Phototriggered protein syntheses by using (7-diethylaminocoumarin-4-yl)methoxycarbonyl-caged aminoacyl tRNAs.

The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. Here we report caged aminoacyl-tRNAs (aa-tRNAs) synthesized using a (7-diethylaminocoumarin-4-yl)methoxycarbonyl (DEACM)-cage compound. DEACM-caged aa-tRNA does not spontaneously deacylate for at least 4 h in neutral aqueous solution, and does not bind to the elongation factor Tu.

Photo-dependent protein biosynthesis using a caged aminoacyl-tRNA.

Translation systems with four-base codons provide a powerful strategy for protein engineering and protein studies because they enable site-specific incorporation of non-natural amino acids into proteins. In this study, a caged aminoacyl-tRNA with a four-base anticodon was synthesized. The caged aminoacyl-tRNA contains a photocleavable nitroveratryloxycarbonyl (NVOC) group.

Site-specific incorporation of arginine analogs into proteins using arginyl-tRNA synthetase.

Arginine analogs were incorporated site-specifically into proteins using an in vitro translation system. In this system, mRNAs containing a CGGG codon were translated by an aminoacyl-tRNA(CCCG), which was charged with arginine analogs using yeast arginyl-tRNA synthetase. N(G)-monomethyl-L-arginine, L-citrulline and L-homoarginine were incorporated successfully into proteins using this method.

Synthesis of a cyclic peptide/protein using the NEXT-A reaction followed by cyclization.

By using the NEXT-A reaction, we introduced a non-natural amino acid at the N-terminus of a peptide/protein that contained a cysteine unit. The side chain of the introduced amino acid spontaneously reacted with the cysteine to afford a cyclic peptide/protein.

Antisense effect of pyrrolidine-based oxy-peptide nucleic acids in Escherichia coli.

To investigate the antisense effect of a pyrrolidine-based oxy-peptide nucleic acid (POPNA), we carried out the LacZ reporter assay using a 12-mer trans-l-POPNA conjugated with a cell-penetrating peptide (antisense reagent). The antisense effect of the conjugated POPNA (inhibition of LacZ activity) was comparable to that shown by a Nielsen-type peptide nucleic acid. Furthermore, the conjugated POPNA could switch the LacZ activity over a wide range of ambient temperatures.