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Case Western Reserve University

1. Lin, Hsuan-Chun. Development and Application of High Throughput Methods for Interrogating RNA Binding Specificity.

Degree: PhD, Biochemistry, 2017, Case Western Reserve University

Structure-function studies of RNA binding and RNA-processing reactions, in which one measures the effects of specific variations in sequence on specific reaction parameters such as binding kinetics, equilibrium binding affinity and catalytic rate, have provided deep insights into biological function. Nonetheless, the perspective offered by structure-function studies is severely limited by the relatively small number of sequence variants that can typically be analyzed. Using RNase P processing of pre-tRNA as an experimental system I contributed to the development of a set of tools, which are collectively named HTS-Enzymology, based on high-throughput sequencing and competitive reaction kinetics. High-throughput Sequencing KINestics (HTS-KIN) allows the accurate and simultaneous determination of kinetic rate constants for large pools of RNA substrates. I developed a similar approach to determine equilibrium binding constants by analyzing the distribution of sequences in free and bound populations separated by electrophoretic mobility shift assay (EMSA) using simple competitive binding models termed High-Throughput Sequencing EQilibrium binding (HTS-EQ). The resulting high-density structure-function data sets provide unique insights into patterns of molecular recognition and the nature of specificity in RNA-protein interactions. Since HTS-KIN and HTS-EQ provide the entire affinity distribution (that is, the full range of effects of mutation on functional affinity), classical methods such as sequence logos and position weight matrix provide restricted views of specificity that ignore information regarding the context dependent effects of sequence variation. Therefore, I applied multivariable regression methods that consider the interaction between different positions in the binding site. In combination these approaches provided a comprehensive understanding of how substrate sequence and structure affect binding affinity, association kinetics and catalysis for a highly conserved RNA processing enzyme, RNase P. Furthermore, HTS-Enzymology was used to decode the novel rules for the broad specificity of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Together these advances helped to put into place a methodological framework for interrogating RNA-protein binding specificity, and illustrated their application in two distinct experimental systems. The result reveal the important contribution of ground state RNA secondary structure and the effects of local sequence contexts on observed sequence specificity. Advisors/Committee Members: Harris, Michael (Advisor).

Subjects/Keywords: Biochemistry; HTS-Enzymology, HiTS-EQ, HiTS-Kin, RNase P, Enzymology, Internal competition, hnRNP A1

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APA (6th Edition):

Lin, H. (2017). Development and Application of High Throughput Methods for Interrogating RNA Binding Specificity. (Doctoral Dissertation). Case Western Reserve University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=case1481206951509556

Chicago Manual of Style (16th Edition):

Lin, Hsuan-Chun. “Development and Application of High Throughput Methods for Interrogating RNA Binding Specificity.” 2017. Doctoral Dissertation, Case Western Reserve University. Accessed June 24, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1481206951509556.

MLA Handbook (7th Edition):

Lin, Hsuan-Chun. “Development and Application of High Throughput Methods for Interrogating RNA Binding Specificity.” 2017. Web. 24 Jun 2017.

Vancouver:

Lin H. Development and Application of High Throughput Methods for Interrogating RNA Binding Specificity. [Internet] [Doctoral dissertation]. Case Western Reserve University; 2017. [cited 2017 Jun 24]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1481206951509556.

Council of Science Editors:

Lin H. Development and Application of High Throughput Methods for Interrogating RNA Binding Specificity. [Doctoral Dissertation]. Case Western Reserve University; 2017. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1481206951509556

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