Browsing by Subject "Aptamer"

Now showing 1 - 6 of 6
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    Aptamer selection using capillary electrophoresis-SELEX
    (2008-10) Mosing, Renee Karen
    SELEX is a method used for the combinatorial selection of aptamers, or single stranded nucleic acid sequences that bind with high affinity and specificity to target molecules. Although successful, SELEX is a very time consuming, laborious process. We introduced a modification to this process called capillary electrophoresis-SELEX. This protocol proved to be significantly more efficient, which greatly decreased the time requirement of the process from weeks to days. This improvement was observed despite the lower loading capacity and resolution limited injections on CE which introduced approximately 1013 sequences to the separation instead of the 1015 sequences introduced in more traditional selections protocols. In fact, ssDNA aptamers with picomolar affinity for HIV-1 RT were identified in 4 rounds. Further sequence/structure characterization of these sequences demonstrated no homology, indicating that several sequences can bind with high affinity to the target. Interestingly, the aptamers were 10 fold more selective for the original target (HIV-1 RT) than other reverse transcriptases. Despite this result, the aptamers did not demonstrate inhibition of reverse transcriptase activity. The success of these collections prompted investigation of more challenging targets such as mitochondria and bacteria. These targets are difficult to purify and have surface chemistries that are constantly changing. Aptamers for these targets must identify a feature on the surface that is consistent in order to be conserved throughout the process. Our experiments indicated that the aptamers may have bound to features on the surface that are not very abundant, making affinity characterization cumbersome. Future experiments aim to determine if the aptamers are becoming more refined for specific features rather than just focusing on increase in affinity. Finally, initial experiments were performed for a model that will drive selections toward a specific binding site on the surface of the HIV assembly protein, capsid. Further experiments are proposed to allow aptamer binding to specific sites, and for aptamer characterization.
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    Development and characterization of aptamer-amphiphiles against fractalkine for targeted drug delivery
    (2013-12) Waybrant, Brett M.
    A foundation of modern diagnostics and therapeutics is the ability to non-covalently bind to a molecule of interest. These affinity molecules are behind a broad array of products ranging from therapeutics to HIV tests. Currently, antibodies are used as the affinity molecule. Despite the success of antibodies, alternatives are needed due to high development and production costs, and issues with stability. Aptamers are an exciting alternative to antibodies. Aptamers are short sequences of single stranded DNA or RNA that bind molecular targets with high affinity and specificity. Aptamers are inexpensive to produce, are very stable, have long shelf lives, and could potentially replace antibodies in a number of applications. One potential application of aptamers is targeted drug delivery. The goal of targeted drug delivery is to selectively deliver a therapeutic payload to the site of action thereby increasing efficacy and decreasing side effects. Fractalkine is a cell surface protein expressed at sites of inflammation. It is expressed on several types of cancerous tissues and it is involved in the patheogenisis of arthritis, asthma, and atherosclerosis. This work describes the development and characterization of an aptamer that binds fractalkine with high affinity. The aptamer was modified with a hydrophobic tail, creating an aptamer-amphiphile, for use in a model drug delivery vesicle called a liposome. The aptamer-amphiphile was optimized for a high affinity interaction with fractalkine by adding a spacer molecule between the aptamer headgroup and the hydrophobic tail. The optimized amphiphile had high affinity for fractalkine and self-assembled into micelles and an interesting nanotape morphology. Finally, as a proof of concept, the optimized aptamer-amphiphile was incorporated into a liposome and targeted to fractalkine expressing cells. This work highlights the development of aptamers as affinity ligands, and demonstrates their use as potential drug delivery agents.
