The Use of A Small Molecule To Improve The Thermostability Of DNA Junctions

The short-term goal of this research project is to employ small molecules as a means to stabilize four-way DNA junctions (4WJs) composed of natural DNA and chimeric nucleic acids. The long-term goal of the project is utilizing the 4WJs as extracellular therapeutic inhibitors of DNA binding proteins [i.e. Histones and High Mobility Group Protein B (HMGB1b)]. A number of studies have shown that classical intracellular DNA-binding proteins have a variety of deleterious side-effects when present in the extracellular milieu. In order to develop a successful 4WJ therapeutic, we are focused on using modified nucleic acids to enhance the stability of the resulting 4WJ. The nucleic acid of interest is PNA (peptide nucleic acid). PNA was selected because it is known to form DNA-PNA duplex/triplex structures with elevated thermo- and nuclease stability. 4WJs are prepared using fluorescently labeled DNA strands and a single PNA strand. Small molecules are currently being investigated as tools to potentially link the PNA-DNA strands to form 4WJs composed of multiple PNA strands. One molecule of interest is [Ru(bpy)2(dpp)PtCl2]Cl2. Electrophoretic mobility shift assays (EMSAs) have shown that stable 4WJs form in the presence of this molecule. The junctions were visualized using polyacrylamide gels. Circular dichroism studies will be employed to characterize the structural properties of hybrid of interest. Once a stable 4WJ structure is identified, the hybrid was used to study binding and inhibition of HMGB1 in cell-based assays

Supporting Data for the Characterization of PNA-DNA Four-Way Junctions

Holliday or DNA four-way junctions (4WJs) are cruciform/bent structures composed of four DNA duplexes. 4WJs are key intermediates in homologous genetic recombination and double-strand break repair. To investigate 4WJs in vitro, junctions are assembled using four asymmetric DNA strands. The presence of four asymmetric strands about the junction branch point eliminates branch migration, and effectively immobilizes the resulting 4WJ. The purpose of these experiments is to show that immobile 4WJs composed of DNA and peptide nucleic acids (PNAs) can be distinguished from contaminating labile nucleic acid structures. These data compare the electrophoretic mobility of hybrid PNA–DNA junctions vs. i) a classic immobile DNA 4WJ, J1 and ii) contaminating nucleic acid structures

Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and mutations in myosin-15 cause hereditary deafness. In addition to transporting actin regulatory machinery to stereocilia tips, myosin-15 directly nucleates actin filament (“F-actin”) assembly, which is disrupted by a progressive hearing loss mutation (p.D1647G, “jordan”). Here, we present cryo–electron microscopy structures of myosin-15 bound to F-actin, providing a framework for interpreting the impacts of deafness mutations on motor activity and actin nucleation. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin’s D-loop, which mediates inter-subunit contacts, while the jordan mutant locks the D-loop in a single conformation. Adenosine diphosphate–bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin’s structural plasticity in a myosin nucleotide state–dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height

Minimization of Cas9 and Perspectives on Genetically Engineered Microorganisms and Their Regulation

