[PubMed] [Google Scholar] 249. been used to incorporate ncAAs into proteins in mammalian cell lines at gm/L scale employing transient expression methods.100-102 Viral vectors have allowed the ncAA machinery to be delivered efficiently into primary cells, as well as tissues,96, 103, 104 where it was used among other applications Epha5 to monitor voltage-sensitive changes in response to membrane depolarization events in neural cells.100 2.1.3. Recent Advances A variety of strategies have been reported to further improve the efficiency and specificity of ncAA incorporation into proteins, including mutations to the aaRS, tRNA, ribosomal peptidyl transferase and elongation factor.13, 17, 104C110 Moreover, aaRS and tRNA expression levels have been modulated in order to facilitate high-level expressions of proteins containing ncAAs.13, 104, 105, 111C113 These alterations have led to ncAA-incorporation on multigram/L levels in large scale bacterial fermentation, and gram/L scale in stable CHO cell lines as demonstrated in the production of ncAA containing pegylated proteins and antibody-drug conjugates (ADCs).111 An exciting recent advance Duocarmycin is the ability to incorporate more than one ncAA into a protein sequence with the ultimate challenging goal of the mRNA template-directed biosynthesis of monodisperse biopolymers made up of synthetic Duocarmycin building blocks. Toward this end several and eRF1 in eukaryotes) that terminate polypeptide synthesis in response to specific nonsense codons, in order to improve suppression efficiencies.75, 114C116 Orthogonal bacterial ribosomes that are directed to an orthogonal message, by the incorporation of a mutant 16S rRNA into their small subunit (and therefore not essential to the cell) have also been created (Figure 3).117, 118 One such orthogonal ribosome that no longer recognizes RF1 was discovered by directed evolution, and enables the efficient incorporation of an ncAA in response to amber codons at multiple sites in a single polypeptide.119 Another approach involves recoding the genome such that some or all of the amber codons have been replaced by the ochre nonsense codon TAA in an effort to remove potential read-through of endogenous termination signals.120C122 These strains, which have TAG or TAGN (N=A, G, C, T) uniquely assigned to the ncAA, have been shown to enhance ncAA incorporation in response to the quadruplet codon TAGA, which is derived from and competes with RF1 recognition of the amber codon (TAG).5 Open in a separate window Figure 3. Generation of an orthogonal ribosome. A) A non-orthogonal ribosome allows for cross talk between the two mRNAs, not providing efficient incorporation of ncAAs. B) An orthogonal ribosome where the endogenous system (grey) and the engineered ribosome and mRNA (green) exhibit no cross-reactivity. C) Crystal structure of the rRNA (orange), mRNA (purple) and tRNA (yellow), illustrating the key 530 loop within the ribosome that was subjected to mutagenesis to afford an orthogonal ribosome.119 There is also interest in the incorporation of multiple distinct amino acids into a single protein, which requires aaRS/tRNA pairs that are mutually orthogonal and orthogonal to the host aaRS/tRNA pairs.9 Recently, a new expression cassette was engineered for bacterial expression that affords two aaRS/tRNA pairs (and and in living cells with minimal perturbation to protein structure. These ncAAs include residues with altered pKas for mechanistic studies, isotopic labels for infrared and NMR studies, photocrosslinkers for mapping biomolecular interactions in living cells, heavy atoms for X-ray crystallography, and spin labels and fluorescent side Duocarmycin chains for EPR and optical applications, respectively. While ncAAs probes have been used in numerous studies, below we highlight instructive examples of their use. 3.1.1 Altering pKa and redox potential Electron-withdrawing or donating substituents allow one to alter the acidity, basicity and redox potential of canonical amino acids (Figure 4).36, 61, 62, 140C146 For example fluorinated tyrosine analogues served as effective EPR probes to monitor long-lived tyrosyl radicals in the complex mechanism of ribonucleotide reductase, and better understand the role of conserved tyrosine residues in the prevention of undesirable radical chemistry.36, 147 These studies complemented previous semisynthetic studies employing nitrotyrosine140 and aminotyrosine,143 which were used to investigate the kinetics of radical intermediate formation within these ribonucleotide reductases. Open in a separate window Figure 4. Modulation of pKa and redox potential of tyrosine residues. A) The ribonucleotide reductase reaction converting ribose to deoxyribose relies upon a catalytic cysteine radical. The generation of this radical is dependent on radical formation on several key tyrosine.