In Vitro Selection of RNA Binding Peptides

Antitermination;In Vitro Selection;RNA Binding Peptides;RNA Display;RNA Protein Fusions;SELEX;Telomerase

Takahashi, Terry Torao ; Roberts, Richard W.

University:California Institute of Technology

Department:Chemistry and Chemical Engineering

关键词: Antitermination;    In Vitro Selection;    RNA Binding Peptides;    RNA Display;    RNA Protein Fusions;    SELEX;    Telomerase;   

Others  :  https://thesis.library.caltech.edu/2099/9/EntireThesis.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

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.