The cutting edge of chemical science research lies in the ability to manipulate and control single molecules. Molecular processes hold the key to developing advanced functional materials and for understanding fundamental life processes. A major challenge in the field of soft materials is to develop a direct link between molecular-scale phenomena and emergent behavior in complex materials. Our research addresses these problems by pioneering a unique and powerful brand of molecular engineering that allows for the precise interrogation of single molecules. In recent years, our work has uncovered new physical mechanisms in complex polymeric systems and has provided fundamentally new methods to study, manipulate, and assemble molecular materials.
Our recent work has focused on: (1) extending the field of single polymer dynamics to new materials, including topologically complex polymers such as ring polymers, branched polymers, and copolymers, (2) development and application of the Stokes trap to study soft material dynamics, including vesicle fusion and coalescence, (3) studying the self-assembly properties of optoelectronic materials such as pi-conjugated oligopeptides, (4) single molecule conductance measurements and precise studies of electron transport at the single polymer level, and (5) single molecule biophysics and direct observation of TALE protein dynamics.
Single Polymer Dynamics
A major goal of our research is to understand the dynamics of architecturally complex polymers and functional materials using single molecule techniques, thereby establishing a connection between emergent macroscale properties and molecular-scale phenomena in soft materials. In recent years, our group has advanced the field of single molecule studies in several new directions to study polymers with complex architectures (stars, combs, bottlebrushes, rings), different backbone chemistries (single stranded DNA and PNIPAM-DNA copolymers), semi-dilute concentration regimes such as those encountered in 3-D printing applications, and entangled solutions.
Until recently, the field of single polymer dynamics has almost exclusively focused on linear polymers in dilute solutions, but our work has extended the field in fundamentally new directions to consider more complex polymers. Our results provide a molecular basis for understanding how molecular topology of branched polymers and rings impacts conformational dynamics far-from-equilibrium. We also developed new methods to determine fundamental materials properties such as elasticity from non-equilibrium dynamics using recent advances in statistical mechanics. This work transforms the field beyond traditional methods of measuring polymer elasticity, such as AFM or optical trapping. Single molecule techniques allow for the direct observation of dynamic microstructure far from equilibrium, thereby revealing molecular sub-populations, distributions in molecular behavior, and real-time dynamics. Our work capitalizes on these features to provide a molecular basis for understanding of complex materials.
Single molecule studies of comb polymers
The molecular topology of polymers is known to influence the bulk properties of these materials, both at equilibrium and in flow. Synthetic polymers used in commercial applications generally have exceedingly complex topologies, including high grafting densities of side chains, hierarchical branching, and dangling ends. Chain branching results in complex flow properties that differ substantially from linear polymers under similar conditions, such as strain hardening in uniaxial extensional flow under relatively low strain rates. Given the importance of polymeric materials in society, it is critical to achieve a molecular-level understanding of polymer dynamics in the context of non-linear chain topologies. Despite this need, however, the field of single polymer dynamics has almost exclusively been focused on linear double stranded DNA in dilute solution flows.
In recent work, we synthesized and directly observed DNA-based comb polymers using single molecule techniques. We are studying the relaxation dynamics and conformational stretching dynamics of single comb polymers in flow using microfluidics. Our initial results have shown that the molecular topology of individual branched polymers plays a direct role on the relaxation dynamics of polymers with complex architectures. In this work, we first synthesize DNA combs using a hybrid enzymatic-synthetic approach, wherein chemically modified DNA branches and DNA backbones are generated in separate polymerase chain reactions, followed by a ‘graft-onto’ reaction via strain-promoted [3+2] azide-alkyne cycloaddition. This method allows for the synthesis of branched polymers with nearly monodisperse backbone and branch molecular weights. Single molecule fluorescence microscopy is then used to directly visualize branched polymers, such that the backbone and side branches can be tracked independently using single- or dual-color fluorescence labeling.
Single molecule studies of ring polymers
Circular macromolecules play a key role in biology and biotechnology, including DNA replication and maintenance of circular genomes, DNA looping, plasmid-based DNA vaccines, and biologically active macrocycles as drugs. Circular macromolecules call into question our understanding of polymer dynamics because the absence of free ends alters flow behavior, diffusion, and ordering transitions compared to linear macromolecules. For these reasons, achieving a clear understanding of circular polymer dynamics in nonequilibrium conditions has been a major task. In recent work, we observed the conformational and orientational dynamics of large circular DNA molecules in extensional flow using single molecule techniques. Our results show that circular DNA molecules show a shifted coil-to-stretch transition and less diverse “ molecular individualism” behavior as evidenced by their conformational stretching pathways.
