DNA origami nanotechnology has evolved rapidly since its conception eleven years ago. Both two-dimensional and three-dimensional nanostructures have been created with potential applications in targeted drug delivery, “smart” diagnostic technology, and the study of cell behavior. By annealing “staple” oligonucleotide strands to a single-stranded DNA scaffold we can effectively fold the DNA onto itself to build the nanostructures of interest. One of the primary physical limitations to what one can build is the scaffold. The most commonly used scaffold is derived from the bacteriophage M13mp18 and has a length of 7,249 nucleotides. Its length has previously been varied; however, an overlooked limitation is the secondary structure DNA naturally exhibits. These are sites in which the scaffold binds to itself, thus creating competition for staples to bind during folding reactions. To predict the impact that a designed sequence with little secondary structure could have, we analyzed the first 6,000 bases of the M13mp18 DNA sequence using NUPACK, a nucleic acid sequence analyzer, for their minimum free energy (MFE) at storage, manipulation, and maximum folding reaction temperature. Preliminary data shows M13mp18 exhibits less secondary structure at a high temperature (65°C) than at a low temperature (4°C) and increasing the concentration of divalent salts linearly increases the amount of secondary structure. Additionally, alternative, shorter sequences have been engineered and their secondary structure is being analyzed at varying conditions. To further determine the effects on yield and stability, structures will be folded using the designed sequence and the standard sequence as a scaffold. These will be compared through agarose gel electrophoresis and transmission electron microscopy. The results from this preliminary data could help us move us toward using a scaffold with decreased secondary structure present at folding temperatures which could potentially result in higher yields, shorter folding reactions, and increased stability.

Characterizing biological functions on a single molecule scale increases understanding of biological functions by providing information about the indiviual contributions which combine to create larger scale functions. Single molecule measurements are a crucial part of characterizing molecular interactions. Although atomic force microscopy (AFM) and magnetic tweezers are able to measure the response of single molecules to mechanical force, it is challenging to ensure single molecules are being measured. In this project, a precise DNA Origami structure was used to space molecules for single molecule force measurements. Base pair association between DNA nucleotides allowed specific nanostructures to be designed and fabricated. Molecules of interest self-assemble to specific sites of the structure. AFM was used for imaging and obtaining force measurements. This research investigates the strength of adhesion for double stranded DNA when subjected to different loading rates as a proof of concept. In the future, this structure will be used to determine force properties of diverse molecular interactions like platelet and bacterial adhesions.

RNA Polymerase (RNAP) is found in all cellular organisms and synthesizes RNA from DNA through a process known as transcription. Using a single-molecule tool, Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT), developed by Gundlach’s Nanopore Lab at the UW, we can track the motion of individual RNAP molecules along DNA at unprecedented spatial and temporal resolution. In this technique, a single nanopore, a biological transmembrane protein, is isolated in a phospholipid bilayer separating two wells both filled with salt solutions. An applied electric field drives ions through the pore, and the ion current is measured. Negatively charged DNA is also driven through the nanopore, partially blocking the ion current. The DNA is sequenced based on the changing ion current as each of the four DNA nucleotides sequentially pass through the pore and block the flow of ions. In SPRNT, the motion of a known sequence of DNA through the nanopore is controlled by a molecular motor (like RNAP), in order to understand how an enzyme moves along DNA. Using this technique, we can detect single base pair steps of RNAP along DNA as brief as 1 ms. In this work, we expand upon initial SPRNT experiments tracking RNAP and analyze the kinetics of RNAP during transcription in biological relevant conditions (1 mM NTPs, 37C) on a long DNA template (up to 6 kbp). We calculate the rate of NTP incorporation and stepping behavior (probabilities of different step types) at many different DNA template sequences using this technique. The results demonstrate how DNA sequences as well as applied force can affect the stepping behavior (probabilities of different step types) and stepping rate of RNAP. A complete understanding of RNAP and its transcriptional regulatory behaviors will enable scientific and medical communities to better understand a variety of genetic diseases associated with transcription.

