Expanding and reprogramming the genetic code of cells and animals.

Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the number of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase/tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (C. elegans and D. melanogaster) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-transfer RNA (tRNA) synthetase/tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging.

[1]  Susan E. Cellitti,et al.  In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy. , 2008, Journal of the American Chemical Society.

[2]  Peng R. Chen,et al.  Site-specific incorporation of photo-cross-linker and bioorthogonal amino acids into enteric bacterial pathogens. , 2011, Journal of the American Chemical Society.

[3]  Carsten Schultz,et al.  Amino acids for Diels-Alder reactions in living cells. , 2012, Angewandte Chemie.

[4]  Wenjiao Song,et al.  Selective functionalization of a genetically encoded alkene-containing protein via "photoclick chemistry" in bacterial cells. , 2008, Journal of the American Chemical Society.

[5]  J. Chin,et al.  Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. , 2010, Nature chemical biology.

[6]  J. Chin,et al.  Genetically encoded photocontrol of protein localization in mammalian cells. , 2010, Journal of the American Chemical Society.

[7]  P. Schultz,et al.  Addition of the keto functional group to the genetic code of Escherichia coli , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[8]  J. Sulston,et al.  Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. , 1977, Developmental biology.

[9]  P. Schimmel,et al.  Aminoacyl-tRNA synthetases: potential markers of genetic code development. , 2001, Trends in biochemical sciences.

[10]  D. Söll,et al.  Expanding the Genetic Code of Escherichia coli with Phosphoserine , 2011, Science.

[11]  L. Isaksson,et al.  A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for tRNA nonsense suppressors , 2004, Molecular and General Genetics MGG.

[12]  M. Chan,et al.  A pyrrolysine analogue for site-specific protein ubiquitination. , 2009, Angewandte Chemie.

[13]  Peter G Schultz,et al.  Protein conjugation with genetically encoded unnatural amino acids. , 2013, Current opinion in chemical biology.

[14]  J. Chin,et al.  Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion , 2007, Nature Biotechnology.

[15]  S. Yokoyama,et al.  Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. , 2008, Biochemical and biophysical research communications.

[16]  P. Schultz,et al.  A Genetically Encoded ε‐N‐Methyl Lysine in Mammalian Cells , 2010, Chembiochem : a European journal of chemical biology.

[17]  Shigeyuki Yokoyama,et al.  Efficient Decoding of the UAG Triplet as a Full-Fledged Sense Codon Enhances the Growth of a prfA-Deficient Strain of Escherichia coli , 2012, Journal of bacteriology.

[18]  R. Plasterk,et al.  Mechanistic insights and identification of two novel factors in the C. elegans NMD pathway. , 2007, Genes & development.

[19]  Andrew B. Martin,et al.  Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[20]  P. Schimmel,et al.  A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase , 1990, Molecular and cellular biology.

[21]  O. Uhlenbeck,et al.  Uniform Binding of Aminoacyl-tRNAs to Elongation Factor Tu by Thermodynamic Compensation , 2001, Science.

[22]  J. Chin,et al.  Genetically encoding N(epsilon)-methyl-L-lysine in recombinant histones. , 2009, Journal of the American Chemical Society.

[23]  J. Chin,et al.  Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA Synthetase/tRNA(CUA) pair and click chemistry. , 2009, Journal of the American Chemical Society.

[24]  Qing Lin,et al.  Rapid, photoactivatable turn-on fluorescent probes based on an intramolecular photoclick reaction. , 2011, Journal of the American Chemical Society.

[25]  Total chemical synthesis of di-ubiquitin chains. , 2010, Angewandte Chemie.

[26]  J. Chin,et al.  Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells , 2011, Journal of the American Chemical Society.

[27]  Peter G Schultz,et al.  A genetically encoded photocaged tyrosine. , 2006, Angewandte Chemie.

[28]  B. E. Kimmel,et al.  Optimized clinical performance of growth hormone with an expanded genetic code , 2011, Proceedings of the National Academy of Sciences.

[29]  Peter G Schultz,et al.  A genetically encoded fluorescent amino acid. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[30]  B. M. Honda,et al.  Differential Expression of Individual Suppressor tRNATrp Gene Family Members In Vitro and In Vivo in the Nematode Caenorhabditis elegans , 1998, Molecular and Cellular Biology.

[31]  E. Strieter,et al.  Nonenzymatic polymerization of ubiquitin: single-step synthesis and isolation of discrete ubiquitin oligomers. , 2012, Angewandte Chemie.

[32]  E. Lemke,et al.  Genetic Encoding of a Bicyclo[6.1.0]nonyne‐Charged Amino Acid Enables Fast Cellular Protein Imaging by Metal‐Free Ligation , 2012, Chembiochem : a European journal of chemical biology.