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    DNA Nanotechnology: Developing and Analyzing a New Tool for Sensing Allergens
    (2016-06) Brumann Clemente, Ana Paula
    Allergens are a major problem especially concerning public health and economy. There are more than 150 foods that can initiate allergic reactions, these reactions can elicit a mild response or a dangerous life threatening condition and in some extreme cases death. Milk and milk ingredients are one of the 8 foods that are responsible for about 90% of all food related allergic reactions. Food containing undeclared allergens in the label are misbranded and adulterated, and in accordance with the FSMA law must be recalled. It is estimated that the food industry can spend up to $10 millions dollars in direct costs from a recall. It was hypothesized that aptamer-amphiphile, a synthesis product from ssDNA aptamer and a hydrocarbon tale, in conjunction with liquid-crystal could be used as a sensor for detection of -lactoglobulin, an allergenic whey protein. The sensor was based on the self-alignment properties of liquid crystals based on the environment that it is exposed and on the capabilities of DNA aptamers to specific binding to targets. Results of this work showed that the aptamer-amphiphile of choice, amphiphile synthesized without a spacer between the DNA head group and the hydrocabon tail, had a great affinity to target, Kd= 45 ± 1.68 nM. In addition to it, it was possible to demonstrate that the interaction of the aptamer-amphiphile with the target protein, -lactoglobulin, using the sensor assembly resulted in images that can be easily identified under the polarizing microscope, sensor exposed to the aptamer-amphiphile alone gave a black image, once the protein was introduced the image was bright. Furthermore, the sensor developed has a limit of detection of 18.4ng of -lactoglobulin. It was also able to selectively identify the target protein, since when aptamer-amphiphile supported on the sensor was exposed to a random protein the image did not change as it did with -lactoglobulin. In conclusion, this sensor developed proves the concept that aptamer-amphiphile and the liquid crystal can potentially be used as a sensor technique in food plants to detect allergens in food contact surfaces.
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    Enhancing selection and biological applications of oligonucleotide affinity reagents
    (2021-08) Dembowski, Sean
    Aptamers are unique sequences of single-stranded DNA or RNA that exhibitsignificant affinity toward a particular target of interest. They have a number of advantages over antibodies – ease of modification, cell-free synthesis, and spontaneous renaturation – yet the preference of most researchers for antibodies continues due to prevalence of commercial antibody sources, hesitance adjusting existing research protocols, and a subconscious association of binding behavior with proteins rather than nucleic acids. While aptamers have already shown their technical advantages in the literature, they will also need to demonstrate their practicality via a broader range of targets and useful applications in order for aptamers to become more widespread in bioanalytical research. One challenge aptamers have faced is the inconsistency of SELEX, the combinatorial method by which new aptamers are identified, as well as a small pool of potential aptamer targets. This dissertation demonstrates the feasibility of aptamer selection against intact membrane proteins using a capillary electrophoresis-based SELEX technique, an achievement not yet demonstrated in the literature that opens SELEX to a much wider range of biologically significant potential targets. In addition to high affinity binding (Kd < 10 nM), these aptamers should undergo cellular internalization with their membrane receptor target, allowing hybrid aptamers to reach previously inaccessible intracellular targets as well. Additionally, this dissertation serves to increase the body of work demonstrating the bioanalytical capabilities of aptamers and other oligonucleotide affinity reagents. First, progress is shown toward aptamers targeting mouse leptin for use in a real-time aptamer- based microfluidic assay to quantify leptin secretion in cultured adipocytes. This proposed device would allow measurement of leptin, an important metabolic protein related to obesity, on a sub-minute time scale in response to metabolic stimuli. Quantification involves on-line separation of aptamers and aptamer-leptin complexes by electrophoresis, a technique that lends itself well to oligonucleotide affinity reagents due to their highly negative charge. Finally, a heart model based on cardiomyocyte differentiation from induced stem cells is demonstrated in preparation for measurement of physiological effects of aptamer-like SPIDRs (small protein-interacting DNAs and RNAs) on cardiomyocyte beating. SPIDRs have been shown to bind to the calcium pump SERCA and its control protein phospholamban, increasing the calcium affinity of SERCA. The cardiomyocyte model will be used to evaluate the hypothesis that this increase in calcium affinity leads to measurable physiological changes in beating, namely the beat rate and fall time, demonstrating the therapeutic potential of SPIDRs. Herein, optimization of stem cell growth and differentiation to cardiomyocytes are shown, as well as preliminary quantitative data regarding the reproducibility of cell impedance measurements.
