Browsing by Subject "deep sequencing"

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    Constrained Diversification Enhances Protein Ligand Discovery and Evolution
    (2017-04) Woldring, Daniel
    Engineered proteins have strongly benefited the effectiveness and variety of precision drugs, molecular diagnostic agents, and fundamental research reagents. A growing demand for new therapeutics motivates the innovative use of natural proteins – improving upon their native properties – as well as discovering proteins with entirely new functionality. Importantly, these are fundamentally separate goals. While evolving improved function can result from making a few carefully chosen mutations, discovering novel function often requires giant leaps to be taken in protein sequence space. Discovering novel function is a notoriously challenging task. The immensity of sequence space (e.g. proteins of length n have 20^n unique options) makes it essentially impossible to experimentally or computationally test all possible protein sequences. Within this space, the landscape is incredibly barren and rugged (i.e. most sequences lack function entirely and making small changes to a protein often damage the activity). Rather than randomly mutating a protein, combinatorial protein libraries provide a systematic and efficient approach for searching sequence space. This method offers precise control over which protein sites are mutated and which amino acids are allowed at the diversified sites. To improve the likelihood of sampling useful sequences, numerous techniques can elucidate the structure-function relationships in proteins. Generally, these techniques have not been applied to combinatorial library design; however, we propose that some, or all, could be greatly beneficial in this area. In this thesis work, protein libraries are designed for the purpose of discovering high affinity, specific binders to a collection of interesting targets. High-throughput sequencing of evolved binders, natural protein-protein interface composition, structural assessment, and computational analysis of stability upon mutation collectively informed sitewise library designs – residues predicted to support function were allowed but destabilizing residues or those not likely to benefit function were avoided. We use multiple small protein scaffolds (affibody and fibronectin) as model systems to test the hypothesis that constrained sitewise diversity will improve the efficiency of novel protein discovery. This hypothesis was experimentally supported by a direct comparison of high-affinity ligand discovery between the sitewise constrained library and a uniformly diversified library (i.e. allowing all 20 residues at each diversified site). The constrained library showed a 13-fold improved likelihood of binder discovery. Moreover, the constrained library variants demonstrated superior thermal stability (Tm 15 °C higher than unbiased variants). This work provides further evidence that sitewise diversification of protein scaffolds can improve the overall quality of combinatorial libraries by offering broad coverage of sequence space without sacrificing stability.
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    tRNA Fragments: Expression and Function in Ovarian Cancer
    (2017-09) Zhou, Kun
    Deep sequencing studies of noncoding RNA in liquid biopsies are revealing a vast repertoire of potential biomarkers. Ovarian cancer is a difficult-to-diagnose disease, urgently requiring novel and readily accessible biomarkers. We hypothesized that urine, one source of liquid biopsy samples, may contain novel noncoding RNAs (ncRNAs) that could serve as biomarkers for ovarian cancer. We proceeded to deep sequence RNA extracted from urine collected from ovarian cancer patients to better understand the repertoire of small RNAs in this type of liquid biopsy sample. The ncRNAs identified in these urine samples were predominantly microRNAs (miRNAs), ribosomal RNA (rRNA) fragments and tRNA fragments (tRFs). tRFs are a group of ncRNAs, which have been found across the biological kingdom and are increasingly being studied for their role in cancer biology. Several tRFs have been studied in cancer, although not previously in ovarian cancer. We have studied the expression of one specific tRF, 5’ fragment of tRNA-Glu-CTC (tRF5-Glu), in five different ovarian cancer cell lines. Several variants of tRF5-Glu were identified and we have now confirmed the expression of tRF5-Glu in ovarian cancer cells by quantitative real-time PCR (qRT-PCR), Northern analysis and ligation PCR. Additionally, we determined that angiogenin (ANG) plays a role in the biogenesis of tRF5-Glu. Furthermore, we have shown that tRF5-Glu targets the mRNA of the Breast Cancer Anti-estrogen Resistance 3 (BCAR3). While BCAR3 is known to regulate cancer cell migration and contributes to anti-estrogen resistance in breast cancer cells, it has not previously been studied in ovarian cancer or shown to be targeted by a tRF. Using synthetic mimics of tRF5-Glu and siRNAs targeting BCAR3, we were able to show that tRF5-Glu expression and the knock down of BCAR3 expression inhibits proliferation in ovarian cancer cells. These studies demonstrate that tRF5-Glu contributes to the regulation of BCAR3 and provides a novel mechanism of the regulation of proliferation in ovarian cancer cell lines.