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Targeting human telomerase RNA via biochemical and in vitro selection methods

Citation

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

Abstract

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
Major Option:Chemistry
Thesis Availability:Mixed availability, specified at file level
Research Advisor(s):
  • Roberts, Richard W.
Thesis Committee:
  • Barton, Jacqueline K. (chair)
  • Dervan, Peter B.
  • Deshaies, Raymond Joseph
  • Roberts, Richard W.
Defense Date:5 September 2006
Author Email:ueda (AT) caltech.edu
Record Number:CaltechETD:etd-09052006-214421
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-09052006-214421
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:3337
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:07 Sep 2006
Last Modified:17 Apr 2013 17:20

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