[33]  Jianghong Rao,et al.  A biocompatible condensation reaction for controlled assembly of nanostructures in live cells , 2010, Nature chemistry.

[34]  P. Schultz,et al.  Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae. , 2003, Journal of the American Chemical Society.

[35]  Michael T. Taylor,et al.  Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes. , 2012, Journal of the American Chemical Society.

[36]  G M Rubin,et al.  A brief history of Drosophila's contributions to genome research. , 2000, Science.

[37]  Andrew B. Martin,et al.  Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. , 2002, Journal of the American Chemical Society.

[38]  J. Chin,et al.  Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. , 2008, Nature chemical biology.

[39]  Shigeyuki Yokoyama,et al.  Codon reassignment in the Escherichia coli genetic code , 2010, Nucleic acids research.

[40]  Qing Lin,et al.  Genetically encoded cyclopropene directs rapid, photoclick-chemistry-mediated protein labeling in mammalian cells. , 2012, Angewandte Chemie.

[41]  J. Chin,et al.  Synthesis of orthogonal transcription-translation networks , 2009, Proceedings of the National Academy of Sciences.

[42]  E. Strieter,et al.  Forging isopeptide bonds using thiol-ene chemistry: site-specific coupling of ubiquitin molecules for studying the activity of isopeptidases. , 2012, Journal of the American Chemical Society.

[43]  Zhiyong Wang,et al.  Catalyst‐Free and Site‐Specific One‐Pot Dual‐Labeling of a Protein Directed by Two Genetically Incorporated Noncanonical Amino Acids , 2012, Chembiochem : a European journal of chemical biology.

[44]  D. Hirsh,et al.  The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans. , 1979, Developmental biology.

[45]  J. Chin,et al.  Photo-cross-linking interacting proteins with a genetically encoded benzophenone , 2005, Nature Methods.

[46]  Zhiyong Wang,et al.  Genetic incorporation of an aliphatic keto-containing amino acid into proteins for their site-specific modifications. , 2010, Bioorganic & medicinal chemistry letters.

[47]  P G Schultz,et al.  Expanding the Genetic Code of Escherichia coli , 2001, Science.

[48]  P. Schultz,et al.  A genetically encoded infrared probe. , 2006, Journal of the American Chemical Society.

[49]  P. Schultz,et al.  Genetic introduction of a diketone-containing amino acid into proteins. , 2006, Bioorganic & medicinal chemistry letters.

[50]  J. Chin,et al.  Synthesizing cellular networks from evolved ribosome-mRNA pairs. , 2006, Biochemical Society transactions.

[51]  T. Umehara,et al.  Genetic-code evolution for protein synthesis with non-natural amino acids. , 2011, Biochemical and biophysical research communications.

[52]  D. Fushman,et al.  Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme. , 2011, Journal of the American Chemical Society.

[53]  P. Schultz,et al.  A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. , 2013, Biochemistry.

[54]  P. Schultz,et al.  Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae. , 2009, Journal of the American Chemical Society.

[55]  R. Weissleder,et al.  Tetrazine-based cycloadditions: application to pretargeted live cell imaging. , 2008, Bioconjugate chemistry.

[56]  Wenjiao Song,et al.  Fast alkene functionalization in vivo by Photoclick chemistry: HOMO lifting of nitrile imine dipoles. , 2009, Angewandte Chemie.

[57]  P. Schultz,et al.  A method to site-specifically introduce methyllysine into proteins in E. coli. , 2010, Chemical communications.

[58]  R. Waterston,et al.  Differential expression of five tRNA(UAGTrp) amber suppressors in Caenorhabditis elegans , 1988, Molecular and cellular biology.

[59]  Jason W. Chin,et al.  Designer proteins: applications of genetic code expansion in cell biology , 2012, Nature Reviews Molecular Cell Biology.

[60]  J. Chin,et al.  Expanding the genetic code of Drosophila melanogaster. , 2012, Nature chemical biology.

[61]  Farren J. Isaacs,et al.  Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement , 2011, Science.

[62]  J. Sulston,et al.  The embryonic cell lineage of the nematode Caenorhabditis elegans. , 1983, Developmental biology.

[63]  Jason W. Chin,et al.  Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome , 2010, Nature.

[64]  K. Anders,et al.  SMG-2 Is a Phosphorylated Protein Required for mRNA Surveillance in Caenorhabditis elegans and Related to Upf1p of Yeast , 1999, Molecular and Cellular Biology.

[65]  Relly Brandman,et al.  Two-dimensional NMR and All-atom Molecular Dynamics of Cytochrome P450 CYP119 Reveal Hidden Conformational Substates* , 2010, The Journal of Biological Chemistry.

[66]  J. Chin,et al.  Orthogonal gene expression in Escherichia coli. , 2011, Methods in enzymology.