CRISPR technology has revolutionized the way biology is conducted. In a few years, scientists have broken through barriers that have hamstrung the field for decades, thanks to the conceptually simple ability to target specific sequences in an organism’s genome. That ability alone has led to enormous leaps in our understanding of complex traits, biological pathways, disease, and evolution. Additionally, genome editing with CRISPR has ushered in a new age of therapeutics and genetic engineering. As new applications of CRISPR technology emerge, we are beginning to push the boundaries of what it is capable of, and new modalities of CRISPR are required to overcome new challenges. For example, despite its reputation of being the “flagship” CRISPR molecule, the S. pyogenes Cas9 (a.k.a. SpCas9), is still too large to be genetically encoded and delivered by adeno-associated viruses (AAVs). As AAVs are one of the predominant delivery mechanisms for gene therapies, precluding SpCas9 from its repertoire is a significant deficiency. SpCas9 has been studied extensively to date. However, certain aspects of the molecule and its mechanisms are still unknown and evade our understanding even as we expand our gene-editing toolkit to include new Cas9s, new CRISPR systems, and new platforms. One of the questions associated with SpCas9 is its multi-domain architecture, which is atypically complex and large for bacterial proteins. While the general molecular mechanism of Cas9 is understood thanks to structural and kinetics studies, the evolution of the overall protein and its domains are less understood. This is especially interesting considering how little sequence identity Cas9s share among orthologs while still possessing a similar three-dimensional architecture.Additionally, SpCas9’s combination of stability, precision, and versatility makes it a prime candidate for protein engineering. Researchers have tried adding new functions to SpCas9, improving its performance, and even making it more suitable for certain use cases using rational design principles. However, making the protein smaller has been an under-utilized concept with limited success. Minimizing SpCas9 while still retaining its DNA targeting function has two main functions: a) understand the essentiality of its domains for DNA targeting, and b) develop a novel protein scaffold that is smaller and more feasible for AAV and other forms of cellular delivery. In Chapter 2 of this thesis, I discuss a project to probe the amino acid deletion landscape of SpCas9, in an effort to biochemically characterize its domains, and also to arrive at a minimal DNA-binding protein module.CRISPR-Cas9 has undoubtedly changed the biotechnology world by advancing genetic engineering and breaking through technical barriers that have plagued the field for decades. Simultaneously, the maturation of several other synthetic biology tools has also converged into a new era of biotechnology. Genome editing, sequencing, bioinformatics, gene synthesis, etc. have brought upon a revolution of new bioengineered products to the market. Leading the charge are genetically engineered microorganisms, or GEMs. GEMs are being developed for the purpose of making biofuels, commodity chemicals, materials, food and food additives, pesticides, and fertilizers. Many of these products have promise as more efficient, eco-friendly, and overall more beneficial alternatives to conventional industries.However, adoption and deployment of these technologies are not as simple as building them. Understandably, bioengineered products require regulatory oversight to ensure safety and efficacy. In the U.S., biotechnology products are regulated by a tri-agency framework consisting of the Food and Drug Administration (FDA), U.S. Department of Agriculture (USDA), and the Environmental Protection Agency (EPA). These agencies have distinct roles and evaluation criteria when assessing new biotech products for market approval. However, one of the key issues with the current regulatory landscape in the U.S. is its complicated and circuitous nature that often delay and/or drive up costs of product development. To maximize the impact of all the innovation and benefit of scientific advances happening in the laboratory, regulation needs to be more streamlined without compromising safety.In Chapter 3 of this thesis, I describe the current state of GEMs in the U.S., from the development and regulation perspectives. I discuss the product areas in which GEMs are emerging as feasible and scalable alternatives to conventional industrial processes, such as fuels, food, and agriculture. I also attempt to explain the current tri-agency regulatory framework as set by the FDA, USDA, and EPA. Finally, I discuss ways in which both GEM regulators and product developers must work hand in hand to enact large-scale solutions to major modern problems like climate change and food insecurity

Characterization of the Structural and Protein Recognition Properties of Hybrid PNA-DNA Four-Way Junctions

The objective of this study is to evaluate the structure and protein recognition properties of hybrid four-way junctions (4WJs) composed of DNA and peptide nucleic acid (PNA) strands. We compare a classic immobile DNA junction, J1, vs. six PNA–DNA junctions, including a number with blunt DNA ends and multiple PNA strands. Circular dichroism (CD) analysis reveals that hybrid 4WJs are composed of helices that possess structures intermediate between A- and B-form DNA, the apparent level of A-form structure correlates with the PNA content. The structure of hybrids that contain one PNA strand is sensitive to Mg+2. For these constructs, the apparent B-form structure and conformational stability (Tm) increase in high Mg+2. The blunt-ended junction, b4WJ-PNA3, possesses the highest B-form CD signals and Tm (40.1 °C) values vs. all hybrids and J1. Protein recognition studies are carried out using the recombinant DNA-binding protein, HMGB1b. HMGB1b binds the blunt ended single-PNA hybrids, b4WJ-PNA1 and b4WJ-PNA3, with high affinity. HMGB1b binds the multi-PNA hybrids, 4WJ-PNA1,3 and b4WJ-PNA1,3, but does not form stable protein-nucleic acid complexes. Protein interactions with hybrid 4WJs are influenced by the ratio of A- to B-form helices: hybrids with helices composed of higher levels of B-form structure preferentially associate with HMGB1b

Supporting data for the characterization of PNA–DNA four-way junctions

Holliday or DNA four-way junctions (4WJs) are cruciform/bent structures composed of four DNA duplexes. 4WJs are key intermediates in homologous genetic recombination and double-strand break repair. To investigate 4WJs in vitro, junctions are assembled using four asymmetric DNA strands. The presence of four asymmetric strands about the junction branch point eliminates branch migration, and effectively immobilizes the resulting 4WJ. The purpose of these experiments is to show that immobile 4WJs composed of DNA and peptide nucleic acids (PNAs) can be distinguished from contaminating labile nucleic acid structures. These data compare the electrophoretic mobility of hybrid PNA–DNA junctions vs. i) a classic immobile DNA 4WJ, J1 and ii) contaminating nucleic acid structures

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics