A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe.

A small and highly fluorescent non-natural amino acid that contains an anthraniloyl group (atnDap) was incorporated into various positions of streptavidin. The positions were directed by a CGGG/CCCG four-base codon/anticodon pair. The non-natural mutants were obtained in excellent yields and some of them retained strong biotin-binding activity. The fluorescence wavelength as well as the intensity of the anthraniloyl group at position 120 were sensitive to biotin binding. These unique properties indicate that the atnDap is the most suitable non-natural amino acid for a position-specific fluorescent labeling of proteins that is highly sensitive to microenvironmental changes.

Incorporation of nonnatural amino acids into proteins by using various four-base codons in an Escherichia coli in vitro translation system.

Incorporation of nonnatural amino acids into proteins is a powerful technique in protein research. Amber suppression has been used to this end, but this strategy does not allow multiple incorporation of nonnatural amino acids into single proteins. In this article, we developed an alternative strategy for nonnatural mutagenesis by using four-base codons. The four-base codons AGGU, CGGU, CCCU, CUCU, CUAU, and GGGU were successfully decoded by the nitrophenylalanyl-tRNA containing the complementary four-base anticodons in an Escherichia coli in vitro translation system. The most efficient four-base decoding was observed for the GGGU codon, which yielded 86% of the full-length protein containing nitrophenylalanine relative to the wild-type protein. Moreover, highly efficient incorporation of two different nonnatural amino acids was achieved by using a set of two four-base codons, CGGG and GGGU. This work shows that the four-base codon strategy is more advantageous than the amber suppression strategy in efficiency and versatility.

Five-base codons for incorporation of nonnatural amino acids into proteins.

Extension of the genetic code for the introduction of nonnatural amino acids into proteins was examined by using five-base codon-anticodon pairs. A streptavidin mRNA containing a CGGUA codon at the Tyr54 position and a tRNA(UACCG) chemically aminoacylated with a nonnatural amino acid were added to an Escherichia coli in vitro translation system. Western blot analysis indicated that the CGGUA codon is decoded by the aminoacyl-tRNA containing the UACCG anticodon. HPLC analysis of the tryptic fragment of the translation product revealed that the nonnatural amino acid was incorporated corresponding to the CGGUA codon without affecting the reading frame adjacent to the CGGUA codon. Another 15 five-base codons CGGN(1)N(2), where N(1) and N(2) indicate one of four nucleotides, were also successfully decoded by aminoacyl-tRNAs containing the complementary five-base anticodons. These results provide a novel strategy for nonnatural mutagenesis as well as a novel insight into the mechanism of frameshift suppression.

Control of tRNA function by antisense PNA derivatives.

The interaction of antisense peptide nucleic acid (PNA) with yeast tRNA(Phe) was investigated. A 6-mer PNA complementary to the 3'-terminal sequence including the 73ACCA end hybridized to the tRNA. While the PNA with a single mismatch did not. PNA is a promising candidate for controlling tRNA functions by the sequence-specific hybridization.

Effect of single base insertion into anticodon loop of frameshift suppressor tRNA.

Frameshift suppressor tRNAs containing an additional nucleotide at anticodon loop were prepared and their translational activity was evaluated in an E. coli in vitro translation system. The mutated tRNA(CCCG) was chemically aminoacylated with nitrophenylalanine, and added to the in vitro translation system together with a streptavidin mRNA containing CGGG codon. Western blot analysis indicates that the mutated tRNAs can decode the four-base codon, except 32.1C and 33.1G mutants.

Site-specific cleavage of a protein via introducing a hydroxy acid derivative in the main chain by using four-base codon/anticodon pairs.

Site-specific incorporation of the tyrosine isostere (2-Hydroxy-3-(4-hydroxy-phenyl)-propionic acid) into streptavidin was accomplished by in vitro frameshift suppression of a CGGG 4-base mutation with a chemically acylated frameshift suppressor tRNA(cccg). The mutant, in which the backbone amide linkage is replaced by an ester linkage, is hydrolyzed under neutral condition. The cleavage rate of the protein backbone at a single-predetermined site was strongly depended on the incorporated position of the isostere.

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs.

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.

Sense codon-dependent introduction of unnatural amino acids into multiple sites of a protein.

Cell-free protein synthesis, driven by a crude S30 extract from Escherichia coli, has been applied to the preparation of proteins containing unnatural amino acids at specific positions. We have developed methods for inactivating tRNA(Asp) and tRNA(Phe) within a crude E. coli tRNA by an antisense treatment and for digesting most of the tRNA within the S30 extract without essential damage to the ribosomal activity. In the present study, we applied these methods to the substitution of Asp and Phe residues of the HIV-1 protease with unnatural amino acids. With 10 mM Mg(2+), the translation efficiency was higher than that with the other tested concentration, and the misreading efficiency was low. The protease mRNA was translated in the presence of an antisense DNA-treated tRNA mixture and 2-naphthylalanyl- and/or p-phenylazophenylalanyl-tRNA. The results suggest that a good portion of the translation products are substituted at all of the seven positions originally occupied by Asp or Phe.

Site-directed incorporation of fluorescent nonnatural amino acids into streptavidin for highly sensitive detection of biotin.

Fluorescent nonnatural amino acids were incorporated into specific positions of streptavidin. The positions of the nonnatural amino acids were directed by a CGGG/CCCG four-base codon/anticodon pair. The nonnatural mutants with a single 2-anthrylalanine at the 22nd, 43rd, 54th, and 120th positions, respectively, were found to bind biotin, indicating that the mutants retained active conformation. The fluorescence intensities of the anthryl groups were relatively insensitive to the positions and the biotin binding when excited at 265 nm. When the anthryl group at the 120th position was excited through energy transfer from tryptophan units, the fluorescence intensity markedly decreased with biotin binding, because of a suppression of the energy transfer. Amino acids carrying 7-methoxycoumarine fluorophore were also incorporated at the 120th position. Their fluorescence quantum yields were very sensitive to the biotin binding. The high sensitivity of the coumarine-labeled streptavidin exemplifies potential applications of fluorescent nonnatural mutants for detecting specific molecules at very low concentrations.

Random insertion and deletion mutagenesis for construction of protein library containing nonnatural amino acids.

A new method was developed for the generation of a library of mutant proteins that contained nonnatural amino acids. The method, "random insertion and deletion (RID) mutagenesis", is based on the deletion of an arbitrary number of bases at random positions and, at the same time, the insertion of an arbitrary sequence into the same position. By using this method, randomly selected three consecutive bases in the gene of green fluorescence protein (GFP) were replaced by a CGGT 4-base codon. When this DNA library was expressed in E. coli, about 80% of colonies lost the fluorescence. The non-fluorescent colonies were picked up and the genes were sequenced. Replacement of three consecutive bases by CGGT 4-base codon was found in two of the four mutated genes.

Incorporation of nonnatural amino acids into proteins by using five-base codon-anticodon pairs.

A novel strategy for the incorporation of nonnatural amino acids into proteins was developed by using five-base codon-anticodon pairs. The streptavidin mRNA containing five-base codon CGGUA and the chemically aminoacylated tRNA with five-base anticodon UACCG were prepared, and added into E. coli in vitro translation system. As a result, the nonnatural amino acid was successfully incorporated into desired position of the protein. Other five-base codons CGGN1N2, where N1 and N2 indicate one of four nucleotides, were also available for the incorporation of the nonnatural amino acid.

Incorporation of two nonnatural amino acids into proteins through extension of the genetic code.

A novel method of the in vitro incorporation of two nonnatural amino acids into proteins through extension of the genetic code was developed. The streptavidin mRNA containing AGGU and CGGG, and chemically aminoacylated tRNA(ACCU) and tRNA(CCCG) were prepared, then they were added into E. coli in vitro protein synthesizing system. As a result, two nonnatural amino acids were successfully incorporated into desired sites of streptavidin.

Site-specific incorporation of photofunctional nonnatural amino acids into a polypeptide through in vitro protein biosynthesis.

Nonnatural amino acids with photofunctional groups were incorporated site-specifically into a polypeptide by using in vitro protein synthesizing system. The nonnatural amino acids were attached to tRNA(CCU) through chemical misacylation method, and added to the in vitro system with a mRNA containing a single AGG codon. L-p-Phenylazophenylalanine, L-2-anthrylalanine, L-1-naphthylalanine, L-2-naphthylalanine and L-p-biphenylalanine were successfully incorporated into a polypeptide, but l-1-pyrenylalanine was not. The polypeptides containing the nonnatural amino acids showed photofunctionalities.

Adaptability of nonnatural aromatic amino acids to the active center of the E. coli ribosomal A site.

3'-N-Aminoacyl analogs of puromycin with nonnatural aromatic amino acids were synthesized and their inhibitory activity in E. coli in vitro protein synthesizing system was evaluated. The analogs with L-2-naphthylalanine, L-p-biphenylalanine, L-2-anthrylalanine and trans-L-p-phenylazophenylalanine were found to inhibit the protein synthesis with high efficiency. The inhibition suggests that these nonnatural amino acids are accepted by the active center of the E. coli ribosomal A site. A model for the adaptability of nonnatural aromatic amino acids to the active center is proposed.