20.109(S11):TR Yellow Research Proposal
Selection of peptide binding sequences to human alpha thrombin using mRNA display evolution
Statement of Purpose
Using directed evolution, we want to find peptides that bind and hopefully inhibit human alpha thrombin. Previous research has shown the GGF motif to exist in all sequences that were found to bind to thrombin, so we want to generate a library based on this motif, and then use mRNA display evolution to find the best possible binder through multiple iterations of library generation and selection. Finding new binders and inhibitors of alpha thrombin can serve as candidates for new anti-thrombotic therapeutics.
By harnessing the power of natural selection to modify a protein’s properties through directed evolution, a library of mutant proteins must first be created. If this process were performed in vivo, the size of the library would have to be limited to between 10^4-10^9 members, and if it were performed in vitro, the size of the library could be expanded to over 10^12 members. The protocol described in this proposal, called “mRNA display,” uses mRNA-displayed proteins, which are proteins that have the strand of mRNA that codes for the protein covalently attached. This creates a link between genotype (the mRNA strand) and phenotype (the attached protein), and therefore enables direct amplification of the desired sequence. The linkage enables several rounds of amplification and selection in libraries larger than 10^12 in one experiment. (Seelig-see resources)
mRNA display has been used to isolate small, functional peptides that bind to and inhibit human alpha thrombin. The sequences of these peptides were analyzed and compared, and out of the 45 sequences that bound to thrombin, more 60% of the sequences contained the conserved four amino acid sequence motif, DPGR. From this, it was concluded that this amino acid sequence plays a crucial role in the binding of the peptide sequence to alpha thrombin. (Raffler et al- see resources)
- Generate a library based around the DPGR motif
- mRNA display evolution to select for binders
- Identify motif
- Generate new library based around new motif
- Test for thrombin activity. Assay?
Deliver p53 mRNA to tumor cells, to inhibit cell proliferation. How? 1) Bind to end of aptamer that gets internalized by a specific cell type 2) Put the sequence in the aptamer (good ribosome binding site?)
Library? Bind onto an aptamer that already is known to internalize and then show that the aptamer is still taken up when bound to the mRNA and that mRNA is expressed in the cell to p53 protein/ shows tumor phenotype?
Sequence with specific primers once in the cell to make sure still the mRNA you wanted (not mutated). Do an assay to make sure it's being converted to protein by ribosomes as well as the phenotypic assay (cell speed/proliferation/apoptosis)
http://onlinelibrary.wiley.com/doi/10.1002/chem.200701330/abstract ^^ IMPORTANT- shows aptamer that internalizes into leukemia T cells, that aptamers can release what they're binded to once in the cell.
From article: "Ted et al. designed a disulfide link- age between the anti-PSMA aptamer and siRNA, so that the disulfide linkage should be cleaved upon entering the en- dosome and releasing the siRNA for processing or func- tion." (T. C. Chu, K. Y. Twu, A. D. Ellington, M. Levy, Nucleic Acids Res. 2006, 34, e73. )
http://www.ncbi.nlm.nih.gov/pubmed/18533682 Different internalized aptamer.
http://www.ncbi.nlm.nih.gov/pubmed/18566214 transformation of E.coli with normal p53 protein