The genetic code of every known organism, from bacteria to humans, encodes the same twenty common amino acids. Different combinations of the same twenty natural amino acids form proteins that carry out virtually all the complex processes of life, from photosynthesis to signal transduction and the immune response. In order to study and modify protein structure and function, scientists have attempted to manipulate both the genetic code and the amino acid sequence of proteins. However, it has been difficult to remove the constraints imposed by the genetic code that limit proteins to twenty genetically encoded standard building blocks (with the rare exception of selenocysteine (see, e.g., A. Bock et al., (1991), Molecular Microbiology 5:515-20) and pyrrolysine (see, e.g., G. Srinivasan, et al., (2002), Science 296:1459-62).
Some progress has been made to remove these constraints, although this progress has been limited, and the ability to rationally control protein structure and function is still in its infancy. For example, chemists have developed methods and strategies to synthesize and manipulate the structures of small molecules (see, e.g., E. J. Corey, & X.-M. Cheng, The Logic of Chemical Synthesis (Wiley-Interscience, New York, 1995)). Total synthesis (see, e.g., B. Merrifield, (1986), Science 232:341-7 (1986)), and semi-synthetic methodologies (see, e.g., D. Y. Jackson et al., (1994) Science 266:243-7; and, P. E. Dawson, & S. B. Kent, (2000), Annual Review of Biochemistry 69:923-60), have made it possible to synthesize peptides and small proteins, but these methodologies have limited utility with proteins over 10 kilo Daltons (kDa). Mutagenesis methods, though powerful, are restricted to a limited number of structural changes. In a number of cases, it has been possible to competitively incorporate close structural analogues of common amino acids throughout proteins. See, e.g., R. Furter, (1998), Protein Science 7:419-26; K. Kirshenbaum, et al., (2002), Chem Bio Chem 3:235-7; and, V. Doring et al., (2001), Science 292:501-4.
Early work demonstrated that the translational machinery of E. coli would accommodate amino acids similar in structure to the common twenty. See Hortin, G., and Boime, I. (1983) Methods Enzymol. 96:777-784. This work was further extended by relaxing the specificity of endogenous E. coli synthetases so that they activate unnatural amino acids as well as their cognate natural amino acid. Moreover, it was shown that mutations in editing domains could also be used to extend the substrate scope of the endogenous synthetase. See Doring, V., et al., (2001) Science 292:501-504. However, these strategies are limited to recoding the genetic code rather than expanding the genetic code and lead to varying degrees of substitution of one of the common twenty amino acids with an unnatural amino acid.
Later it was shown that unnatural amino acids could be site-specifically incorporated into proteins in vitro by the addition of chemically aminoacylated orthogonal amber suppressor tRNAs to an in vitro transcription/translation reaction. See, e.g., Noren, C. J., et al. (1989) Science 244:182-188; Bain, J. D., et al., (1989) J. Am. Chem. Soc. 111:8013-8014; Dougherty, D. A. (2000) Curr. Opin. Chem. Biol. 4, 645-652; Cornish, V. W., et al. (1995) Angew. Chem. Int. Ed. 34:621-633; J. A. Ellman, et al., (1992), Science 255:197-200; and, D. Mendel, et al., (1995), Annual Review of Biophysics and Biomolecular Structure 24:435-462. These studies show that the ribosome and translation factors are compatible with a large number of unnatural amino acids, even those with unusual structures. Unfortunately, the chemical aminoacylation of tRNAs is difficult, and the stoichiometric nature of this process severely limited the amount of protein that could be generated.
Unnatural amino acid tRNA complexes have been microinjected into cells. For example, unnatural amino acids were introduced into the nicotinic acetylcholine receptor in Xenopus oocytes (e.g., M. W. Nowak, et al. (1998), In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system, Method Enzymol. 293:504-529) by microinjection of a chemically misacylated Tetrahymena thermophila tRNA (e.g., M. E. Saks, et al. (1996), An engineered Tetrahymena tRNAGln for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression, J. Biol. Chem. 271:23169-23175), and the relevant mRNA. See also D. A. Dougherty (2000), Unnatural amino acids as probes of protein structure and function, Curr. Opin. Chem. Biol. 4:645-652. Unfortunately, this methodology is limited to proteins in cells that can be microinjected, and because the relevant tRNA is chemically acylated in vitro, and cannot be re-acylated, the yields of protein are very low.