Single polymer dynamics in semi-dilute solutions
The dynamics of semi-dilute polymer solutions is an intriguing yet particularly challenging problem in soft materials and rheology. Dilute polymer solutions are characterized by the rarity of overlap of single chains, whereas concentrated solutions and melts are governed by topological entanglements and dense polymer phases. Unentangled semi-dilute solutions, however, are characterized by coil-coil interpenetration at equilibrium, albeit in the absence of intermolecular entanglements under quiescent conditions. From this view, semi-dilute polymer solutions are known to exhibit large fluctuations in concentration, which precludes the straightforward treatment of polymer dynamics in these solutions using a mean-field approach. Given these complexities, we still do not fully understand non-equilibrium dynamics in semi-dilute solutions. In recent work, we used single molecule techniques to investigate the dynamics semi-dilute solutions of DNA in extensional flow, including polymer relaxation from high stretch, transient stretching dynamics in step-strain experiments, and steady-state stretching in flow. Our results are consistent with a power-law scaling of the longest polymer relaxation time. We further studied the non-equilibrium stretching dynamics of semi-dilute polymer solutions, including transient and steady-state stretching dynamics in extensional flow using an automated microfluidic trap. Interestingly, we observe a unique set of molecular conformations during the transient stretching process for single polymers in semi-dilute solutions, which suggests that the transient stretching pathways for polymer chains in semi-dilute solutions is qualitatively different compared to dilute solutions due to intermolecular interactions.
Single polymer dynamics in large amplitude oscillatory extensional flow (LAOE)
Understanding the rheological behavior of complex fluids is essential for controlling and engineering the properties of functional materials. To this end, small amplitude oscillatory shear (SAOS) has been used as a common method to probe the response of complex fluids in the limit of small deformations. However, SAOS probes only the linear viscoelastic properties of materials, which is usually insucient to fully understand the non-linear properties of fluids with complex micro- or nanostructures. To address this issue, large amplitude oscillatory shear (LAOS) was developed and widely adopted in recent years to characterize the nonlinear rheological behavior of complex fluids. In LAOS, the non-linear stress response of a material is no longer a simple rst order sinusoidal function, rather, it typically appears as a complex distorted shape with higher order harmonics that depend on the material structure. There is a general need to study these processes at the single molecule level, however, the vast majority of prior single polymer studies have employed simple on/off step functions for imposing flow forcing functions for both transient and steady-state experiments. In recent work, we studied the dynamics of single DNA molecules in large amplitude oscillatory extensional (LAOE) flow, including results from experiments and Brownian dynamics simulations. Our results show that polymers experience periodic cycles of compression, re-orientation, and extension in LAOE. Based on these data, we construct a series of single polymer Lissajous curves over Pipkin space to characterize both the linear and nonlinear responses as functions of dimensionless strength (Weissenberg number Wi) and probing frequency (Deborah number De).
The ability to confine and manipulate single particles and molecules has revolutionized several fields of science, with common methods including optical traps and magnetic tweezers. Hydrodynamic trapping offers an attractive method for particle manipulation in free solution without the need for optical, electric, acoustic, or magnetic fields. Recently, our lab developed the Stokes trap, which is a new method for trapping and manipulating multiple particles using only fluid flow. The Stokes trap enables the simultaneous manipulation of two particles in a simple microfluidic device using model predictive control. We have used this technique for the fluidic-directed assembly of multiple particles in solution, and we are further using the Stokes trap to study interactions between soft particles, collisions, and vesicle fusion. From a broad perspective, this technique opens new vistas for fundamental studies of particle-particle interactions and provides a new method for the directed assembly of colloidal particles.
Supramolecular Assembly & Pi-conjugated Oligopeptides
A grand challenge in supramolecular assembly is to design and create functional optoelectronic materials that can assemble into precise structures. Despite recent progress, understanding the design rules for molecular assembly remains a central problem in the field. Our group has addressed this challenge by pursuing a new class of materials that combine elements of synthetic polymers (pi-conjugated materials) and biopolymers (proteins and nucleic acids), thereby generating precisely defined electronically active materials. In recent work, we directly studied the sol-gel transition and optical/structural properties of pi-conjugated synthetic oligopeptides using a combination of microrheology and optical spectroscopy. In ongoing work, we are studying the fluidic-directed assembly of these materials.
