Exploration into the use of aptamers as cross-reactive receptors was the focus of this work. Cross-reactivity is of interest for developing assays to identify complex targets and solutions. By exploiting the simple chemistries of aptamers, we hope to introduce a new class of receptors to the science of molecular discrimination. This manuscript first addresses the use designed aptamers for the identification of variants of HIV-1 reverse transcriptase. In this research aptamers were immobilized on a platform and were used to discriminate four variants of HIV-1 reverse transcriptase. It was found that not only could the array discriminate HIV-1 reverse transcriptase variants for which aptamers were designed, it would also discriminate variants for which no aptamers exist. A panel of aptamers was used to discriminate four separate cell lines, which were chosen as examples of complex targets. This aptamer panel was used to further explore the use of aptamers as cross-reactive sensors. Forty-six aptamers were selected from the literature that were designed to be specific to cells or molecules expected to be in the surface of cells. This panel showed differential binding patterns to each of the cell types, displaying cross-reactive behavior. During the course of this research, we also developed a novel ratiometric method of using aptamer count derived from next-generation sequencing as a method for discrimination. This is in lieu of the more commonly used fluorescent signals. Finally the use of multiple signals for pattern recognition routines was further explored by running various models using artificial data. Various situations were applied to replicate different possible situation which might arise when working with macromolecular interactions. The purpose of this was to advance the communities understanding and ability to interpret results from the pattern recognition methods of PCA and LDA. / text

<p>Thrombosis is associated with the occlusion of a blood vessel and can be triggered by a number of types of injury, such as the rupture of an atherosclerotic plaque on the artery wall, changes in blood composition, or blood stasis. The resulting thrombosis can cause major diseases such as myocardial infarction, stroke, and venous thromboembolic disorders that, collectively, account for the most common cause of death in the developed world. Anticoagulants are used to treat and prevent these thrombotic diseases in a number of clinical and surgical settings. Although commonly prescribed, currently approved anticoagulants have a major limitation of severe drug-induced bleeding that contributes to the high levels of morbidity and mortality associated with use. The "holy grail" for antithrombotic therapy is to identify a drug that inhibits thrombus formation without promoting bleeding. Understanding the differences between thrombosis and hemostasis in the vascular system is critical to developing these safe and effective anticoagulants, as this depends on striking the correct balance between inhibiting thrombus formation (efficacy) and reducing the risk of severe bleeding (safety). While it is commonly thought that the same factors play a similar role in hemostasis and thrombosis, recent evidence points to differing functions for FXI and FXII in each of these settings. Importantly, these factors seem to contribute to pathological thrombus formation without being involved in normal hemostasis.</p><p> The overall goal of this project was to evaluate the inhibition of the intrinsic pathway of coagulation as a potential anticoagulant strategy utilizing the aptamer platform. Aptamers are short, highly structured nucleic acids that act as antagonists by binding to large surface areas on their target protein and thus tend to inhibit protein-protein interactions. High affinity binding aptamers have been isolated that specifically target a diverse range of proteins, including transcription factors, proteases, viral proteins, and growth factors, as well as other coagulation factors. As synthetic molecules, aptamers have a small molecular weight, are highly amenable to modifications that can control their bioavailability, and have not been found to elicit an immune response, thus making them ideal drug candidates. Importantly, aptamers can be rapidly and effectively reversed with either a sequence specific antidote that recognizes the primary sequence of the aptamer or a universal antidote that binds to their backbone and reverses all aptamer activity independent of sequence. This ability lends itself well to their therapeutic application in coagulation, as rapid reversal of a drug upon the onset of bleeding is a key property for increasing the safety of this class of drugs.</p><p> Aptamers targeting FXI/FXIa and FXII/FXIIa were isolated in two separate SELEX (systematic evolution of ligands by exponential enrichment) procedures: the FXII aptamer was isolated in a convergent SELEX approach and the FXIa aptamer was isolated from a purified protein selection. In both processes, 2'fluoropyrimindine modified RNA with a 40-nucleotide random region was incubated with either the plasma proteome (in initial rounds of the convergent SELEX) or the purified protein target (FXII or FXIa). The nucleic acids that did not bind to the target were separated from those that bound, and these molecules were then amplified to generate an enriched pool with increased binding affinity for the target. This process was repeated under increasingly stringent conditions to isolate the aptamer that bound with the highest affinity to the purified target protein. Utilizing biochemical and in vitro coagulation assays, specific, high-affinity binding and functional anticoagulant aptamers were identified for both protein targets, and the mechanism of anticoagulation was ascertained for each aptamer. </p><p> Overall, both aptamers bound to an exosite on their target protein that was able to inhibit downstream activation of the next protein in the coagulation cascade. In order to specifically examine aptamer effects on several parameters of thrombin generation, a new assay was developed and fully characterized using aptamer anticoagulants targeting other coagulation factors. Aptamer inhibition of both FXI and FXII was able to decrease thrombin generation in human plasma. However, limited cross-reactivity in other animal species by both aptamers hindered our ability to assess aptamer inhibition in an in vivo setting. Moving forward, screening aptamers against a larger selection of animal plasmas will hopefully allow us to identify an animal species in which we can analyze aptamer inhibition of the intrinsic pathway for effectiveness and safety in inhibiting thrombosis. The further characterization and use of these aptamers in plasma and blood based settings will allow us to study the diverging functions of the intrinsic pathway in thrombosis and hemostasis.</p><p> A critical need exists for safe and effective anticoagulants to treat and prevent numerous thrombotic procedures and diseases. An ideal anticoagulant is one that strikes the correct balance between inhibiting thrombus formation and reducing drug-induced bleeding. Inhibition or depletion of factors XI and XII of the intrinsic pathway of coagulation have shown reduced thrombus formation without interruption of normal hemostasis in several models of thrombosis. By developing novel RNA aptamer anticoagulants to these factors, we have set the stage for evaluating the net therapeutic benefit of intrinsic pathway inhibition to effectively control coagulation, manage thrombosis, and improve patient outcome. As well as developing a safe anticoagulation, these agents can lead to important biological discoveries concerning the fundamental difference between hemostasis and thrombosis.</p> / Dissertation

