Implementing locked nucleic acids as a bioinspired colloidal assembly and disassembly tool
Eze, Ngozi A.
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Oligonucleotides are popular recognition-based biomaterials assembly and disassembly tools due to their specificity and ease of control. Their susceptibility to degradation by nucleases and false positive signals under certain conditions, however, has led to great interest in chemically modified oligonucleotides such as locked nucleic acids (LNA) that enhance both nuclease resistance and target specificity. This dissertation extends prior work with DNA sequences to investigate incorporating locked nucleic acid (LNA), a synthetic oligonucleotide, in isothermal colloidal assembly and disassembly schemes as well as on hybridization kinetics between single-stranded and double-stranded probes immobilized on microspheres. Incorporation of LNA nucleotides into DNA sequences is of particular interest as a means of enhancing the performance of DNA in a biomaterials context due to the increased resistance of LNA to nuclease degradation, its greater intrinsic affinity for oligonucleotide targets, and low cytotoxicity effects. The effects of LNA modification, target sequence length, sequence fidelity, and salt concentration are key variables explored. After providing an overview of DNA and its properties, synthetic oligonucleotides, colloidal particles, and previous applications of DNA and LNA in colloidal assembly schemes, this work then discusses the selection and characteristics of appropriate pairs of hybridization partners for reversible colloidal assembly scenarios. A comparative investigation of the in situ primary hybridization kinetics between select LNA or DNA targets and single-stranded probes immobilized on colloidal surfaces is performed. To support the disassembly studies, the in situ competitive displacement kinetics of hybridized LNA primary targets by either LNA or DNA secondary targets is discussed. For these in situ studies, flow cytometry was used to quantify the hybridization reactions as they occur on microsphere surfaces. While comparable rate constants were typically observed between target and single-stranded probes, LNA typically exhibited more extensive primary and secondary hybridization activity. Optimizing hybridization parameters, such as duplex concentration, sequence fidelity, and LNA content in the probe and target strands, allows for the extent of colloidal disassembly to be tuned, an important step in developing a multifunctional colloid-based biomaterial system.