[67]  D. Fushman,et al.  Nonenzymatic assembly of branched polyubiquitin chains for structural and biochemical studies. , 2013, Bioorganic & medicinal chemistry.

[68]  Baoyan Bai,et al.  Organization of the Caenorhabditis elegans small non-coding transcriptome: genomic features, biogenesis, and expression. , 2005, Genome research.

[69]  Peter G Schultz,et al.  Adding new chemistries to the genetic code. , 2010, Annual review of biochemistry.

[70]  Peter G Schultz,et al.  Synthesis of site-specific antibody-drug conjugates using unnatural amino acids , 2012, Proceedings of the National Academy of Sciences.

[71]  J. Chin,et al.  Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a Pyrrolysyl-tRNA Synthetase/tRNA Pair , 2010, Journal of the American Chemical Society.

[72]  E. Lemke,et al.  Genetically Encoded Copper-Free Click Chemistry , 2011, Angewandte Chemie.

[73]  S. Yokoyama,et al.  Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells. , 2002, Nucleic acids research.

[74]  Peter G Schultz,et al.  Control of protein phosphorylation with a genetically encoded photocaged amino acid. , 2007, Nature chemical biology.

[75]  P. Schimmel,et al.  A single base pair dominates over the novel identity of an Escherichia coli tyrosine tRNA in Saccharomyces cerevisiae. , 1991, Molecular and cellular biology.

[76]  P. Schultz,et al.  The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination , 2004, Nature Biotechnology.

[77]  T. Sixma,et al.  Nonhydrolyzable ubiquitin-isopeptide isosteres as deubiquitinating enzyme probes. , 2010, Journal of the American Chemical Society.

[78]  F. Blattner,et al.  Emergent Properties of Reduced-Genome Escherichia coli , 2006, Science.

[79]  J. Noel,et al.  Genetically encoding unnatural amino acids for cellular and neuronal studies , 2007, Nature Neuroscience.

[80]  C. James,et al.  A New UAG-Encoded Residue in the Structure of a Methanogen Methyltransferase , 2002, Science.

[81]  J. Hodgkin.  Novel nematode amber suppressors. , 1985, Genetics.

[82]  T. Sakmar,et al.  Tracking G-protein-coupled receptor activation using genetically encoded infrared probes , 2010, Nature.

[83]  Peter G Schultz,et al.  An enhanced system for unnatural amino acid mutagenesis in E. coli. , 2010, Journal of molecular biology.

[84]  Alexander Deiters,et al.  Photocontrol of tyrosine phosphorylation in mammalian cells via genetic encoding of photocaged tyrosine. , 2012, Journal of the American Chemical Society.

[85]  P. Schultz,et al.  In vivo incorporation of an alkyne into proteins in Escherichia coli. , 2005, Bioorganic & medicinal chemistry letters.

[86]  A. Slusarczyk,et al.  De novo generation of mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. , 2010, Journal of the American Chemical Society.

[87]  R. Waterston,et al.  Genetic and molecular analysis of eight tRNA(Trp) amber suppressors in Caenorhabditis elegans. , 1990, Journal of molecular biology.

[88]  Joseph M. Fox,et al.  Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity. , 2008, Journal of the American Chemical Society.

[89]  J. Chin,et al.  Genetically encoding protein oxidative damage. , 2008, Journal of the American Chemical Society.

[90]  P. Schultz,et al.  A genetically encoded fluorescent amino acid. , 2006, Journal of the American Chemical Society.

[91]  Farren J. Isaacs,et al.  Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion , 2012, FEBS letters.

[92]  V. Ambros,et al.  A new kind of informational suppression in the nematode Caenorhabditis elegans. , 1989, Genetics.

[93]  S. Briggs,et al.  Expanding the genetic code of Caenorhabditis elegans using bacterial aminoacyl-tRNA synthetase/tRNA pairs. , 2012, ACS chemical biology.

[94]  S. Yokoyama,et al.  Genetic encoding of non‐natural amino acids in Drosophila melanogaster Schneider 2 cells , 2010, Protein science : a publication of the Protein Society.

[95]  J. Chin,et al.  Traceless and Site-Specific Ubiquitination of Recombinant Proteins , 2011, Journal of the American Chemical Society.

[96]  G. Rubin,et al.  Genetic transformation of Drosophila with transposable element vectors. , 1982, Science.

[97]  A. Deiters,et al.  Site-specific incorporation of fluorotyrosines into proteins in Escherichia coli by photochemical disguise. , 2010, Biochemistry.

[98]  Guifang Wang,et al.  Protein (19)F NMR in Escherichia coli. , 2010, Journal of the American Chemical Society.

[99]  H. Lashuel,et al.  Highly efficient and chemoselective peptide ubiquitylation. , 2009, Angewandte Chemie.

