Matching Items (45)

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Role of RAG2 C-terminal region in enforcing appropriate recombination cleavage directed at legitimate DNA targets

Description

V(D)J Recombination is the mechanism responsible for generating diversity in the repertoire of antigen receptors of T and B cells. This recombination process proceeds in two steps: site-specific cleavage mediated

V(D)J Recombination is the mechanism responsible for generating diversity in the repertoire of antigen receptors of T and B cells. This recombination process proceeds in two steps: site-specific cleavage mediated lymphocyte-specific recombinase known as Recombination Activating Genes 1 and 2 complex (RAG) at the junction of coding gene segments and their flanking recombination signal sequence (RSS) and then followed by rejoining of the double strand broken DNA by the non-homologous end joining (NHEJ) complex. Mutations and truncations of the RAG-recombinase have been found associated with genomic instability and chromosomal translocation. It has been hypothesized that these RAG mutants may have abnormality in their interactions with recombination intermediates, ultimately causing premature release of the ends for aberrant joining. Additionally, these mutations have an increase in targeting non-B type DNA instead of legitimate recombination substrates that contain RSSs. To directly test these hypotheses, we have developed a fluorescence-based detection system to monitor in real time the recombination cleavage reaction from the pre-cleavage to the post-cleavage stages and to compare RAG-DNA interactions between wild type and mutant RAG1/2 during this process. Our study provides important insight into the ability of the C-terminus of RAG to regulate RAG recombinase activity.

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  • 2014-12

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Exploration of Enzymatic Efficiency in Double-Stranded DNA by Uracil-DNA Glycosylase and Optimization of Glycosylation Reaction of DNA Precursor

Description

The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and

The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and its relationship to enzymatic efficiency. Chapter 2 centers itself on threose nucleic acid and optimization of a step in the path to its synthesis. While Chapter 1 discusses DNA and Uracil-DNA Glycosylase with regards to the base excision repair pathway, Chapter 2 focuses on chemical synthesis of an intermediate in the pathway to the synthesis of TNA, an analogous structure with a different saccharide in the sugar-phosphate backbone.
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.

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Date Created
  • 2016-05

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Synthesis of Hybrid DNA-Protein Nanostructures

Description

While DNA and protein nanotechnologies are promising avenues for nanotechnology on their own, merging the two could create more diverse and functional structures. In order to create hybrid structures, the

While DNA and protein nanotechnologies are promising avenues for nanotechnology on their own, merging the two could create more diverse and functional structures. In order to create hybrid structures, the protein will have to undergo site-specific modification, such as the incorporation of an unnatural amino, p-azidophenylalanine (AzF), via Shultz amber codon suppression method, which can then participate in click chemistry with modified DNA. These newly synthesized structures will then be able to self-assemble into higher order structures. Thus far, a surface exposed residue on the aldolase protein has been mutated into an amber stop codon. The next steps are to express the protein with the unnatural amino acid, allow it to participate in click chemistry, and visualize the hybrid structure. If the structure is correct, it will be able to self-assemble.

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  • 2017-05

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Aptamer-functionalized Hydrogel for the Capture and Release of CCRF-CEM Leukemia Cancer Cells

Description

The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of

The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of an aptamer-functionalized hydrogel thin film at the bottom of a microfluidic channel, which changes its film thickness as the temperature of the fluid in the system changes. The functionalized hydrogel film has been created as the primary steps to creating the microfluidic device that could capture and release leukemia cells by turning the temperature of the fluid and length of exposure. Circulating tumor cells have recently become a highly studied area since they have become associated with the likelihood of patient survival. Further, circulating tumor cells can be used to determine changes in the genome of the cancer leading to targeted treatment. First, the aptamers were attached onto the hydrogel through an EDC/NHS reaction. The aptamers were verified to be attached onto the hydrogel through FTIR spectroscopy. The cell capture experiments were completed by exposing the hydrogel to a solution of leukemia cells for 10 minutes at room temperature. The cell release experiments were completed by exposing the hydrogel to a 40°C solution. Several capture and release experiments were completed to measure how many cells could be captured, how quickly, and how many cells captured were released. The aptamers were chemically attached to the hydrogel. 300 cells per square millimeter could be captured at a time in a 10 minute time period and released in a 5 minute period. Of the cells captured, 96% of them were alive once caught. 99% of cells caught were released once exposed to elevated temperature. The project opens the possibility to quickly and efficiently capture and release tumor cells using only changes in temperature. Further, most of the cells that were captured were alive and nearly all of those were released leading to high survival and capture efficiency.

