Development of a new method for nonnatural amino acid mutagenesis of proteins based on a ribozyme technology
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Site specific mutagenesis is a technique that allows for the substitution of nonnatural amino acids at a specific position in a protein. The technique relies on misacylated tRNAs to incorporate these desired substitutions both in vitro and in vivo. In vitro efforts conducted by the Hecht and Schultz groups utilized a dinucleotide, pdCpA, to be aminoacylated with the desired amino acid that would then be ligated onto a truncated tRNA(-CA). However, this technique requires multiple purification steps and is quite laborious and costly. In vivo efforts conducted by Schultz focuses on reprogramming aminoacyl-tRNA synthetases (ARSs) from a different species to be orthogonal to the host species in order to successfully incorporate the desired nonnatural amino acid at a specified position. This technique only allows for one amino acid to be incorporated at a time and the ARS has to be reprogrammed each time a different amino acid substitution is desired. An artificial RNA catalyst (ribozyme) capable of charging the 3 ' terminus of tRNA with a phenylalanine substrate was isolated using in vitro evolution. The ribozyme that was initially isolated was a cis -acting catalyst was shown to maintain function when separated from the tRNA making it a trans catalyst. The ribozyme recognized the aromatic side chain of the amino acid making it specific to aromatic amino acids. The ribozyme was reevolved into a more flexible catalyst capable of charging various tRNAs with a variety of phenylalanine analogs. To take advantage of this flexible attribute the ribozyme was immobilized onto a solid support thus making the ribozyme reusable and aminoacyl-tRNA could be easily prepared. The aminoacyl-tRNA product could then be used directly for in vitro translation thus simplifying the process of site specific mutagenesis. To enhance the versatility of ribozyme, studies were undertaken to make the catalyst capable of multiple turnover. A fluorescently-labeled substrate was prepared to be used with the catalyst as part of a collaborative effort using compartmentalized selection to isolate the ribozyme with the greatest number of multiple turnovers.