Single Molecule Conductance
Understanding electron transport through sequence-defined oligomers and polymers is a crucial step for designing new functional materials for energy storage and for building integrated electronic devices. Recent advances in molecular electronics have brought us closer towards achieving the ultimate limits in miniaturization and spatial and functional control over electronic performance. Despite recent progress, however, our knowledge of molecular-scale electron transport is limited by the inability to explore the vast chemical sequence space using existing synthetic methods. A full understanding of the effects of chemical sequence on electron transport properties will aid in the development of new materials for energy storage and capture.
To address these challenges, we study electron transport and conductance at the single molecule level. We recently built a custom scanning tunneling microscope-break junction instrument (STM-BJ) for measuring single polymer conductance of complex materials, including a new class of synthetic pi-conjugated nucleic acids (DNA/RNA). Our work focuses on the fundamental design rules of nucleic acid-synthetic oligomers, where assembly is driven by directional molecular interactions. Taken together, our work aims to uncover new classes of materials that hold promise for electronic applications via directional and cooperative interactions.
Single Molecule Studies of TALE(N) Proteins
Recent advances in genome engineering offer the potential to dramatically alter the treatment of human disease. Achieving this potential, however, is a major challenge due to the high degrees of precision and accuracy required for modifying large, intact genomes. Genome editing techniques based on programmable nucleases, including zinc-finger nucleases, the CRISPR/Cas9 system, and transcription activator-like effector nucleases (TALENs), are finding widespread use for genomic editing in plants, bacteria, and mammalian cells. Despite recent progress, however, the molecular mechanisms underlying the DNA search process for TALEs are not fully understood. In recent work, we have used single molecule techniques to directly study the non-specific search process for TALEs along DNA templates. We use a series of single molecule experiments to study TALE search along DNA, including size-dependent protein-probe diffusion and a hydrodynamic flow assay. Our results have revealed that TALEs utilize a two-state ‘search and check’ model for finding target sites along DNA. Moreover, our results further show that TALEs utilize a rotationally decoupled mechanism for non-specific DNA search, despite remaining associated with DNA templates during the search process. In this way, TALE search is largely absent of rotationally coupled sliding. Our results suggest that the helical structure of TALEs enables these proteins to adopt a loose wrapped conformation around DNA templates during non-specific search, thereby facilitating rapid one-dimensional (1-D) diffusion under a wide range of solution conditions. Taken together, our results suggest that the search mechanism for TALEs appears to be unique amongst the broad class of sequence-specific DNA binding proteins and supports efficient 1-D search along DNA.
Advanced Imaging Probes
Fluorescent dendrimer nanoconjugates
Advanced imaging techniques in the biological and chemical sciences critically rely on bright and photostable probes. We developed a new class of fluorescent molecules, fluorescent dendrimer nanoconjugates (FDNs), based on the covalent attachment of multiple fluorescent dyes onto a single dendritic scaffold. This results in extremely bright, nanometer scale, single-molecule probes that we have used for both traditional fluorescence microscopy, as well as for the burgeoning field of super-resolution microscopy where brightness is directly related to achievable resolution. In addition, we have engineered the photophysical properties of these probes by attaching the “photo-protective” triplet-state quencher Trolox directly onto the scaffold, an anti-fade agent normally used in solution. Trolox-conjugated probes are extremely photostable with vastly decreased amounts of transient dark states compared with single fluorescent dyes, and by modulating the amount of conjugated Trolox, we can exceed enhancement in photostability provided by adding large concentrations of Trolox in solution.
Flavin-binding Fluorescent Proteins (FbFPs)
Recently, a new class of oxygen-independent fluorescent reporters was reported based on flavin-binding photosensors from Bacillus subtilis, Pseudomonas putida, and Arabidopsis thaliana. Flavin-binding fluorescent proteins (FbFPs) can function in the absence of oxygen, whereas the widely used green fluorescent protein (GFP) and related analogs strictly require oxygen for maturation of fluorescence. In our lab, we have applied the tools of directed evolution to isolate new and spectrally improved variants of FbFPs. Overall, we anticipate that spectrally enhanced AFP variants will find pervasive use as reporter proteins for gene expression, subcellular localization and protein interactions in obligate and facultative anaerobes and in hypoxic niches of the human body (e.g. malignant tumors)