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  • 2016-12

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The Interaction of Rubisco and Rubisco Activase: A FRET-based Study

Description

Rubisco is a very important protein which catalyzes the addition of CO2 to ribulose-1,5-bisphosphate (RuBP) to form two molecules of 3-phosphoglycerate in photosynthesis. Rubisco activase is the protein which

Rubisco is a very important protein which catalyzes the addition of CO2 to ribulose-1,5-bisphosphate (RuBP) to form two molecules of 3-phosphoglycerate in photosynthesis. Rubisco activase is the protein which functions to uninhibit Rubisco, however proof of a physical interaction has never been shown. A possible method for determining the interaction of the two proteins is by Förster Resonance Energy Transfer (FRET) based analysis of the two proteins. Attempts to get a FRET signal from these two proteins have been unsuccessful. To get better results, Ficoll 70, a crowding agent, was used. Analysis suggests that Ficoll 70 does not affect the fluorescence of Alexa-fluor 488 and Alexa-fluor 647 used to label the two proteins. Further analysis also suggests that while the Alexa label on Rubisco activase does not affect the ATPase activity of the protein, the protein also does not have a high rate of ATP turnover.

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Date Created
  • 2015-05

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Early Detection of MicroRNA Biomolecular Markers using CRISPR-Cas12a

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Extensive efforts have been made to develop efficient and low-cost methods for diagnostics to identify molecular biomarkers that are linked to a wide array of conditions, including cancer. A highly

Extensive efforts have been made to develop efficient and low-cost methods for diagnostics to identify molecular biomarkers that are linked to a wide array of conditions, including cancer. A highly developed method includes utilizing the gene-editing enzyme CRISPR-Cas12a (Cpf1), which demonstrates double-stranded DNase activity with RuvC catalytic domain with high sensitivity and specificity. This DNase activity is RNA-guided and requires a T-rich PAM site on the target sequence for functional cleavage. There have been recent efforts to utilize this DNase activity of Cas12a by combining it with isothermal amplification and analysis by lateral strip tests. This project examined CRISPR-based early detection of microRNA biomarkers. MicroRNA are short RNA molecules that have large roles in post-transcriptional gene regulation. However, due the short length of microRNA and its single-stranded nature, it is challenging to use Cas12a for microRNA detection using existing methods. Thus, this project investigated the potential of two microRNA detection strategies for recognition by CRISPR-Cas12a. These methods were microRNA-splinted ligation with polymerase chain reaction (PCR) and MicroRNA-specific reverse transcriptase PCR (RT-PCR). Gel imaging demonstrated effective amplification of ligated DNA through microRNA-splinted ligation with PCR/RPA. In addition, lateral strips tests showed effective cleavage of the target sequences by Cas12a. However, RT-PCR method demonstrated low amplification by PCR and inefficient poly(A) elongation. This project paves the way for the detection of an extensive range of microRNA biomarkers that are linked to an array of diseases. Future directions include analysis and modifications of RT-PCR method to improve experimental results, extending these detection methods to a larger range of microRNA sequences, and eventually utilizing them for detection in human samples.

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Date Created
  • 2019-05

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A Smart Shipping Incubator for Biodosimetry Logistics in Radiation Countermeasure Operations

Description

Mass nuclear catastrophe is a serious concern for society at large when considering the rising threat of terrorism and the risks associated with harnessing nuclear energy. In the case of

Mass nuclear catastrophe is a serious concern for society at large when considering the rising threat of terrorism and the risks associated with harnessing nuclear energy. In the case of a mass nuclear/radiological event that requires hundreds of thousands of individuals to be assessed for radiation exposure, a rapid biodosimetry triage tool is crucial [1]. The Cytokinesis Block Micronucleus Assay (CBMN) is a promising cytogenetic biodosimetry assay for triage [2]; however, it requires shipping samples to a central laboratory (1-3 days) followed by a lengthy cell culture process (~3 days) before the first dose estimate can be available. The total ~ 1 week response time is too long for effective medical care intervention. A shipping incubator could cut the response time in half (~3 days) by culturing samples in transit; however, possible shipping delays beyond 2 days without the addition of a necessary reagent (Cyto-B) would ruin the integrity of the samples—for accurate CBMN assay endpoint observation, Cyto-B must be added within a 24-44 hour window after sample culture is initiated. Here, we propose a “Smart” Shipping Incubator (SSI) that can add Cyto-B while samples are in transit through a centrifugal system equipped with microfluidic capillary valve caps. The custom centrifugal system was constructed with CNC machined and 3D printed plastic parts, controlled by a custom printed circuit board (PBC) microcontroller, and housed inside a commercial shipping incubator (iQ5 from MicroQ Technologies). Teflon-coated, pre-pulled glass micropipettes (FivePhoton BioChemicals) were used as microfluidic capillary valve caps. Release of Cyto-B was characterized by a desktop centrifugal system at different tip sizes and relative centrifugal forces (RCFs). A theoretical model of Cyto-B release was also deduced to aid the optimization of the process. The CBMN assay was conducted both in the SSI with centrifugal Cyto-B release and in a standard CO2 incubator with manual addition of Cyto-B as the control. The expected mechanical shock during shipment was measured to be less than 25g. Optimal Cyto-B release was found to be at 35g RCF with a Teflon-coated 40 µm tip. Similar CBMN dose curves of micronuclei per binucleated cells (MN/BN) vs. exposed radiation (Gy) were produced for samples assessed conventionally and with the SSI. The similarities between the two methods suggest that centrifugation does not significantly affect the CBMN assay.

