Dynamics between organic molecules and surface enhanced Raman scattering (SERS) hot spots are extracted from far-field optical images by two experimental methods presented in this thesis: orientational imaging and super-resolution optical imaging. We introduce SERS orientational imaging as an all-optical technique able to determine the three-dimensional orientations of SERS-active Ag nanoparticle dimers. This is accomplished by observing lobe positions in SERS emission patterns formed by the directional polarization of SERS emission along the longitudinal axis of the dimer. We further extend this technique to discriminate nanoparticle dimers from higher order aggregates by observing the wavelength-dependence of SERS emission patterns, which are unchanged in nanoparticle dimers, but show differences in higher order aggregates involving two or more nanoparticle junctions. Dynamic fluctuations in the SERS emission pattern lobes are observed in aggregates labeled with low dye concentrations, as molecules diffuse into regions of higher electromagnetic enhancement in multiple nanoparticle junctions. In order to investigate these dynamic interactions between single organic molecules and nanoparticle hot spots we present the first super-resolution optical images of single-molecule SERS (SM-SERS), introducing super-resolution imaging as a powerful new tool for SM-SERS studies. Mapping the dynamic movement of SM-SERS centroid positions with +/- 5 nm resolution reveals the position-dependent SERS intensity as the centroid samples different positions in space. We have proposed that the diffusion of the SERS centroid is due to diffusion of a single molecule on the surface of the nanoparticle, which leads to changes in coupling between the scattering dipole and the optical near field of the nanoparticle. Finally, we combine an isotope-edited bi-analyte SERS spectral approach with super-resolution optical imaging and atomic force microscopy (AFM) structural analysis for a more complete picture of molecular dynamics in SERS hot spots. We demonstrate the ability to observe multiple molecule dynamics in a single hot spot and show that in addition to the single-molecule regime, a "few" molecule regime is able to report on position-dependent SERS intensities in a hot spot. Furthermore, we are able to identify multiple local hot spots in single nanoparticle aggregates. / text

Effective tissue engineering scaffolds should mimic the physical and chemical attributes of native tissue. Native tissues have intricate patterns, a multitude of porosities, and large water contents that are each directly associated with their ability to regulate and support life function. Therefore, the physical architecture of scaffolds intended to mimic these tissues for engineering applications plays an important role in scaffold performance both in vitro and in vivo. Self-assembling molecules organize into intricate patterns with a complexity that resembles that of native tissue. Hyaluronic acid (HA) hydrogels are widely used in tissue engineering for a variety of applications but fail to offer physical architecture beyond the inherent hydrogel porosity. To address this issue, a novel method to impose architecture within thin HA-based films using crystal nucleation was developed in the Schmidt lab [1]. The work described herein extends this method for use in three-dimensional matrices, with the main vii goal being the creation of hydrogels with a complex macroarchitecture. Four in situ self-assembling molecules were used: glycine, guanidine, urea and potassium dihydrogen phosphate. The crystallization of each molecule creates a specific porous network within the hydrogel that is the negative imprint of the crystalline geometry. The novel restriction of aqueous polymer into the molecule interstitial crystalline space allows hydrogels to embody complex geometric lumen architectures. The hydrogels were characterized in terms of their internal architectures, swelling, bulk moduli, biodegradability, cytotoxicity and in vitro cellular response. The unique structure-property relationships displayed by hydrogels templated by each of the crystallizing molecules were characterized in regards to mechanical properties. The need for complex microscopic architecture is conserved over many tissue engineering applications and templated scaffolds were evaluated for two unique applications. Crystal-templated hydrogels were investigated for use as an artificial stem cell niche environment to expand undifferentiated neural progenitor cells. Additionally, the templated hydrogels were evaluated for the in vitro study of myelin expression from Schwann cells. A hydrogel that combines the biocompatible properties of HA and the architectural complexity of native tissue may prove beneficial for biomedical applications. / text

