Characterization and application of a novel composite nanomaterial comprised of porous protein crystals and synthetic DNA

Stuart, Julius D., author
Snow, Christopher D., advisor
Kennan, Alan J., committee member
Shores, Matthew P., committee member
De Long, Susan K., committee member
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Composite nanomaterials are systems comprised of multiple components boasting enhanced properties over those exhibited by the individual constituents when isolated. Such systems are highly tunable, allowing one to vary component types (e.g., polymer, metal, ceramic) for influencing performance in various contexts. Moreover, composite nanomaterials can be further modified using biofunctionalization for use in biological settings. Composite nanomaterials have been tested in applications including, but not limited to, textile, defense, food, energy and biomedical engineering. A sub-domain within composite nanomaterials involves porous protein crystals soaking, or, separately, encapsulating various guest molecules. Porous protein crystals are ordered, insoluble assemblies forming a network of nanopores capable of allowing inward diffusion of guest molecules. Moreover, recombinant protein variants can be engineered for probing guest molecule binding to host crystal nanopores further highlighting the tunability of this novel composite nanomaterial. The goal of this work is to evaluate a novel composite nanomaterial comprised of host porous protein crystals and guest double stranded DNA. We show that guest DNA loads into host crystals predominantly along the axial nanopores. Equilibrium adsorption isotherm results suggest guest DNA unbinds from host crystals relatively slowly. Computational modeling and Fluorescence Recovery After Photobleaching (FRAP) studies suggest intra-nanopore guest diffusion is attenuated relative to bulk diffusion. We also show that porous protein crystals loading with synthetic DNA barcodes can be used for tracking mosquitoes. Fluorescently labeled crystals can be ingested by mosquito larvae and adults, followed by detection using fluorescence confocal microscopy. Crystal-bound DNA can be liberated from host crystals by incubation with solution containing deoxynucleotide triphosphates (dNTPs). Previously ingested barcode-loaded crystals can be recovered using standard molecular biological techniques. Lastly, we show a DNA barcode sequence construction strategy that is modular, economical and scalable. Computational sequence design and scoring allowing identification of top candidates for experimental validation. Analysis of next-generation sequencing datasets informs barcode construction specificity while simultaneously reinforcing the multiplexing capabilities boasted by modular DNA barcodes.
Includes bibliographical references.
2022 Fall.
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Embargo Expires: 01/09/2025
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