Single Polymer Dynamics
In our group, we have extended the field of single polymer dynamics to study chains with complex architectures (rings, stars, combs), different backbone chemistries (single stranded DNA), and increasing solution concentrations (semi-dilute and entangled solutions). Single molecule techniques allow for the direct observation of dynamic microstructure, thereby revealing molecular sub-populations, distributions in molecular behavior, and real-time dynamics and kinetics. This is particularly powerful method to study the dynamics of polymers and soft 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.
Single Molecule Studies of TALE 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)