Ueda, Christine Terumi (2007) Targeting human telomerase RNA via biochemical and in vitro selection methods. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-09052006-214421
Telomerase is an enzyme responsible for the maintenance of eukaryotic chromosome ends. The two main components required for activity are a protein subunit, the human telomerase reverse transcriptase (hTERT), and an RNA subunit, the human telomerase RNA (hTR). While telomerase is not active in most normal human cells, roughly 85% to 90% of oncogenic cells display increased telomerase activity. An understanding of the biochemistry of telomerase will aid in the development of molecules that will lead to antitumor-specific therapies. The first part of the work presented here describes a biochemical analysis of an RNA-RNA interaction between two catalytically important domains of hTR. The interactions were characterized via mobility-shift assay, mutation analysis, and UV cross-linking experiments. The data argue for a revised model for the structure of hTR, and point to possible three-dimensional contacts present in the telomerase complex. The next part of this thesis describes an in vitro selection against the catalytically important RNA stem-loop P6.1 in hTR. The in vitro selection was performed using mRNA display, which allows us to isolate RNA-binding peptides from libraries containing trillions of unique sequences. Unexpectedly, the selected peptide binds with high affinity and specificity to the relatively rare dimeric form of the P6.1 stem-loop rather than the more abundant monomeric conformation. Characterization of this novel RNA-peptide interaction was performed via circular dichroism, steady-state fluorescence, mobility-shift assay, and surface plasmon resonance. The data highlight the power of mRNA display to isolate high affinity ligands from large libraries of molecules.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||human telomerase RNA (hTR); in vitro selection; mRNA display; RNA-peptide interaction; RNA-RNA interaction|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Mixed availability, specified at file level|
|Defense Date:||5 September 2006|
|Author Email:||ueda (AT) caltech.edu|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||07 Sep 2006|
|Last Modified:||17 Apr 2013 17:20|
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