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    Microfluidic assays for assessing oligonucleotide catalyst abundance and monitoring biomolecule concentration in real time
    (2023-10) Douma, Cecilia
    Microfluidic platforms control and manipulate very small volumes of liquid, typically at the microliter or nanoliter scale. By replacing pipettes and flasks with microfluidic channels and chambers, routine laboratory processes can be scaled down and sped up. Microfluidic platforms can mix, react, incubate, separate, extract, and detect solutions with high throughput and reproducibility, measuring the natural world at physical scales and timescales that would be inaccessible using traditional laboratory techniques. This thesis describes the development of microfluidic assays to address two bioanalytical challenges. First, a droplet microfluidic platform was developed to quantify the abundance of catalytic molecules in pools of random-sequence DNA. Although catalytic oligonucleotides are attractive as sensors and therapeutic agents, the full scope of their catalytic activity is largely unknown. The microfluidic platform described here encapsulates a library of DNA sequences in droplets with a fluorogenic substrate. Droplets that contain a catalytic sequence will become fluorescent after a period of incubation, while droplets without a catalyst will remain dark. The frequency of catalysts in the original library can be calculated from the ratio of fluorescent and non-fluorescent droplets. This thesis describes the technical design of a droplet microfluidic platform, its performance in library screening experiments, and its application for the detection of a known DNA catalyst. A versatile microfluidic platform for oligonucleotide library screening could assess catalyst abundance across a wide variety of reactions and conditions, creating a new framework for understanding the catalytic potential of oligonucleotides. Second, an aptamer affinity assay was developed for continuous cytokine quantification using micro free-flow electrophoresis (µFFE). Affinity assays are a prominent tool for biomolecule quantification because of their excellent sensitivity and specificity. However, traditional affinity assays use discrete samples and are poorly suited for measuring dynamic changes in an analyte’s concentration. The ultimate aim of the aptamer assay is to continuously quantify cellular cytokine secretion in real time using µFFE, a continuous separation technique that can detect free aptamer and bound aptamer complexes in a flowing sample stream. This thesis describes the characterization of µFFE devices fabricated in cyclic olefin copolymer as well as initial development of a µFFE aptamer assay for continuous quantification of tumor necrosis factor α (TNFα).
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    Microfluidic selection of DNA aptamers using capillary electrophoresis and micro free flow electrophoresis systematic evolution of ligands by exponential enrichment
    (2012-08) Jing, Meng
    SELEX is the process used to generate aptamers, which are ssDNA or RNA molecules that can bind specific targets with high affinity. Although aptamers show great potential in clinical applications, the generation process is currently tedious and inefficient, being a limiting step. Thus, understanding, developing, and applying advanced partitioning platform are pivotal. CE is an advanced separation method in SELEX and has been successfully used to generate aptamers toward multiple targets. However, there are still interesting questions unanswered, making our understanding in CE-SELEX lag behind its applications. We applied high-throughput sequencing on CE-SELEX selected pools against rhVEGF and obtained sequencing information of more than 104 sequences per pool, which allowed characterization on diversity of individual pools. This study revealed the coexistence of high diversity and fast enrichment rate of CE-SELEX. To further improve the separation platform, we integrated a μFFE device into the SELEX process. Using this device, 1014~1015 sequences were introduced and analyzed within 30 min, which was a 370 fold improvement compared to CE. As a proof of concept, four cycles of selection were performed to the target human IgE, and high affinity ligands were generated even after the first round of selection, proving the feasibility and high efficiency of μFFE in SELEX. Later, μFFE-SELEX was applied to generate aptamers for a membrane protein SERCA, whose selection has never been achieved in conventional SELEX due to the technical difficulty in target immobilization. High nM Kd pool was generated toward SERCA solubilized in C12E8 at the fifth round of selection, demonstrating that this separation strategy is more compatible with membrane proteins due to the free solution based separation, lower electric field, faster separation speed, and straightforward fraction collection. The success of this application opens a door for high-throughput generation of aptamers toward complex targets in the future. Besides SELEX, the function and activity of the ATPase SERCA were also explored in this thesis. It was discovered that nonspecific ssDNA sequences can bind to the endogenous regulator of SERCA, PLN, in a length dependent way. A highly sensitive and reproducible SERCA activity assay, which cut the use of SERCA by 2,000 folds, was developed to directly detect the product ADP via TR-FRET.