To overcome these limitations, new components, e.g., orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases and pairs thereof, were added to the protein biosynthetic machinery of the prokaryote Escherichia coli (E. coli) (see, e.g., L. Wang, et al., (2001), Science 292:498-500), which allowed genetic encoding of unnatural amino acids in vivo. A number of new amino acids with novel chemical, physical or biological properties, including photoaffinity labels and photoisomerizable amino acids, photocrosslinking amino acids (see, e.g., Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:11020-11024; and, Chin, J. W., et al., (2002) J. Am. Chem. Soc. 124:9026-9027), keto amino acids (see, e.g., Wang, L., et al., (2003) Proc. Natl. Acad. Sci. U.S.A. 100:56-61), heavy atom containing amino acids, and glycosylated amino acids have been incorporated efficiently and with high fidelity into proteins in E. coli in response to, e.g., the amber codon (TAG), using this methodology.
Several other orthogonal pairs have been reported. Glutaminyl (see, e.g., Liu, D. R., and Schultz, P. G. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:4780-4785), aspartyl (see, e.g., Pastrnak, M., et al., (2000) Helv. Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S., et al., (1998) J. Biochem. (Tokyo. Jpn.) 124:1065-1068; and, Kowal, A. K., et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) systems derived from S. cerevisiae tRNAs and synthetases have been described for the potential incorporation of unnatural amino acids in E. coli. In addition, a leucine system, which includes an archaeal derived tRNA and a synthetase derived from, e.g., Methanobacterium thermoautotrophicum, has also been described. See Anderson and Schultz, (2003), Biochemistry, 42(32):9598-608. Systems derived from the E. coli glutaminyl (see, e.g., Kowal, A. K., et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) and tyrosyl (see, e.g., Edwards, H., and Schimmel, P. (1990) Mol. Cell. Biol. 10:1633-1641) synthetase have been described for use in S. cerevisiae. The E. coli tyrosyl system has been used for the incorporation of 3-iodo-L-tyrosine in vivo, in mammalian cells. See Sakamoto, K., et al., (2002) Nucleic Acids Res. 30:4692-4699.
Unnatural amino acids that have been incorporated into proteins, e.g., using variants of the tyrosyl-tRNA synthetase, are aryl derivatives, including p-azido- (see, e.g., Chin et al., (2002) Addition of p-azido-L-phenylalamine to the genetic code of Escherichia coli, J. Am. Chem. Soc., 124:9026-9027), p-benzoyl- (see, e.g., Chin et al., (2002) Addition of a photocrosslinkiing amino acid to the genetic code of Escherchia coli, PNAS, USA, 99:11020-11024), p-amino- (see, e.g., Santoro et al., (2002), An efficient system for the evolution of aminoacyl-tRNA synthetase specificity, Nat. Biotechnology, 20:1044-1048), p-isopropyl- (see, e.g., Santoro et al., (2002), An efficient system for the evolution of aminoacyl-tRNA synthetase specificity, Nat. Biotechnology, 20:1044-1048), m-acetyl- (see, e.g., Wang et al., (2003), Addition of the keto functional group to the genetic code of Escherichia coli, PNAS, USA, 100:56-61), and p-acetyl-phenylalanine (Wang et al., (2003), Addition of the keto functional group to the genetic code of Escherichia coli, PNAS, USA, 100:56-61); O-methyl- (see, e.g., Wang et al., (2001), Expanding the genetic code of Escherichia coli, Science 292:498-500) and O-allyl-tyrosine (see, e.g., Santoro et al., (2002), An efficient system for the evolution of aminoacyl-tRNA synthetase specificity, Nat. Biotechnology, 20:1044-1048; and Zhang et al., (2002) The selective incorporation of alkenes into proteins in Escherichia coli, Angew Chem. Int. Ed. Engl., 41:2840-2842); 3-(2-naphthyl)alanine (see, e.g., Wang et al., (2002) Adding L-3-(2-Naphthyl)alanine to the genetic code of E. coli, J. Am. Chem. Soc., 124:1836-1837); and a p-propargyloxy phenylalanine (see, e.g., Deiters et al., (2003) Adding Amino Acids with Novel Reactivity to the Genetic Code of Saccharomyces Cerevisiae, in press). To further expand the genetic code and increase the diversity of unnatural amino acid structures that can be incorporated into proteins in a cell, there is a need to develop improved and/or additional components of the biosynthetic machinery, e.g., orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases and/or unique codons. This invention fulfills these and other needs, as will be apparent upon review of the following disclosure.