<p>Current standard cancer treatments such as chemotherapeutics, and radiation therapy are nearly as likely to kill the patient as cure the cancer. Therapies that have such a narrow window of efficacy are necessary for the treatment of aggressive diseases, but safer alternatives must be created. By discovering novel therapeutics that target specific disease processes within specific diseased cells, while leaving healthy cells unaltered, we can improve the lives of millions of cancer sufferers and their families. A therapeutic's window of efficacy can be measured by the therapeutic index. For many anti-cancer therapeutics, the therapeutic index is very small, the dose of treatment that kills cancer cells and shrinks tumors is nearly the dose that causes toxicity. In cancer patients, this toxicity causes many serious conditions such as gastrointestinal distress, organ damage, and death. </p><p>Recently, the model of cancer treatment has evolved from non-specific cytotoxic agents to more selective therapeutics that target cellular processes necessary for cancer cell survival. If a therapy can be targeted to selectively bind and internalize targeted cells, its toxicity would only impact the targeted cells and healthy cells in the immediate vicinity, which would greatly reduce the toxic effects on the rest of the body. Targeting cancer cells can be done through cancer biomarkers, which are cell surface proteins, expressed exclusively, or are much more abundant on the surface of cancer cells than on somatic cells. </p><p>Advances in antibody and aptamer technology have enabled researchers to design those molecules to bind specifically to cancer cells, and deliver drugs that alter specific cellular processes. An aptamer designed to bind PSMA, a prostate cancer biomarker, only bound to a specific subset of cancer cells, and delivered a therapeutic siRNA that prevented a specific survival process from occurring. While this technology is promising, it is currently limited to targeting small subsets of cancer types. To generate an aptamer therapeutic that would have greater utility and efficacy, I have examined the properties of a nucleolin aptamer-mediated delivery system that targets multiple types of cancer cells, and delivers various oligonucleotide therapeutics. </p><p>The nucleolin aptamer targeted cancer cells by binding to membrane&ndash;associated nucleolin. Nucleolin, a conserved protein found in all eukaryotes, shuttles from the nucleus, through the cytoplasm to the cell membrane. Cancer cells express a far greater amount of membrane&ndash;associated nucleolin than somatic cells, making nucleolin an ideal cancer biomarker. The shuttling, and oligonucleotide binding attributes of the protein enable it to deliver aptamer chimeras from the cell surface to the nucleus. Therefore the nucleolin aptamer has unique access to the nuclei of cancer cells, and can deliver therapeutic oligonucleotide cargoes through nucleolin binding.</p><p>The nucleolin aptamer delivered splice&ndash;switching oligonucleotides, a form of antisense technology, improving their efficacy, and potentially increasing their therapeutic viability. The ability to deliver antisense oligonucleotides to the nuclei of cancer cells has the potential for other therapeutic possibilities including the inhibition of transcription with antisense triplexes.</p><p>The nucleolin aptamer can also deliver therapeutic aptamers. The nucleolin aptamer&ndash;&beta;&ndash;arrestin aptamer chimera prevented the stem cell renewal phenotype necessary for leukemia progression in human patient tissue samples. The ability to effectively deliver therapeutic aptamers may lead to clinical applications for many of the aptamers that have been selected against intracellular targets including transcriptional activators.</p><p>Oligonucleotide research continues to advance our understanding of potentially therapeutic oligonucleotides. Long non&ndash;coding RNAs for example, may impact epigenetics, and transcription. Additionally, locked nucleic acids have been developed to improve binding affinity, thus increasing the efficacy of antisense oligonucleotides. In order to bring these discoveries into the clinic, they must be safely and specifically delivered to their target cells. </p><p>This work demonstrated that the nucleolin aptamer could deliver oligonucleotide therapeutics to specific cancer cells. Nucleolin aptamer chimeras have the potential to develop into safe and effective cancer therapies, thus improving the treatment options for cancer sufferers.</p> / Dissertation

