Graduate Thesis Or Dissertation
 

Innovative Bioanalytical Tools & Methods for Combinatorial Non-Coding RNA Analysis

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/sq87c0572

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  • Recently researchers have discovered that groups of small non-coding RNAs (ncRNAs) play regulatory roles in gene expression and participate in various biological processes. For example, pathogenesis of many diseases, cell cycle regulation, and signaling pathways. Intracellular and live cell imaging of small ncRNA groups will reveal their relative expression levels and provide unparalleled detail on spatial and temporal heterogeneities within a single cell. The ability to measure heterogeneities will help define cell types and cell states for differentiating cell populations. Having analytical tools that reveal heterogeneities among cell populations will provide unique insights on the physiological changes and processes (e.g. aging and apoptosis) of each cell and how the cells work together to maintain homeostasis or drive disease progression. To profile small ncRNAs expression, one popular analytical tool is known as programmable molecular logic sensors. Relying on nucleic acids, a natural building block8, molecular logic sensors are constructed for computing what groups of small ncRNAs are in a cell. As a model system to design innovative molecular logic sensors around, I picked microRNAs. MicroRNAs (miRs) are small non-coding single-stranded RNAs that are approximately 22 nucleotides in length.9 The roles of miRs are to regulate gene expression, mainly post-transcriptionally, during messenger-RNA translation. Current nucleic-acid-based in situ sensors that are capable of revealing a cells miR pattern suffer from 1) low multiplexing ability (up to two miR inputs per sensor), 2) poor selectivity, and 3) false signals due to sensor degradation by nucleases. Therefore, I conducted research to design, characterize, optimize, and apply two different designs of logic sensors to overcome some of the bottlenecks facing current in situ sensors. My research in the field of molecular logic centered around contributing innovative designs and establishing design principles for constructing nanodevices. The term “AND” means the sensor’s signal only turns ON when all miR inputs are present. My first logic sensor design, published in Nanoscale, is called a nano-assembly logic gate (NALG). My contribution to the molecular logic field is a unique multi-hairpin motif designed. The purpose the multi-hairpin motif was to improve input number (multiplexing ability), selectivity, and robustness to false signal generation. Furthermore, the motif’s design will serve as the base building block for a modular design for scaling up the multiplexing ability. NALG was designed for three miRs: miR27a, miR96, and miR182. The signal transduction mechanism of NALG was based on Frster Resonance Energy Transfer (FRET) enhancement. The results showed that NALG had: (1) low nanomolar (nM) limits of detection (LOD), (2) selectivity against off-analyte cocktails (sequence similariaty ranged from 13% to 27%), (3) no false-positive signal from nuclease degradation, and (4) the ability to respond to three miRs in a matrix mimicking the cellular environment (i.e. crude MCF-7 cell lysate). However, NALG needed refinement to improve its ability to differentiate input numbers because it showed signal response in the presence of two out of three miRs. In order to reduce NALG’s signal response from two miRs, I studied how to fine-tune the multi-hairpin motif to better resist biochemical and biophysical interactions with two miRs. The manuscript for this work is currently under review at Analytical Chemistry. Three new motif types were developed based on the original motif. The motif designs were assessed based on the following design metrics: (1) the location of the inputs’ complementary sequence, (2) the predicted number of Hydrogen-bonds formed in the motif, (3) the predicted change in thermodynamic values of the motif after the addition of the inputs, and (4) the predicted molarity percentage of motif forming complex with different numbers of inputs (two versus three). We measured the fluorescence response from these motifs in the presence of inputs and discovered gaps between the predicted and experimental results. Our findings provide a noteworthy improvement to the design process of molecular logic sensors for measurement science. To overcome the limitations in the first logic sensor (NALG) design and applying what I learned about the design process, I came up with an innovative design that I call: autowalk AND logic operator (AALO). AALO was designed for a three-miR combination: miR27a, miR24, and miR210. Different from current nucleic-acid-based sensors that recognize analyte miRs through a single toehold-mediated strand displacement reaction (TMSDR), AALO relies on a cascading (five-step) TMSDRs. The cascading TMSDRs mechanism exposes one toehold per step to initiate successive TMSDRs. The toehold (~3-6 nucleotides sequence) in the gate strand initiates binding with an incoming strand and subsequently displaces a pre-bound strand from the gate. Such a recognition mechanism requires the presence of all miR inputs to complete the cascading process and achieve signal change. AALO’s lower signal change in the presence of two miRs (19% from AALO compared to 53% from NALG) means that AALO’s recognition mechasim was able lower the false response from incomplete miR combinations. The five-step TMSDRs were thus able to improve the logic sensors’ differentiating-input-number ability. Compared to NALG, AALO showed increased selectivity against off-analyte miRs with sequence similiarity ranging from 41% to 95%. We have prelimitary data that shows AALO was transfected into the cell line HEK 293T through nucleofection.
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  • Ongoing Research
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  • 2018-10-31 to 2020-12-02

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