Staphylococcus aureus is responsible for large numbers of patient morbidity worldwide. However, studies of this important pathogen have suffered from the lack of a robust toolset for scalable genetic and genomic manipulation in the organism. Our goal for this study was to develop an efficient, simple, and inexpensive system for precision genome engineering in S. aureus. Our strategy employs ssDNA oligonucleotide recombineering coupled with CRISPR/Cas9-mediated counterselection to address these needs. We first identified a single-stranded recombinase protein, EF2132, derived from Enterococcus faecalis, which can successfully integrate single-stranded DNA oligonucleotides into the S. aureus genome. Our results showed that EF2132 readily mediates recombineering across many different laboratory strains and primary clinical strains, normally yielding thousands of recombinants per transformation. We proceeded to expand the generality of our genome editing system by incorporating lethal counterselection, enabling efficient recovery of recombineered isolates lacking a selectable phenotype. We constructed a temperature sensitive, two-vector system that enables conditional recombineering and CRISPR/Cas9- mediated counterselection in S. aureus without permanently introducing exogenous genetic material or unintended genetic lesions. Our findings indicate that our system effectively targets and edits genes with both specific mutations and large-scale knockouts by utilizing inexpensive, commercially synthesized oligonucleotides as substrates for inducing and counterselecting precise genomic changes. Since our system utilizes cheap synthetic DNA oligonucleotides as substrates for recombineering and counterselection, it provides a scalable, precise and inexpensive tool for producing isogenic strain in S.aureus. Because of this, our system can be used to effectively facilitate future studies to investigate the genetic mechanisms of many different biological phenomena. As our study progresses, we hope to introduce refinements to further improve the scalability and efficiency of this methodology.

Gene amplification can be associated with genetic disorders such as cancer and varying types of autism. One such form of amplification is a neochromosome where a chromosomal segment is amplified and these two segments are joined at an inverted repeat. There are two models to explain how this event could occur: double stranded break and repair and origin dependent inverted repeat amplification (ODIRA). ODIRA hypothesizes that a replication fork error occurring near short inverted repeats could cause the leading strands to erroneously become ligated to the lagging strands and produce an extrachromosomal palindromic DNA intermediate. Because both models produce identical neochromosomes, I have worked to find which pathway produces them. To do so I utilized CRISPR/cas9 to induce breaks at commonly observed junctions, and analyzed the resulting intermediates using gel analysis of whole yeast chromosomes. If the neochromosome were to occur via the double stranded break and repair model, I expected that providing the break should increase the frequency with which these neochromosomes arise. Alternatively, if the neochromosomes were occurring due to the ODIRA model, I expected the frequency to remain unchanged. I followed this initial analysis with DNA sequencing to give further support to either outcome. In particular I verified that the CRISPR system was working by sequencing across the junctions where cas9 was cutting. If cas9 was indeed cutting, then when comparing the sequence from the cas9 survivors to that of the yeast genome, the survivors had an altered cut site. My work provides insight to the etiology to this fascinating class of chromosome rearrangements.

DNA replication is critical to cellular reproduction. In bacterial and eukaryotic cells alike, DNA replication occurs in immobile, discrete complexes (so-called “factories”) enriched for replication proteins. However, the size of these replication complexes is well below the optical diffraction limit (~250 nm), making them difficult to study using conventional microscopy. Using Escherichia coli as a model organism, the goal of my project is to create an ultrastructural model (50-nm resolution) of the bacterial replication complex. To this end, I have developed an expansion microscopy (ExM) protocol that is compatible with bacterial cells. In short, E. coli cells harboring fluorescent fusions to replication proteins are embedded in a swellable polymer gel and physically expanded by a factor of four. The expanded specimen is subsequently visualized at high-resolution using a conventional fluorescence microscope. Unlike more traditional super-resolution methods, ExM is compatible with simultaneous imaging of up to four replication proteins, which I expect will be critical in determining the overall organization of the replication complex. Although ExM is established for eukaryotic cells, I am the first to apply the method to visualize the interiors of bacterial cells. I expect that this project will not only contribute a new super-resolution-microscopy method for bacterial cells, but also reveal the structure of the replication complex with unprecedented detail, perhaps providing insight into the mechanism of the fundamental process of DNA replication. This, in-turn, could lead to new therapies for combating diseases associated with replication defects, including cancer.

