Development of a new method for nonnatural amino acid mutagenesis of proteins based on a ribozyme technology

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.