No description available.

This work was designed as a proof-of-principle concept or prototype to show the effect of protein-assisted targeting of DNA to specific genomic loci. Two strategies were employed to deliver the DNA with the aim that once inside the cell the DNA would be delivered to the target sequence by the assistance of a protein. In our case, the chosen protein was the site-specific meganuclease I-SceI. The first strategy described herein was to bind the targeting DNA to I-SceI by the use of a fusion protein between I-SceI and a known DNA-binding domain, the GAL4-DBD. The second strategy involved using a DNA aptamer to I-SceI to link the targeting DNA and I-SceI. Testing in vivo revealed that in our human cells (HEK-293) single-stranded DNA was more efficient at gene targeting than double-stranded DNA. In order for the first strategy to work, we needed to have some region of double-stranded DNA. We found that in human cells, it was better for gene targeting to have that double-stranded DNA on the 5’ side of our targeting DNA. We also used gel shift assays to confirm binding by our candidate DNA-binding domain, the GAL4-DBD. We were unable to detect expression of the fusion protein of I-SceI and the GAL4-DBD. For the second strategy we were able to construct an aptamer to I-SceI using a variant of the systematic evolution of ligands by exponential enrichment (SELEX). The I-SceI aptamer was synthesized as part of a longer DNA molecule containing homology to a target locus. Using this chimeric oligonucleotide (part aptamer, part DNA repair region) testing was done in both yeast and human cells. Aside from instances where the aptamer’s secondary structure may have been compromised, the aptamer containing oligonucleotide stimulated repair at a rate 2 to 15-fold higher than the non-selected control sequence. These experimental results show that by delivering targeting DNA within close proximity to the site of modification, gene targeting frequencies can be increased.

Universiẗat, Diss., 2004--Bonn.