Antibiotic resistance is an intractable clinical challenge that disproportionally affects much of the world’s most impoverished populations. A critical driver of resistance stems from DNA mutations. Therefore, insights into how mutations arise in the genome is critical to understanding the development of antibiotic resistance. Our lab is currently identifying factors and environmental conditions that promote mutagenesis and bacterial evolution. Utilizing mutation rate analysis, we have identified factors and conditions that promote mutagenesis in divergent bacterial species. Additionally, using short-term evolution experiments, we have found that these factors promote the development of antibiotic resistance over time. This work provides novel mechanistic insights into mutagenesis and antibiotic resistance development in bacteria.

For more than a century, Drosophila melanogaster (fruit flies) have been an invaluable and versatile tool to further our understanding of cell signaling and survival mechanisms. To this day, they continue to shed light on the endogenous pathways that cancer cells can hijack in order to proliferate, metastasize, and recur following remission. The molecular conservation of these pathways invites parallels between the germline stem cells in D. melanogaster and the cancer stem-like cells in human carcinoma. In the same way that a tumor can relapse following a period of dormancy, Drosophila germline stem cells are capable of repopulating their niche after insult from Ionizing Radiation (IR). Utilizing this powerful model, we have conducted a small molecule drug screen of 512 compounds that we have narrowed down to eight candidate drugs that appear to increase cell death in Drosophila germline stem cells. Having already characterized the wild type Drosophila germline stem cell response to IR-induced DNA damage, we  probed how drug treatment and gene knockdown affected the germline stem cells' ability to recover from insult. Previous work in the Ruohola-Baker Lab has demonstrated the critical importance of the mechanistic target of rapamycin (mTOR) and the Tie receptor pathways in regulating regeneration after insult in the Drosophila germline. The Tuberous Sclerosis Complex (TSC), a heterodimer comprised of Tsc1 and Tsc2, is a known negative regulator of mTOR. Additionally, the Tie receptor is central to anti-apoptotic signal transduction in the Drosophila ovary. We have screened four candidate drugs to see if they effectively increase stem cell death in Tsc1-knockdown and Tie-null flies, in order to ascertain whether our drugs affect stem cell survival mechanisms through mTOR and/or Tie signaling. Our findings may shed light onto how to mitigate the quiescent threat of tumor relapse.

Small RNA molecules called microRNAs (miRNAs) are translational repressors and are involved in a variety of processes important for normal cell development and function. Previous studies in our lab showed that miRNAs are required in early (embryonic) stages of retinal development. After depletion of miRNAs in early retinal progenitor cells (RPCs), late RPCs and their progeny, such as Müller glia (MG) which are the primary glial in the retina, are not generated. Instead, only early born neurons are produced. The depletion of miRNAs can be obtained through deletion of an enzyme that generates mature miRNAs called Dicer. Since we know that miRNAs are crucial for early retinal development, this study investigated if miRNAs are also important for proper late (postnatal) retinal development. We used a Sox2 reporter mouse which allows visualizing late retinal progenitors (Sox2 is a gene expressed in RPCs) and selectively deleted Dicer in RPCs at postnatal day 2-6. The tissue was analyzed 2, 4 and 7.5 weeks after Dicer deletion and compared to normal retinas using immunofluorescent staining of retinal cross sections and confocal microscopy. We found massive disruptions in the retinal architecture such as the formation of rosette/ bubble-like structures, indicating that miRNAs are required for proper formation of the retinal layers. Since MG play a role in maintaining the retinal structure, in a second series of experiments we induced Dicer deletion specifically in MG at postnatal day 11 –14, when the retina is almost completely developed. The tissue was analyzed 8 and 20 weeks after Dicer-deletion and compared to normal tissue. We found an atypical location of MG and disruptions in the layered retinal structure, indicating that miRNAs are also required for accurate MG location in the retina, which is essential for normal retinal architecture.