Caltech Library System

About | Browse | Search | Caltech Student Instructions

Type of Document	Dissertation
Author	Takahashi, Terry Torao
Author's Email Address	ttakahashi99 AT yahoo.com
URN	etd-05262005-163739
Persistent URL	http://resolver.caltech.edu/CaltechETD:etd-05262005-163739
Title	In vitro selection of RNA binding peptides
Degree	PhD
Option	Biochemistry and Molecular Biophysics
Advisory Committee	Advisor Name Title Douglas C. Rees Committee Chair Peter B. Dervan Committee Member Richard W. Roberts Committee Member Stephen L. Mayo Committee Member
Keywords	Antitermination RNA Display RNA Binding Peptides In Vitro Selection SELEX Telomerase RNA Protein Fusions
Date of Defense	2005-05-11
Availability	mixed
Abstract RNA is recognized to play an increasing number of roles in the cell: transcription regulation, translation, and catalysis. Peptides that bind RNA would therefore be useful as biochemical tools and lead compounds for therapeutics. Existing genetic methods of isolating RNA binding peptides are prone to biases and can only search millions of sequences. In vitro selections using mRNA display provide an avenue to discover specific, high affinity peptides that bind to any RNA target from libraries composed of trillions of molecules. Here, we describe initial experiments to optimize the mRNA display selection cycle for the isolation of RNA binding peptides. We use this optimized cycle to show that enrichment of specific sequences is possible using mRNA display, and select mutants of the lambda N peptide which bind in a different conformation than wild-type. Characterization of these peptides demonstrates that affinity is not enough for in vivo activity; binding in a correct conformation is also important. Based on these experiments, we designed a strategy to isolate RNA binding peptides to targets for which no natural ligand is known. We test this strategy and isolate peptides that bind to functionally important domains of telomerase RNA with nanomolar affinity and high specificity. Using mutagenic PCR and additional rounds of selection, we increase the specificity of several peptides for telomerase RNA and also isolate other peptides which bind an important domain of the Hepatitis C Virus internal ribosome entry site.
Files	Filename       Size       Approximate Download Time (Hours:Minutes:Seconds)     28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access    0_TitleIntro.pdf 413.84 Kb 00:01:54 00:00:59 00:00:51 00:00:25 00:00:02   1_Chapter1_Overview.pdf 189.49 Kb 00:00:52 00:00:27 00:00:23 00:00:11 00:00:01   2_Chapter2.pdf 1.09 Mb 00:05:04 00:02:36 00:02:16 00:01:08 00:00:05   3_Chapter3.pdf 1.78 Mb 00:08:13 00:04:13 00:03:42 00:01:51 00:00:09   4_Chapter4.pdf 3.18 Mb 00:14:42 00:07:34 00:06:37 00:03:18 00:00:16   5_Chapter5.pdf 3.34 Mb 00:15:28 00:07:57 00:06:57 00:03:28 00:00:17 There are 3 files which have been withheld at the author's request. indicates that a file or directory is accessible from the campus network only and must not be distributed to non-campus persons.