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.

Quantitative screening of EGF receptor-binding peptides by using a peptide library with multiple fluorescent amino acids as fluorescent tags.

EGF receptor-binding peptides could be found by a peptide screening method using fifteen fluorescent amino acids as fluorescent tags. Of 225 peptides, we found an 8-mer peptide containing a dipeptide unit, Y-F, which was the strongest binding peptide to the EGF receptor.

Use of EF-Tu mutants for determining and improving aminoacylation efficiency and for purifying aminoacyl tRNAs with non-natural amino acids.

We present three methods relating to tRNA aminoacylation with non-natural amino acids using an Escherichia coli EF-Tu E215A/D216A mutant that can bind tightly to aa-tRNAs carrying either non-natural or natural amino acids: (i) a method for improving aminoacylation efficiency, (ii) a rapid method for analysing aminoacylation efficiency without the use of radioisotope labelling and (iii) a method for purifying aminoacyl-tRNAs. Although the EF-Tu mutant may be incompatible with some kinds of non-natural amino acids, we confirmed that the EF-Tu mutant could efficiently bind to aa-tRNAs carrying various amino acids (Arg, Ser, O-methyltyrosine, Bodipy FL-aminophenylalanine and 2-acrydonylalanine). These methods may be used for the efficient in vitro synthesis of proteins containing various non-natural amino acids.

Lactobacillus-mediated RNA interference in nematode.

We engineered Lactobacillus paracasei to produce a dsRNA that would trigger RNAi-induced silencing of an essential gene in the nematode Caenorhabditis elegans. The dsRNA-expressing L. paracasei can be used in experiments conducted on culture plates and may also be used as an orally administrable dsRNA carrier for humans and other mammals.

A novel method for screening peptides that bind to proteins by using multiple fluorescent amino acids as fluorescent tags.

We describe a new screening method for simultaneously detecting peptides that bind to a target protein by fluorescence obtained from fluorescent amino acid-modified peptides.

Detection of bioactive small molecules by fluorescent resonance energy transfer (FRET) in RNA-protein conjugates.

Bioactive small molecules such as metabolites and drugs play important roles in regulating biological functions. A technique for visualizing such small molecules is very useful to understand their molecular mechanisms. In this study, an RNA-protein conjugate, which consists of an RRE-RNA sensor protein (EYFP-Rev-ECFP) and an altered RRE-RNA, was constructed to detect bioactive small molecules by fluorescent resonance energy transfer (FRET).

The NEXT-A (N-terminal EXtension with Transferase and ARS) reaction.

L/F-transferase is known to catalyze transfer of hydrophobic amino acids from aminoacyl tRNA to the N-terminus of a protein possessing lysine or arginine as the N-terminus. Combining L/F-transferase with E. coli phenylalanyl-tRNA synthetase (ARS), we achieved non-ribosomal N-terminal-specific introduction of various kinds of nonnatural amino acids to a protein.

Carrier PNA for shRNA delivery into cells.

A peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (carrier PNA) was used as 'bridge-builder' to connect a CPP with an shRNA. The carrier PNA successfully formed a hybrid with an shRNA bearing complementary dangling bases and the shRNA was introduced into cells by the carrier PNA, and RNAi was induced by the shRNA.

A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates.

We developed novel substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide solubility and increased the molecular weight, enabling sensitive detection by FCS.

Spatial regulation of specific gene expression through photoactivation of RNAi.

In this study we describe the spatial regulation of RNA interference (RNAi) using an RNA-carrier protein labeled with a fluorescent dye and a light source to trigger the RNAi. We demonstrate photo-dependent gene silencing using several dyes with different excitation wavelengths. Additionally, we use light from a halogen lamp and a photomask to produce photopatterned RNAi, and laser light to trigger single-cell RNAi on cell culture plates.

Isolation of small RNAs using biotinylated PNAs.

In this study, an RNA isolation method was developed using a biotinylated peptide nucleic acid (PNA) that is complementary to the target RNA. Using the biotinylated PNA method, we successfully isolated several RNAs from Escherichia coli and from human total RNA in pure form. Damage to the RNA appears to be negligible by this method because the method is rapid and does not require a high temperature treatment to facilitate RNA-PNA binding.

Cellular siRNA delivery mediated by a cell-permeant RNA-binding protein and photoinduced RNA interference.

HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the U1A RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5'-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay.

Delivery of dsRNA with lactic acid bacteria for RNA interference.

RNA interference (RNAi) mechanism is promising for gene-targeted therapy. In this study, Lactobacillus casei were engineered to produce long double-stranded RNA (dsRNA) or short hairpin RNA (shRNA) for RNAi-delivery to the intestine. We prepared two kinds of L.

Photo inducible RNA interference using cell permeable protein carrier.

In this study, we constructed fluorescently labelled protein carrier constructed from cell-penetrating peptide (CPP) and RNA binding domain (RBD) for intracellular shRNA or siRNA delivery. The protein carrier specifically bound to an RNA cargo containing short extended sequence tag for protein binding, and internalized into CHO cells together with the RNA. Although the internalized protein/RNA complexes showed cytoplasmic punctuate distributions, they widely spread into cytosol by photo irradiation.

Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system.

Nonnatural amino acids have been introduced into proteins using expanded protein biosynthesis systems. However, some nonnatural amino acids, especially those containing large aromatic groups, are not efficiently incorporated into proteins. Reduced binding efficiency of aminoacylated tRNAs to elongation factor Tu (EF-Tu) is likely to limit incorporation of large amino acids.

Design of carrier tRNAs and selection of four-base codons for efficient incorporation of various nonnatural amino acids into proteins in Spodoptera frugiperda 21 (Sf21) insect cell-free translation system.

Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system was expanded to include nonnatural amino acids. Orthogonal tRNAs that work as carriers of nonnatural amino acids in the insect system were explored. Four-base codons for assigning the positions of nonnatural amino acids were also selected.

Leucyl/phenylalanyl(L/F)-tRNA-protein transferase-mediated aminoacyl transfer of a nonnatural amino acid to the N-terminus of peptides and proteins and subsequent functionalization by bioorthogonal reactions.

We report here a new strategy for derivatizing peptides and proteins at the N-terminus. To achieve this, a nonnatural amino acid was charged onto a tRNA and then enzymatically transferred to a lysine (Lys) unit at the N-terminus of a peptide or a protein by using L/F-tRNA-protein transferase. By using the chemoenzymatic technique, beta-(2-quinolyl)-L-alanine, p-azido-L-phenylalanine, and p-acetyl-L-phenylalanine were introduced to the N-terminus.

Efficient incorporation of a nonnatural amino acid into a protein in an insect cell-free translation system.

Recently, we have succeeded in incorporating various nonnatural amino acids into proteins by using four-base codon-anticodon pairs in Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system. Here, the reaction was conducted under various conditions in order to optimize the incorporation efficiency. The optimal concentration of aminoacyl-tRNA, reaction temperature, and reaction time were 2 nM, 25 degrees C, and 1.

Synthesis of a newly peptide nucleic acid that contains tertiary amino groups.

A thymine monomer of pyrrolidine-based peptide nucleic acid that contains a tertiary amino group in the backbone has been synthesized for use in the synthesis of novel peptide nucleic acid.