Morphology is the origin of life and function. Defining and designing morphology, understanding the relationship between morphology and function, is an essential theme in a number of research areas. In conjugated polymer research, the major obstacles to achieving these goals are the heterogeneity and complexity of conjugated polymer films. In the study presented in this dissertation, various single molecule spectroscopy techniques were used as an approach to minimize the complexity of these problems. By using excitation polarization spectroscopy, it was discovered that single chains of poly[2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) assume a highly ordered rod conformation despite the fact that the morphology of bulk films is known to be amorphous. The comparison of results from experiments and a coarse grained bead-on-a-chain simulation suggested that single chains have the ability to use a thermally induced defect to maximize [pi]-[pi] stacking and adopt a rod conformation as a stable conformation. Bias-induced centroid spectroscopy (BIC) on highly ordered single chains demonstrated that the energy transfer scale could be an order of magnitude larger than the value typically measured for bulk films. It was further demonstrated that such an extraordinary long energy transfer was not a unique property of single chains but was also observed in aggregates as long as the morphology was ordered. These studies were extended to another model compound poly(3-hexylthiophene) (P3HT) to generalize the mechanism of morphology formation and the structure-property relationship. For P3HT, it was shown that side-chains were a very important factor in determining single chain conformation, while the conformation of MEH-PPV was not affected by side-chains. By controlling the side-chains, both ordered and disordered P3HT chains were obtained. The comparison of results from experiments and an energy transfer model simulation quantified that energy transfer was at least twice as efficient in ordered chains as in disordered chains. In aggregates, the difference between the energy transfer efficiency of ordered and disordered morphology was even larger than that in the case of single chains. These results could suggest that there is a very fast energy transfer mechanism that occurs through interchain interactions when chains are packed in ordered fashion. / text

Telomerase and telomere play crucial roles in the maintenance of genomic stability. Through its ability to extend chromosome ends with G-rich telomeric sequence, telomerase solves the end-replication problem of linear chromosomes and allows complete replication of the genetic information. Telomere along with its protein partners solves the end-protection problem and guards the chromosome ends against aberrant DNA damage response. In this thesis, I present two single-molecule fluorescence-based studies that determined the functional structure of telomerase RNA within active telomerase holoenzyme and probed the structure of telomere and its dependence on telomere binding proteins. In the first study, we developed a single-molecule Förster resonance energy transfer (FRET) assay to interrogate the structure of telomerase RNA within active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity. FRET signals from individual enzyme molecules during active binding events were then used to determine the organization of telomerase RNA within active telomerase. Using this assay, we have identified an active conformation of telomerase in which the conserved telomerase RNA pseudoknot is properly folded. In the second study, we used super-resolution fluorescence technique STochastic Optical Reconstruction Microscopy (STORM) to probe the structure of mammalian telomere. We showed that previously described telomere loop structures are detected by STORM imaging. Removal of telomere-binding protein TRF2 significantly reduces the fraction of telomeres found in loops. Furthermore, this reduction of telomere loops occurs in the absence of ATM-dependent DNA damage signaling and non-homologous end joining mediated chromosome fusion, suggesting a direct role of TRF2 in the formation or maintenance of telomere loops.

DNA replication is a fundamental cellular process. However, the structure and dynamics of the eukaryotic DNA replication machinery remain poorly understood. A soluble extract system prepared from Xenopus eggs recapitulates eukaryotic DNA replication outside of a cell on a variety of DNA templates. This system has been used to reveal many aspects of DNA replication using a variety of ensemble biochemical techniques. Single-molecule fluorescence imaging is a powerful tool to dissect biochemical mechanisms. By immobilizing or confining a substrate, its interaction with individual, soluble, fluorescently-labeled reactants can be imaged over time and without the need for synchrony. These molecular movies reveal binding parameters of the reactant and any population heterogeneity. Moreover, if the experiments are imaged in wide-field format, the location or motion of the labeled species along the substrate can be followed with nanometer accuracy. This dissertation describes the use and development of novel single-molecule fluorescence imaging techniques to study eukaryotic DNA replication. A biophysical characterization of a replication fork protein, PCNA, revealed both helical and non-helical sliding modes along DNA. Previous experiments demonstrate that the egg extracts efficiently replicate surface-immobilized linear DNA. This finding suggested replication of DNA could be followed as motion of the replication fork along the extended DNA. However, individual proteins bound at the replication fork could not be visualized in the wide-field due to the background from the high concentration of the fluorescent protein needed to compete with the extract’s endogenous protein. To overcome this concentration barrier, I have developed a wide-field technique that enables sensitive detection of single molecules at micromolar concentrations of the labeled protein of interest. The acronym for this method, PhADE, denotes three essential steps: (1) Localized PhotoActivation of fluorescence at the immobilized substrate, (2) Diffusion of unbound fluorescent molecules to reduce the background and (3) Excitation and imaging of the substrate-bound molecules. PhADE imaging of flap endonuclease I (Fen1) during replication revealed the time-evolved pattern of replication initiation, elongation and termination and the kinetics of Fen1 exchange during Okazaki fragment maturation. In the future, PhADE will enable the elucidation of the dynamic events at the eukaryotic DNA replication fork. PhADE will also be broadly applicable to the investigation of other complex biochemical process and low affinity interactions. It will be especially useful to those researchers wishing to correlate motion with binding events.

