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Highly multiplexed single-cell in situ RNA and DNA analysis with biorthogonal cleavable fluorescent oligonucleotides

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The understanding of normal human physiology and disease pathogenesis shows great promise for progress with increasing ability to profile genomic loci and transcripts in single cells in situ. Using biorthogonal cleavable fluorescent oligonucleotides, a highly multiplexed single-cell in situ RNA

The understanding of normal human physiology and disease pathogenesis shows great promise for progress with increasing ability to profile genomic loci and transcripts in single cells in situ. Using biorthogonal cleavable fluorescent oligonucleotides, a highly multiplexed single-cell in situ RNA and DNA analysis is reported. In this report, azide-based cleavable linker connects oligonucleotides to fluorophores to show nucleic acids through in situ hybridization. Post-imaging, the fluorophores are effectively cleaved off in half an hour without loss of RNA or DNA integrity. Through multiple cycles of hybridization, imaging, and cleavage this approach proves to quantify thousands of different RNA species or genomic loci because of single-molecule sensitivity in single cells in situ. Different nucleic acids can be imaged by shown by multi-color staining in each hybridization cycle, and that multiple hybridization cycles can be run on the same specimen. It is shown that in situ analysis of DNA, RNA and protein can be accomplished using both cleavable fluorescent antibodies and oligonucleotides. The highly multiplexed imaging platforms will have the potential for wide applications in both systems biology and biomedical research. Thus, proving to be cost effective and time effective.

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2018-05

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Utilization of fluorescent microspheres as a surrogate for Cryptosporidium removal in conventional drinking water treatment

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The purpose of this study was to determine the applicability of fluorescent microspheres as a surrogate to measure the removal of Cryptosporidium oocysts through the coagulation, flocculation, sedimentation, and filtration steps of conventional water treatment. In order to maintain accuracy

The purpose of this study was to determine the applicability of fluorescent microspheres as a surrogate to measure the removal of Cryptosporidium oocysts through the coagulation, flocculation, sedimentation, and filtration steps of conventional water treatment. In order to maintain accuracy and applicability, a local water treatment facility was chosen as the system to model. The city of Chandler Arizona utilizes conventional treatment methodologies to remove pathogens from municipal drinking water and thus the water, coagulant, polymer, and doses concentrations were sourced directly from the plant. Jar testing was performed on four combinations of coagulant, polymer, and fluorescent microsphere to determine if the log removal was similar to that of Cryptosporidium oocysts.

Complications with the material properties of the microspheres arose during testing that ultimately yielded unfavorable but conclusive results. Log removal of microspheres did not increase with added coagulant in the predicted manner, though the beads were seen aggregating, the low density of the particles made the sedimentation step inefficient. This result can be explained by the low density of the microspheres as well as the potential presence of residual coagulant present in the system. Given the unfavorable properties of the beads, they do not appear to be a suitable candidate for the surrogacy of Cryptosporidium oocysts in conventional drinking water treatment. The beads in their current state are not an adequate surrogate; however, future testing has been outlined to modify the experiment in such a way that the microspheres should behave like oocysts in terms of physical transportation.

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Date Created
2015

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Sensing and regulation from nucleic acid devices

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The highly predictable structural and thermodynamic behavior of deoxynucleic acid (DNA) and ribonucleic acid (RNA) have made them versatile tools for creating artificial nanostructures over broad range. Moreover, DNA and RNA are able to interact with biological ligand as either

The highly predictable structural and thermodynamic behavior of deoxynucleic acid (DNA) and ribonucleic acid (RNA) have made them versatile tools for creating artificial nanostructures over broad range. Moreover, DNA and RNA are able to interact with biological ligand as either synthetic aptamers or natural components, conferring direct biological functions to the nucleic acid devices. The applications of nucleic acids greatly relies on the bio-reactivity and specificity when applied to highly complexed biological systems.

This dissertation aims to 1) develop new strategy to identify high affinity nucleic acid aptamers against biological ligand; and 2) explore highly orthogonal RNA riboregulators in vivo for constructing multi-input gene circuits with NOT logic. With the aid of a DNA nanoscaffold, pairs of hetero-bivalent aptamers for human alpha thrombin were identified with ultra-high binding affinity in femtomolar range with displaying potent biological modulations for the enzyme activity. The newly identified bivalent aptamers enriched the aptamer tool box for future therapeutic applications in hemostasis, and also the strategy can be potentially developed for other target molecules. Secondly, by employing a three-way junction structure in the riboregulator structure through de-novo design, we identified a family of high-performance RNA-sensing translational repressors that down-regulates gene translation in response to cognate RNAs with remarkable dynamic range and orthogonality. Harnessing the 3WJ repressors as modular parts, we integrate them into biological circuits that execute universal NAND and NOR logic with up to four independent RNA inputs in Escherichia coli.

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Date Created
2019