Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


Thesis-Hsuan-Chun Lin_Upload.pdf (3.4 MB)

ETD Abstract Container

Abstract Header

Development and Application of High Throughput Methods for Interrogating RNA Binding Specificity

Lin, Hsuan-Chun
http://orcid.org/0000-0003-2220-0823
http://rave.ohiolink.edu/etdc/view?acc_num=case1481206951509556

Abstract Details

2017, Doctor of Philosophy, Case Western Reserve University, Biochemistry.
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.
Michael Harris (Advisor)
169 p.
Biochemistry
HTS-Enzymology, HiTS-EQ, HiTS-Kin, RNase P, Enzymology, Internal competition, hnRNP A1

Recommended Citations

Citations

  • Lin, H.-C. (2017). Development and Application of High Throughput Methods for Interrogating RNA Binding Specificity [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1481206951509556

    APA Style (7th edition)

  • Lin, Hsuan-Chun. Development and Application of High Throughput Methods for Interrogating RNA Binding Specificity. 2017. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1481206951509556.

    MLA Style (8th edition)

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

    Chicago Manual of Style (17th edition)

case1481206951509556
501
© 2017, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.