Over one-third of the world’s population is at risk for infection with dengue virus (DENV), a mosquito-borne virus that can cause a severe febrile disease. There are no specific treatments available for dengue infection, and much remains unknown about how DENV interaction with the host cell leads to a successful infection. This dissertation examines DENV entry using small molecule inhibitors and mutagenesis of the envelope (E) protein, the major protein on the viral surface. This work grew from our initial observation that small molecule GNF-2 is capable of lowering DENV yield when present at two separate points during DENV infection. Treatment of infected cells with GNF-2 post-entry significantly lowered DENV yield, most likely due to GNF-2’s documented activity against Abl kinase. However, we also observed that treatment of virus inocula with GNF-2 prior to cellular infection significantly lowered DENV yield. We discovered that GNF-2 bound directly to the dengue virion and co-localized with DENV envelope protein shortly after cellular infection. Using GNF-2 as a scaffold, we performed a structure-activity relationship study and identified twenty-one compounds that have similar or increased potency as GNF-2 when pre-incubated with virus. Using a subset of compounds from this study, we demonstrated that they block completion of DENV fusion in vitro, suggesting that the compounds inhibit DENV entry by preventing the completion of viral fusion inside cellular endosomes. In experiments complementing the mechanism of action studies, we selected for inhibitor-resistant virus by passaging virus in the presence of small molecules. We identified a single point mutation in the envelope protein located in the domain I/II interface that enhanced viral entry and conferred resistance to virus particles against select compoundsin a single-cycle reporter virus system. Further examination of this E protein “hinge region” found that mutations in this area may affect both release and entry of reporter virus particles. The work presented in this dissertation may inform the design of future small molecule inhibitors of DENV as well as increase our understanding of how point mutations in the DENV E protein can influence viral entry and other steps of the viral life cycle.

Advances in optical imaging techniques have allowed quantitative studies of many biological systems. This dissertation elaborates on our efforts in both developing novel imaging modalities based on detection of optical absorption and applying high-sensitivity fluorescence microscopy to the study of biology. / Chemistry and Chemical Biology

Small molecules play important roles in therapeutics and drug discovery. Significant progress has been made by the chemical biology community to discover small-molecule probes to explore biological processes and to treat disease. This thesis describes both the discovery of novel probes for the Hedgehog (Hh) pathway and the application of small molecules in identifying cancer dependencies. / Chemistry and Chemical Biology

Advances in genome and proteome research have led to a dramatic increase in the number of macromolecular targets of interest to the life sciences. A solution-phase method to simultaneously reveal all ligand-target binding pairs from a single solution containing libraries of ligands and targets could significantly increase the efficiency and effectiveness of target-oriented screening efforts. Here, we describe interaction-dependent PCR (IDPCR), a solution-phase method to identify binding partners from combined libraries of small-molecule ligands and targets in a single experiment. Binding between DNA-linked targets and DNA-linked ligands induces formation of an extendable duplex. Extension links codes identifying the ligand and target into one selectively amplifiable DNA molecule. In a model selection, IDPCR resulted in the enrichment of DNA encoding all five known protein-ligand pairs out of 67,599 possible sequences.

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