assemblies. We found that, contrary to the general assumption that defects lower the melting temperature of DNA, some defects increase the melting temperature of DNA-linked nanoparticle assemblies. The effects of mismatches and deletions were found to depend on the specific base pair, the sequence, and the location of the defects. Our results demonstrate that the surface-bound DNA exhibit hybridization behavior different from that of free DNA. Such findings indicate that a detailed understanding of DNA-nanoparticle assembly phase behavior is required for quantitative interpretation of DNA-nanoparticle aggregation.

Quantifying DNA melting transitions using single-molecule force spectroscopy

We stretched a DNA molecule using atomic force microscope and quantified the mechanical properties associated with B and S forms of double-stranded DNA (dsDNA), molten DNA, and single-stranded DNA (ssDNA). We also fit overdamped diffusion models to the AFM time series and used these models to extract additional kinetic information about the system. Our analysis provides additional evidence supporting the view that S-DNA is a stable intermediate encountered during dsDNA melting by mechanical force. In addition, we demonstrated that the estimated diffusion models can detect dynamical signatures of conformational degrees of freedom not directly observed in experiments.

Melting transition of directly linked gold nanoparticle DNA assembly

DNA melting and hybridization is a fundamental biological process as well as a crucial step in many modern biotechnology applications. DNA confined on surfaces exhibits a behavior different from that in free solutions. The system of DNA-capped gold nanoparticles exhibits unique phase transitions and represents a new class of complex fluids. Depending on the sequence of the DNA, particles can be linked to each other through direct complementary DNA sequences or via a ‘linker’ DNA, whose sequence is complementary to the sequence attached to the gold nanoparticles. We observed different melting transitions for these two distinct systems.

Temperature and chemical denaturant dependence of forced unfolding of titin I27.

Single-molecule force measurement opens a new door for investigating detailed biomolecular interactions and their thermodynamic properties by pulling molecules apart while monitoring the force exerted on them. Recent advances in the nonequilibrium work theorem allows one to determine the free-energy landscapes of these events. Such information is valuable for understanding processes such as protein and RNA folding and receptor-ligand binding. Here, we used force as a physical parameter under the traditional chemical and temperature denaturing environment to alter the protein folding energy landscape and compared the change in the unfolding free-energy barrier of the I27 domain of human cardiac titin. We found that the trends in protein unfolding free-energy barriers are consistent for single-molecule force measurements and bulk chemical and temperature studies. The results suggest that the information from single-molecule pulling experiments are meaningful and useful for understanding the mechanism of folding of titin I27.

Analyzing Single-Molecule Manipulation Experiments

Single‐molecule manipulation studies can provide quantitative information about the physical properties of complex biological molecules without ensemble artifacts obscuring the measurements. We demonstrate computational techniques which aim at more fully utilizing the wealth of information contained in noisy experimental time series. The “noise” comes from multiple sources e.g., inherent thermal motion, instrument measurement error, etc. The primary focus of this paper is a methodology that uses time domain based methods to extract the effective molecular friction from single‐molecule pulling data. We studied molecules composed of eight tandem repeat titin I27 domains, but the modeling approaches have applicability to other single‐molecule mechanical studies. The merits and challenges associated with applying such a computational approach to existing single‐molecule manipulation data are also discussed. Copyright

The reversible phase transition of DNA-linked colloidal gold assemblies

We present direct evidence for a reversible phase transition of DNA-linked colloidal gold assemblies. Transmission electron microscopy and optical absorption spectroscopy are used to monitor the colloidal gold phase transition, whose behavior is dominated by DNA interactions. We use single-stranded DNA-capped colloidal gold that is linked by complementary linker DNA to form the assemblies. We found that, compared to free DNA, a sharp melting transition is observed for the DNA-linked colloidal gold assemblies. The structure of the assemblies is non-crystalline, much like a gel phase, consistent with theoretical predictions. Optical spectra and melting curves provide additional evidence of gelation of the colloidal system. The phase transition and separation are examples of percolation in a dilute solvent.

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.

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.