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Permanent link (DOI): https://doi.org/10.7939/R3FJ29J40

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Specific and programmable release of target molecules via DNA strand displacement reaction Open Access

Descriptions

Other title
Subject/Keyword
Fluoroimmunoassay
DNA strand displacement reaction
Sample cleanup
Transcription factor
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Ramezani, Hamid
Supervisor and department
Harrison, D. Jed (Chemistry)
Examining committee member and department
Corn, Robert M (Chemistry, UC Irvine)
Loppnow, Glen R (Chemistry)
Campbell, Robert E (Chemistry)
Gibbs-Davis, Julianne M (Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2014-09-29T13:44:03Z
Graduation date
2014-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
DNA strand displacement reaction (SDR) is a fast, isothermal, and sequence specific reaction. In SDR, an invading DNA single strand, also known as the “fuel” strand, unzips a partial duplex to form a more thermodynamically stable duplex. The complementary strand to the fuel strand is referred to as the template. SDR releases the initial complement to the template upon formation of the fuel-template duplex. In DNA computation, the initial complement is called the “output” strand. In this work, we demonstrated how the output strand could be programmed to execute different functions such as specific release of biomolecules or interrogating the presence of a particular target molecule in the chemical system. The capture and subsequent release of molecules from a solid phase has far-reaching applications in separation science and chemical analyses. Different molecular properties have been exploited to capture molecules on the solid phase offering a big repertoire of mechanisms for capture. The spectrum of capture mechanisms ranges from very general hydrophobicity-based partitioning of molecules in gas or liquid chromatography to the highly specific molecular recognition between ligands and analytes in the affinity chromatography techniques. The release mechanisms, while very diverse on the generic extreme of the spectrum, are very limited in terms of specificity. Here, we proposed SDR as a specific release mechanism for solid phase extraction that could be triggered only by the presence of a proper DNA fuel strand. We demonstrated that integration of SDR to a fluoroimmunoassay on silica microparticles for a thyroid cancer biomarker, thyroglobulin, offers a very effective in situ cleanup method, especially in the presence of a complex biological fluid such as whole serum. The unique ability of DNA to carry information in its sequence compelled us to further extend the application of SDR to characterization and purification of transcription factors (TFs). TFs are DNA-binding proteins regulating the gene expression levels in cells by binding to the specific regions of genome. The non-specific methods of release for elution of captured TFs from the DNA affinity solid phase result in losing information about the DNA sequences acting as the binding sites for the TFs of interest. It is also very difficult to multiplex purification of TFs for the mentioned reason. We propose an SDR-based strategy called IDCAPT (for Integrative Discovery, Characterization, Assay, and Purification of TFs) that uses multiple sequential SDRs to characterize the potential binding sites for TFs, quantify, and purify them. We first proved the concept of SDR-mediated multiplexing on beads for three different sets of fluorescently labeled DNA capture strands. We then showed that IDCAPT could successfully interrogate the presence of a model TF, NFκβ, in the solution using its simple sequential SDR-based reasoning module. In summary, SDR provides us with an efficient, specific, and sequence-encoded release tool opening up the avenue for many applications requiring multiplexation, sample cleanup, and logical gate-based sensing.
Language
English
DOI
doi:10.7939/R3FJ29J40
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
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