Filtering by
- All Subjects: Biophysics
- Creators: School of Molecular Sciences
- Member of: Theses and Dissertations
- Status: Published
Lyme disease is a common tick-borne illness caused by the Gram-negative bacterium Borrelia burgdorferi. An outer membrane protein of Borrelia burgdorferi, P66, has been suggested as a possible target for Lyme disease treatments. However, a lack of structural information available for P66 has hindered attempts to design medications to target the protein. Therefore, this study attempted to find methods for expressing and purifying P66 in quantities that can be used for structural studies. It was found that by using the PelB signal sequence, His-tagged P66 could be directed to the outer membrane of Escherichia coli, as confirmed by an anti-His Western blot. Further attempts to optimize P66 expression in the outer membrane were made, pending verification via Western blotting. The ability to direct P66 to the outer membrane using the PelB signal sequence is a promising first step in determining the overall structure of P66, but further work is needed before P66 is ready for large-scale purification for structural studies.
for facile, low cost scaling of nanostructures. However, current benchtop experiments have
limitations based on the placement of molecular species that exhibit greater than singlemolecular binding. In addition, reliance upon bottom-up self-assembly of close-packed
nanospheres makes it problematic to resolve images using low-cost light microscopes due to the
spacing limitations smaller in magnitude than light wavelength. One method that is created to
resolve this issue is iterative size reduction (ISR), where repetitive ‘iterative’ processes are
employed in order to increase the precision at which single molecules bind to a given substrate.
ISR enables inherent separation of nanospheres and therefore any subsequent single molecule
binding platforms. In addition, ISR targets and encourages single-molecule binding by
systematically reducing binding site size. Results obtained pursuing iteratively reduced
nanostructures showed that many factors are needed to be taken into consideration, including
functionalization of nanosphere particles, zeta potential, and protonation-buffer reactions.
Modalities used for observation of nanoscale patterning and single-molecule binding included
atomic force microscopy (AFM) and ONI super-resolution and fluorescence microscopy. ISR
was also used in conjunction with zero mode waveguides, which are nanoapertures enabling realtime single molecule observation at zeptoliter volumes. Although current limitations and
obstacles still exist with reproducibility and scalability of ISR, it nonetheless exhibits limitless
potential and flexibility in nanotechnology applications.
θATP-bd exists. The ATP-binding dwell can occur even at saturating ATP concentrations. We report that ω follows a distinct pattern in the vicinity of the ATP-binding dwell, and that the ω(θ) curve contains the same oscillations within it regardless of θATP-bd. We observed that an acceleration/deceleration dependence before and after the ATP-binding dwell, respectively, remained for increasing time intervals as the dwell occurred later in Phase-1, to a maximum of ≈ 40°. The results were interpreted in terms of a model in which the ATP-binding dwell results from internal drag at a variable position on the γε rotor.
Molecular engineering is an emerging field that aims to create functional devices for modular purposes, particularly bottom-up design of nano-assemblies using mechanical and chemical methods to perform complex tasks. In this study, we present a novel method for constructing an RNA clamp using circularized RNA and a broccoli aptamer for fluorescence sensing. By designing a circular RNA with the broccoli aptamer and a complementary DNA strand, we created a molecular clamp that can stabilize the aptamer. The broccoli aptamer displays enhanced fluorescence when bound to its ligand, DFHBI-1T. Upon induction with this small molecule, the clamp can exhibit or destroy fluorescence. We demonstrated that we could control the fluorescence of the RNA clamp by introducing different complementary DNA strands, which regulate the level of fluorescence. Additionally, we designed allosteric control by introducing new DNA strands, making the system reversible. We explored the use of mechanical tension to regulate RNA function by attaching a spring-like activity through the RNA clamp to two points on the RNA surface. By adjusting the stiffness of the spring, we could control the tension between the two points and induce reversible conformational changes, effectively turning RNA function on and off. Our approach offers a simple and versatile method for creating RNA clamps with various applications, including RNA detection, regulation, and future nanodevice design. Our findings highlight the crucial role of mechanical forces in regulating RNA function, paving the way for developing new strategies for RNA manipulation, and potentially advancing molecular engineering. Although the current work is ongoing, we provide current progress of both theoretical and experimental calculations based on our findings.
Here, a mathematical model of dielectrophoretic data is presented to connect analyte properties with data features, including the intercept and slope, enabling DEP to be used in applications which require this information. The promise of DEP to distinguish between analytes with small differences is illustrated with antibiotic resistant bacteria. The DEP system is shown to differentiate between methicillin-resistant and susceptible Staphylococcus aureus. This differentiation was achieved both label free and with bacteria that had been fluorescently-labeled. Klebsiella pneumoniae carbapenemase-positive and negative Klebsiella pneumoniae were also distinguished, demonstrating the differentiation for a different mechanism of antibiotic resistance. Differences in dielectrophoretic behavior as displayed by S. aureus and K. pneumoniae were also shown by Staphylococcus epidermidis. These differences were exploited for a separation in space of gentamicin-resistant and -susceptible S. epidermidis. Besides establishing the ability of DEP to distinguish between populations with small biophysical differences, these studies illustrate the possibility for the use of DEP in applications such as rapid diagnostics.
To read DNA nucleotides with high accuracy in an STM tunnel junction a series of nitrogen-based heterocycles were designed and examined to check their capabilities to interact with naturally occurring DNA nucleotides by hydrogen bonding in the tunnel junction. These recognition molecules are Benzimidazole, Imidazole, Triazole and Pyrrole. Benzimidazole proved to be best among them showing DNA nucleotide classification accuracy close to 99%. Also, Imidazole reader can read an abasic monophosphate (AP), a product from depurination or depyrimidination that occurs 10,000 times per human cell per day.
In another study, I have investigated a new universal reader, 1-(2-mercaptoethyl)pyrene (Pyrene reader) based on stacking interactions, which should be more specific to the canonical DNA nucleosides. In addition, Pyrene reader showed higher DNA base-calling accuracy compare to Imidazole reader, the workhorse in our previous projects. In my other projects, various amino acids and RNA nucleoside monophosphates were also classified with significantly high accuracy using RT. Twenty naturally occurring amino acids and various RNA nucleosides (four canonical and two modified) were successfully identified. Thus, we envision nanopore sequencing biomolecules using Recognition Tunneling (RT) that should provide comprehensive betterment over current technologies in terms of time, chemical and instrumental cost and capability of de novo sequencing.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.