Szostak Lab at Harvard

Szostak Lab at Harvard
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Application of the principles of in vitro selection and directed evolution to peptides and proteins is a powerful tool for investigating protein function and structure and for obtaining insight into the pathways by which enzymes evolve in nature. Our approach has been to generate stable, covalent RNA-protein fusions in a completely in vitro system. We do this by covalently linking puromycin, an antibiotic that mimics an aminoacylated tRNA, to the 3' end of a synthetic mRNA through a DNA linker. A ribosome begins translation of such a template as usual, generating a peptide as it transits the open reading frame. When the ribosome reaches the end of the open reading frame and hits the DNA linker it stalls, allowing the nearby puromycin to enter the A site of the ribosome and accept the nascent peptide chain. The resulting RNA-peptide fusions can be formed efficiently from mRNAs encoding small peptides or large proteins. We have prepared libraries of fusions encoding random peptides and are preparing to begin evolving new binding domains and enzymes. An exciting future application will be the ability to conduct side-by-side comparisons of RNA and protein evolution.
Submitted by elementlist on Feb 19, 2006
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