Over the years, colorimetric reagents and immunology have been widely used in screening tests for illicit drugs; however, the test kits are not always convenient for field use and often require the user to mix and develop a specific set of reagents. In our project, we have been working on alternative platforms based on paper microfluidic devices (uPADs) for field testing. These devices utilize wax channels printed on paper to direct the analyte towards a specific set of chemical reagents. Using the procedure, we have developed a six-channel chip that adapts known colorimetric reagents targeting cocaine, opiates, amphetamines and ketamine for multiplex detection. For more sensitive and specific determinations than the colorimetric reagents, we have also developed a paper device that utilizes the interaction between gold nanoparticles and drug specific aptamers. The µPADs using colorimetric reagent are designed as a six-channel multiplexed system. Sequences of different reagents applied to each channel to produce a series of reactions and the color changes appear at the end of each channel. The entire process takes less than five minutes. The adjusted reagents produce specific color changes for seized drugs on the paper microfluidic devices. Procedures have been developed for the detection of cocaine, ketamine, codeine, ephedrine, morphine, amphetamine, methamphetamine, and MDMA. These devices have been tested for sensitivity, specificity and stability against a variety of potential interferences and test conditions. Gold nanoparticles (AuNPs)/ aptamers µPADs were developed to detect cocaine. The presence of cocaine cause the binding with aptamers, and the gold nanoparitcles produced a salt-indicated aggregations and gave a color change of AuNPs from red to black. The absence of cocaine allowed the aptamers freely to bind gold nanoparticles, and no color change occured. The device had a preliminary validation of sensitvity and specificity against a variety of potential interferences. The use of paper microfluidic devices permits the development of rapid, inexpensive and easily operated tests for drug samples in the field. They present a safe and convenient presumptive tool that can be used in the field.

Du fait de leur haute stabilité et bas coût de production, les aptamères suscitent un intérêt croissant, depuis près de 20 ans, dans le design de biocapteurs en tant qu'élément de reconnaissance idéal. Le but de ce travail de thèse est de démontrer l'intérêt et la pertinence d'un outil tel qu'une biopuce à aptamères, associant les avantages des sondes aptamères à ceux d'une détection par SPRi (Surface Plasmon Resonance imaging), permettant une détection sans marquage et en temps réel d'interactions moléculaires. Dans ce but, deux aptamères anti-thrombine (APT1 = 5′- GGT-TGG-TGT-GGT-TGG -3′ et APT2 = 5′-AGT-CCG-TGG-TAG-GGG-AGG-TTG-GGG-TGA-CT-3′) ont été choisis comme objets d'étude modèles. Ce choix a permis d'orienter différents axes de recherche : utilisés indépendamment comme sondes lors de l'élaboration de notre biopuce, ils ont tout d'abord permis de réaliser une détection cinétique optimisée de la thrombine, avec des performances remarquables pour une détection de ce type, ainsi que le calcul de constantes de dissociation en solution et à la surface des biopuces. Mais au-delà d'un simple biocapteur, la biopuce a également pu être utilisée comme véritable plateforme d'étude de la thrombine et de ses interactions, au sein de structures plus complexes telles que la structure « sandwich » entre les deux aptamères, ou d'autres interactions impliquant la thrombine en tant qu'acteur de la cascade de coagulation (inhibition de la thrombine par l'antithrombine III et le cofacteur II de l'héparine, transformation de la prothrombine au sein du complexe prothrombinase). / For 20 years, aptamers have been raising an increasing interest for biosensor applications as replacements for antibodies, due to their high stability and low cost. The main objective of this Ph.D. thesis is to show the great capacities of an aptamer biochip that combines the advantages of aptamer probes associated with a SPRi (Surface Plasomn Resonance imaging) detection to monitor, in real-time and in a label-free manner, molecular interactions occurring on the surface of the biochip. Two aptamers selected against the thrombin protein (APT1 = 5′- GGT-TGG-TGT-GGT-TGG -3′ and APT2 = 5′-AGT-CCG-TGG-TAG-GGG-AGG-TTG-GGG-TGA-CT-3′) were chosen as models for our study. This choice led to the exploration of different lines of research. First, both aptamers were used independently to develop a kinetic biosensor with remarkable performances for the quantification of thrombin. This tool served to determine independently, and compare, both the solution- and surface-phase affinities of the trombin-APT2 interaction. But more than a simple and effective biosensor, this kind of biochip represents a true platform to study the protein and its interactions within complex structures, such as the sandwich-like architecture with APT1 and APT2, or its interactions with other factors of the coagulation cascade (inhibition of thrombin by antithrombin III and heparin cofactor II, conversion of prothrombin into thrombin by the prothrombinase complex).