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  • 2019-12

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Synthesis of Enzyme-Mimetic Catalysts

Description

The synthesis of the bis(2-diphenylphosphinoethyl)amine chelating ligand (1) was a crucial component in the preparation of non-canonical amino acids (NCAAs) throughout the project. Studies in this project indicated the need

The synthesis of the bis(2-diphenylphosphinoethyl)amine chelating ligand (1) was a crucial component in the preparation of non-canonical amino acids (NCAAs) throughout the project. Studies in this project indicated the need to isolate the ligand from its hydrochloride salt form seen in (1) which led to the synthesis of the brown oil, (Ph2PCH2CH2)2NH, (2). The ligand features a phosphine-nitrogen-phosphine group that is not observed in existing NCAAs. Phosphine groups are rarely seen in existing NCAAs and avoided by biochemists because they tend to oxidize before metal addition. In this project, (1) was used in a 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) mediated method and palladium-catalyzed method to tether an amino acid to the nitrogen atom of the ligand framework. Both methods were monitored through the use of Nuclear Magnetic Resonance (NMR) spectroscopy. While the palladium catalyzed method exhibited little to no coupling, the 31P NMR spectrum obtained for the HATU mediated method did reveal that some coupling had occurred. The unsuccessful attempts to tether an amino acid to (1) led to the hypothesis that the phosphine groups were interfering with the palladium catalyst during the cross-coupling reaction. In an effort to test this hypothesis, (2) was reacted with the dimer, [Rh(nbd)Cl]2, to coordinate the rhodium metal to the free phosphorous arms and the nitrogen atom of the isolated PNP ligand. The PNP-based metal complex was used in the palladium catalyzed method, but cross-coupling was not observed. The new PNP-based metal complex was investigated to demonstrate that it exhibits moisture and air stability.

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Date Created
  • 2018-05

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Exploration of Enzymatic Reactivity of Human Endonuclease Enzyme APE1 in Clustered DNA Damages Involving an Abasic Site

Description

This study was conducted to understand the reactivity of APE1 in repairing abasic sites associated with clustered DNA damages and to determine if the efficiency of APE1 enzyme is affected

This study was conducted to understand the reactivity of APE1 in repairing abasic sites associated with clustered DNA damages and to determine if the efficiency of APE1 enzyme is affected by the type of bases (purines or pyrimidines) neighboring the AP site. DNA damages are always occurring in living cells and if left uncorrected can lead to various problems such as diseases and even cell death. Cells are able to recognize and correct these DNA damages to prevent further damages to the genome, and the Base Excision Repair (BER) pathway is one of the mechanisms used in repairing DNA damages. A former student in the Levitus Lab, Elana Maria Shepherd Stennett, henceforth referred to as Elana worked on this project. She observed that the activity of the APE1 enzyme increased some when the base opposing the abasic site was changed from thymine (T) to adenine (A) while no difference was observed when the surrounding bases were changed. Thus, this experiment was conducted to further study the results she obtained and to possibly validate her findings. The AP sites used in this study are natural abasic sites created by UDG glycosylase enzyme from a double stranded uracil-containing DNA samples ordered from IDT technologies. Each reaction was carried out at physiological temperature (37degrees Celsius) and analyzed using polyacrylamide gel electrophoresis.

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Date Created
  • 2018-05

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Investigating the Stability of DNA Origami Structures in Buffer Solutions

Description

DNA nanotechnology uses the reliability of Watson-Crick base pairing to program and generate two-dimensional and three-dimensional nanostructures using single-stranded DNA as the structural material. DNA nanostructures show great promise for

DNA nanotechnology uses the reliability of Watson-Crick base pairing to program and generate two-dimensional and three-dimensional nanostructures using single-stranded DNA as the structural material. DNA nanostructures show great promise for the future of bioengineering, as there are a myriad of potential applications that utilize DNA’s chemical interactivity and ability to bind other macromolecules and metals. DNA origami is a method of constructing nanostructures, which consists of a long “scaffold” strand folded into a shape by shorter “staple” oligonucleotides. Due to the negative charge of DNA molecules, divalent cations, most commonly magnesium, are required for origami to form and maintain structural integrity. The experiments in this paper address the discrepancy between salt concentrations required for origami stability and the salt concentrations present in living systems. The stability of three structures, a two-dimensional triangle, a three-dimensional solid cuboid and a three-dimensional wireframe icosahedron were examined in buffer solutions containing various concentrations of salts. In these experiments, DNA origami structures remained intact in low-magnesium conditions that emulate living cells, supporting their potential for widespread biological application in the future.

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Date Created
  • 2020-05