Browsing by Subject "Aptamers"
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Item Aptamer selections against bacterial toxins and cells(2006-08) Cockrum, Seth Edward; Ellington, Andrew D.In vitro selection of functional RNA molecules has formed the basis for a new class of molecules termed “aptamers.” Aptamers have been selected against a wide range of molecules, ranging from simple chemical compounds to multi-cellular living organisms. The majority of selections are carried out against targets, such as proteins, that are typically composed of one type of molecule. Targets composed of multiple types of molecules (lipids, proteins, carbohydrates, etc.) are termed “complex,” and examples of successful selections against them include parasites, virions, and red blood cell ghosts. Through various properties inherent in their composition, aptamers have the potential to play a role in everything from therapeutics to broad based detection platforms. Bacterial toxins are a means by which pathogenic bacteria are able to exert an effect on a host organism. Although there are a few aptamer selections that have been carried out against toxins, there have not been any successful selections against whole bacterial cells. As some bacteria are easily grown in laboratory conditions, the possibility of their use as a biological threat agent is relatively high. Therefore, there is a need develop rapid and reliable technologies for the detection of such threats. This work details two aptamer selections carried out against both a bacterial toxin, Bacillus. anthracis protective antigen (PA), and a Bacillus subtilis vegetative cell. The selection against PA resulted in a high affinity aptamer that is capable of inhibiting the cleavage of PA. This cleavage step is the first in the pathway whereby anthrax toxin is able to exert its effect. The selection against B. subtilis vegetative cells is a proof of principle selection. B. subtilis is meant to be a surrogate for B. anthracis, which has long been regarded as a potential bio-weapon. Aptamers selected against these vegetative cells are shown to discriminate between bacterial vegetative cells of the same genus, bacteria of a different genus, and also spores produced by B. subtilis. With these selections as examples, it is hoped that the role of aptamers can continue to be expanded into viable detection systems for biological threat agents.Item Nucleic acid localization in diagnostics and therapeutics(2010-05) Pai, Supriya Sudhakar; Ellington, Andrew D.; Georgiou, George; Williams, Robert O.; Tian, Ming; Sullivan, ChrisAptamers are short nucleic acid ligands generated by the process of iterative selection. Nucleic acid counterparts to protein antibodies, aptamers bind their targets with relatively high affinities by assuming characteristic shapes. Highly thermostable, open to manipulations and non-immunogenic, these olignucleotides can be readily adapted to a variety of diagnostic assays and harvested for their therapeutic potential. We have particularly focused on the unique prospects that stem from their localization patterns both in vitro and in vivo. While several assays exist for protein diagnostics, many of these are limited by the amount of target they can detect. To overcome these limitations it might prove effective to couple protein detection with nucleic acid based amplification. The Proximity Ligation Assay (PLA) is an innovative technique that facilitates protein detection on a zeptomolar range by amplifying a tiny signal via the polymerase chain reaction. PLA is based on the concept that two DNA tags when co-localized adjacent to one another on a protein surface and ligated via a connector nucleotide will form a unique amplicon that can detected using real-time PCR and in turn detect the protein. We have adapted PLA to the peptide based detection of Bacillus spores as well as the RNA aptamer based detection of cancer cells. Highly sensitive and specific, nucleic acid based PLA could serve as a promising tool in diagnostics. Aptamers have also been analyzed for their localization patterns in vivo. Using two anti-prostate specific membrane antigen RNA aptamers, we have demonstrated that there is an inherent bias for some circulating oligonucleotides over others based solely on their sequence. This phenomenon has also been explored in cancer models of mice for persistence of specific aptamers over others in tumors for therapy. An in vivo “Stealth” selection scheme has also been designed and executed to hunt for stable and robust aptamer species that are naturally chosen for survival within a mouse system. Generation of such ligands could benefit several therapeutic ventures such as targeted drug delivery past complex vasculature as in the case of the blood:brain barrier.Item Stimulus-responsive delivery systems for enabling the oral delivery of protein therapeutics exhibiting high isoelectric point(2015-05) Koetting, Michael Clinton; Peppas, Nicholas A., 1948-; Contreras, Lydia M; Ellison, Christopher J; Stachowiak, Jeanne C; Truskett, Thomas MProtein therapeutics offer numerous advantages over small molecule drugs and are rapidly becoming one of the most prominent classes of therapeutics. Unfortunately, they are delivered almost exclusively by injection due to biological obstacles preventing high bioavailability via the oral route. In this work, numerous approaches to overcoming these barriers are explored. PH-Responsive poly(itaconic acid-co-N-vinylpyrrolidone) (P(IA-co-NVP)) hydrogels were synthesized, and the effects of monomer ratios, crosslinking density, microparticle size, protein size, and loading conditions were systematically evaluated using in vitro tests. P(IA-co-NVP) hydrogels demonstrated up to 69% greater equilibrium swelling at neutral conditions than previously-studied poly(methacrylic acid-co-N-vinylpyrrolidone) hydrogels and a 10-fold improvement in time-sensitive swelling experiments. Furthermore, P(IA-co-NVP) hydrogel microparticles demonstrated up to a 2.7-fold improvement in delivery of salmon calcitonin (sCT) compared to methacrylic acid-based systems, with a formulation comprised of a 1:2 ratio of itaconic acid to N-vinylpyrrolidone demonstrating the greatest delivery capability. Vast improvement in delivery capability was achieved using reduced ionic strength conditions during drug loading. Use of a 1.50 mM PBS buffer during loading yielded an 83-fold improvement in delivery of sCT compared to a standard 150 mM buffer. With this improvement, a daily dose of sCT could be provided using P(IA-co-NVP) microparticles in one standard-sized gel capsule. P(IA-co-NVP) was also tested with larger proteins urokinase and Rituxan. Crosslinking density provided a facile method for tuning hydrogels to accommodate a wide range of protein sizes. The effects of protein PEGylation were also explored. PEGylated sCT displayed lower release from P(IA-co-NVP) microparticles, but displayed increased apparent permeability across a Caco-2 monolayer by two orders of magnitude. Therefore, PEG-containing systems could yield high bioavailability of orally delivered proteins. Finally, a modified SELEX protocol for cellular selection of transcellular transport-initiating aptamers was developed and used to identify aptamer sequences showing enhanced intestinal perfusion. Over three selection cycles, the selected aptamer library showed significant increases in absorption, and from an initial library of 1.1 trillion sequences, 5-10 sequences were selected that demonstrated up to 10-fold amplification compared to the naïve library. These sequences could provide a means of overcoming the significant final barrier of intestinal absorption.