abstract: Since Darwin popularized the evolution theory in 1895, it has been completed and studied through the years. Starting in 1990s, evolution at molecular level has been used to discover functional molecules while studying the origin of functional molecules in nature by mimicing the natural selection process in laboratory. Along this line, my Ph.D. dissertation focuses on the in vitro selection of two important biomolecules, deoxynucleotide acid (DNA) and protein with binding properties. Chapter two focuses on in vitro selection of DNA. Aptamers are single-stranded nucleic acids that generated from a random pool and fold into stable three-dimensional structures with ligand binding sites that are complementary in shape and charge to a desired target. While aptamers have been selected to bind a wide range of targets, it is generally thought that these molecules are incapable of discriminating strongly alkaline proteins due to the attractive forces that govern oppositely charged polymers. By employing negative selection step to eliminate aptamers that bind with off-target through charge unselectively, an aptamer that binds with histone H4 protein with high specificity (>100 fold)was generated. Chapter four focuses on another functional molecule: protein. It is long believed that complex molecules with different function originated from simple progenitor proteins, but very little is known about this process. By employing a previously selected protein that binds and catalyzes ATP, which is the first and only protein that was evolved completely from random pool and has a unique &alpha;/&beta;-fold protein scaffold, I fused random library to the C-terminus of this protein and evolved a multi-domain protein with decent properties. Also, in chapter 3, a unique bivalent molecule was generated by conjugating peptides that bind different sites on the protein with nucleic acids. By using the ligand interactions by nucleotide conjugates technique, off-the shelf peptide was transferred into high affinity protein capture reagents that mimic the recognition properties of natural antibodies. The designer synthetic antibody amplifies the binding affinity of the individual peptides by &#8764;1000-fold to bind Grb2 with a Kd of 2 nM, and functions with high selectivity in conventional pull-down assays from HeLa cell lysates. / Dissertation/Thesis / Ph.D. Biochemistry 2013

Riboswitches are regulatory structures present in the 5′-UTR of a wide range of bacterial mRNAs. They consist of a small-molecule binding aptamer domain, which affects the conformation of a nearby expression platform to control gene expression through a transcriptional or translational mechanism. Because of their ability to bind selectively to very small concentrations of ligand, in a protein-independent manner, they have great potential for use as novel small-molecule controllable gene expression systems. This thesis describes how a combination of chemical genetics and genetic selection were used to develop and test a novel riboswitch-based gene-expression system. Several constructs were generated which could respond in vivo to a variety of non-natural small heterocyclic compounds and output via a simple fluorescence based assay in a dose-dependent manner. Methods for controlling the overall protein expression landscape of the riboswitch-based gene-expression system are outlined. In addition, the rational design of mutant riboswitch aptamers with improved ligand-binding capabilities is described alongside attempts to modulate the structural stability of the expression platform. Riboswitches need to be highly discriminatory to function effectively in vivo, binding to one ligand from a cellular pool of thousands. Mutant riboswitches were created that responded specifically to the ligands ammeline or azacytosine, and were found to have no induction in the presence of adenine, the wild-type riboswitch ligand. This in vivo ligand orthogonality was confirmed by subsequent in vitro studies. The ligand-induced structural changes undertaken by the mutant riboswitch aptamer domains were subsequently characterised using a variety of in vitro methods including SHAPE, ITC and x-ray crystallography. Finally, the feasibility of using riboswitch gene-expression systems in fully synthetic applications was demonstrated through the construction and analysis of small synthetic gene clusters and operons. The in vivo expression of two fluorescent proteins under independent riboswitch control was studied under single and dual induction for a range of ligand concentrations. The ability to control the expression of multiple genes is highly desirable in the emerging field of synthetic biology, the results described here indicate that riboswitches are ideally suited to complement current gene expression tools.