Charles Schroeder Research
Single Polymer Dynamics, Soft Materials, Single Molecule Biophysics
We study the dynamics of polymers, proteins, and soft materials using single molecule techniques. A major goal of our research is to understand how microscopic phenomena give rise to the emergent, macroscopic properties of soft materials. 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) single molecule studies of TALE proteins, which are a major tool for gene editing applications, and (4) studying the assembly properties of optoelectronic materials such as pi-conjugated oligopeptides.
Single Polymer Dynamics
Polymers underlie an untold number of technologies ranging from consumer products to electronics. Despite tremendous progress, we still lack a full understanding of the non-equilibrium flow properties of polymer solutions and melts. A major goal of our research is to develop a detailed understanding of single molecule processes that, when ensemble averaged, produce bulk-level properties. How does polymer microstructure result in the emergent, macroscopic properties such as viscosity and bulk stress? The key to answering this question lies at the single polymer level.
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, it is critical to achieve a molecular-level understanding of polymer dynamics in the context of non-linear chain topologies. Until now, 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 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 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 insufficient 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 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. Recently, our lab developed the Stokes trap, which is a new method for trapping and manipulating multiple particles using only fluid flow. Hydrodynamic trapping offers an advantageous method for particle manipulation in free solution without the need for optical, electric, acoustic, or magnetic fields. 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.
Fluidic-directed Assembly of Optoelectronic Materials
A grand challenge in advanced materials engineering is the development of robust strategies for the engineered self-assembly of functional synthetic materials. Biomimetic materials such as synthetic polypeptides and peptide-polymer conjugates serve as model systems that provide insight into the design and engineering of materials. Recently, we showed that fluidic-based assembly of synthetic peptides enables reproducible and reliable fabrication of aligned hierarchical materials that do not form spontaneously in solution. Directed assembly techniques have emerged as potential new routes towards building supramolecular structures consisting of small molecules, oligomers, and polymers. To this end, we used a microfluidic cross-slot device to generate a planar extensional flow, thereby inducing simultaneous assembly and alignment of synthetic peptide materials. In particular, we studied the assembly properties of two oligopeptides, wherein a symmetric backbone of amino acids flanks an internal pi-conjugated oligo(p-phenylenevinylene) (OPV) core. These oligopeptides assemble into unaligned gel-like structures in acidic conditions due to protonation of amino acid residues, pH-triggered screening of charges, formation of H-bonding, and intermolecular pi-pi stacking between adjacent OPV cores. Our work shows that fluidic-directed assembly of supramolecular structures can be achieved with unprecedented manipulation at the nano- and mesoscale, which has the potential to provide rapid and efficient control of functional materials (e.g., electronic materials for charge transport/storage, or optoelectronic materials for energy/light interactions).
Advanced Fluorescent Probes for Microscopy and Imaging
In recent work, we developed a new class of bright and photostable fluorescent probes based on dendritic polymers. Fluorescent dendrimer nanoconjugates (FDNs) are multichromophoric systems, wherein dendrimers are used as molecular scaffolds to assemble multiple dyes (~10-30) and functional groups, including biotin and alkynes for copper-free click chemistry. FDNs are compact (overall size of ~2-5 nm), smaller than quantum dots (~10-20 nm), and brighter and more photostable than single dyes (~10-100x enhanced photobleaching lifetimes). Our group routinely uses single molecule fluorescence microscopy (SMFM) to characterize the photophysical properties of these probes. Importantly, our experiments demonstrate that FDNs can be localized with superior precision compared to single organic dyes, which is extremely useful for high-resolution single molecule localization and sub-cellular center-of-mass tracking. As proof-of-principle demonstration, FDNs have been used to assay single molecule nucleic acid hybridization, to perform immunofluorescence imaging of microtubules in cytoskeletal networks, and to study antibody binding in single molecule protein pull down assays (SiMPull).