[100]  J. Chin,et al.  Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. , 2012, Nature chemistry.

[101]  Peng R. Chen,et al.  A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance. , 2011, Nature chemical biology.

[102]  J. Chin,et al.  A network of orthogonal ribosome·mRNA pairs , 2005, Nature chemical biology.

[103]  Matthew D. Schultz,et al.  RF1 Knockout Allows Ribosomal Incorporation of Unnatural Amino Acids at Multiple Sites , 2011, Nature chemical biology.

[104]  P. Schultz,et al.  The selective incorporation of alkenes into proteins in Escherichia coli. , 2002, Angewandte Chemie.

[105]  Peter G Schultz,et al.  An expanded genetic code with a functional quadruplet codon. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[106]  Kai Johnsson,et al.  How to obtain labeled proteins and what to do with them. , 2010, Current opinion in biotechnology.

[107]  J. Chin,et al.  Expanding the Genetic Code of an Animal , 2011, Journal of the American Chemical Society.

[108]  P. Schultz,et al.  Recombinant expression of selectively sulfated proteins in Escherichia coli , 2006, Nature Biotechnology.

[109]  Ryohei Ishii,et al.  Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. , 2008, Chemistry & biology.

[110]  Peter G Schultz,et al.  An Expanded Eukaryotic Genetic Code , 2003, Science.

[111]  J. Chin,et al.  Functional epitopes at the ribosome subunit interface , 2006, Nature chemical biology.

[112]  Peter G Schultz,et al.  A genetically encoded photocaged amino acid. , 2004, Journal of the American Chemical Society.

[113]  Zhiyong Wang,et al.  A genetically encoded photocaged Nepsilon-methyl-L-lysine. , 2010, Molecular bioSystems.

[114]  T. Muir,et al.  Disulfide-directed histone ubiquitylation reveals plasticity in hDot1L activation. , 2010, Nature chemical biology.

[115]  P. Schultz,et al.  Expanding the genetic code , 2022, Protein science : a publication of the Protein Society.

[116]  T. Muir,et al.  Genetically encoded 1,2-aminothiols facilitate rapid and site-specific protein labeling via a bio-orthogonal cyanobenzothiazole condensation. , 2011, Journal of the American Chemical Society.

[117]  Peter G Schultz,et al.  Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli , 2012, Proceedings of the National Academy of Sciences.

[118]  David H Russell,et al.  A facile system for genetic incorporation of two different noncanonical amino acids into one protein in Escherichia coli. , 2010, Angewandte Chemie.

[119]  J. Chin,et al.  Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels–Alder Reactions , 2012, Journal of the American Chemical Society.

[120]  D. Raleigh,et al.  Interpretation of p-cyanophenylalanine fluorescence in proteins in terms of solvent exposure and contribution of side-chain quenchers: a combined fluorescence, IR and molecular dynamics study. , 2009, Biochemistry.

[121]  S. Hahn,et al.  The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex , 2007, Nature Structural &Molecular Biology.

[122]  N. Perrimon,et al.  Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. , 1993, Development.

[123]  Qing Lin,et al.  Discovery of new photoactivatable diaryltetrazoles for photoclick chemistry via 'scaffold hopping'. , 2011, Bioorganic & medicinal chemistry letters.

[124]  S. Yokoyama,et al.  Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid , 2005, Nature Methods.

[125]  D. Hamelberg,et al.  Clicking 1,2,4,5-tetrazine and cyclooctynes with tunable reaction rates. , 2012, Chemical communications.

[126]  J. Chin,et al.  Cellular logic with orthogonal ribosomes. , 2005, Journal of the American Chemical Society.

[127]  Wei Zhang,et al.  A biosynthetic route to photoclick chemistry on proteins. , 2010, Journal of the American Chemical Society.

[128]  Matthew D. Schultz,et al.  Release Factor One Is Nonessential in Escherichia coli , 2012, ACS chemical biology.

[129]  P. Schimmel,et al.  An Escherichia coli tyrosine transfer RNA is a leucine-specific transfer RNA in the yeast Saccharomyces cerevisiae. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[130]  J. Chin,et al.  Genetically encoding an aliphatic diazirine for protein photocrosslinking , 2011 .

[131]  Qian Wang,et al.  New methods enabling efficient incorporation of unnatural amino acids in yeast. , 2008, Journal of the American Chemical Society.

[132]  M. Chan,et al.  A pyrrolysine analogue for protein click chemistry. , 2009, Angewandte Chemie.

[133]  P. Schultz,et al.  Progress toward an expanded eukaryotic genetic code. , 2003, Chemistry & biology.

[134]  Peter G Schultz,et al.  Synthesis of bispecific antibodies using genetically encoded unnatural amino acids. , 2012, Journal of the American Chemical Society.