Quantification of the orientations of pyrrolidine-based oxypeptide nucleic acid–DNA hybrid duplexes

We describe the fluorescence quenching-based quantification of complementary parallel and antiparallel hybrids of DNAs with pyrrolidine-based oxypeptide nucleic acids (POPNAs). When BODIPY-modified DNAs as fluorescent probe formed hybrids with complementary POPNAs, fluorescence of the BODIPY was effectively quenched by the guanine unit and the Lys unit on the POPNAs. The orientations of hybrids of POPNA with DNA were estimated by the quenching efficiencies of two BODIPY-modified DNAs . As a result, we clarified that configurations of POPNAs affect the extent of orientation of the hybrid duplexes

In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system

Position-specific incorporation of non-natural amino acids into proteins is a useful technique in protein engineering. In this study, we established a novel selection system to obtain tRNAs that show high decoding activity, from a tRNA library in a cell-free translation system to improve the efficiency of incorporation of non-natural amino acids into proteins. In this system, a puromycin–tRNA conjugate, in which the 3′-terminal A unit was replaced by puromycin, was used. The puromycin–tRNA conjugate was fused to a C-terminus of streptavidin through the puromycin moiety in the ribosome. The streptavidin–puromycin–tRNA fusion molecule was collected and brought to the next round after amplification of the tRNA sequence. We applied this system to select efficient frameshift suppressor tRNAs from a tRNA library with a randomly mutated anticodon loop derived from yeast [Formula: see text]. After three rounds of the selection, we obtained novel frameshift suppressor tRNAs which had high decoding activity and good orthogonality against endogenous aminoacyl-tRNA synthetases. These results demonstrate that the in vitro selection system developed here is useful to obtain highly active tRNAs for the incorporation of non-natural amino acid from a tRNA library

Multiple incorporation of non-natural amino acids into a single protein using tRNAs with non-standard structures

AbstractThe ability to introduce non-natural amino acids into proteins opens up new vistas for the study of protein structure and function. This approach requires suppressor tRNAs that deliver the non-natural amino acid to a ribosome associated with an mRNA containing an expanded codon. The suppressor tRNAs must be absolutely protected from aminoacylation by any of the aminoacyl-tRNA synthetases in the protein synthesizing system, or a natural amino acid will be incorporated instead of the non-natural amino acid. Here, we found that some tRNAs with non-standard structures could work as efficient four-base suppressors fulfilling the above orthogonal conditions. Using these tRNAs, we successfully demonstrated incorporation of three different non-natural amino acids into a single protein

Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids

In vitro selection and directed evolution of peptides from mRNA display are powerful strategies to find novel peptide ligands that bind to target biomolecules. In this study, we expanded the mRNA display method to include multiple nonnatural amino acids by introducing three different four-base codons at a randomly selected single position on the mRNA. Another nonnatural amino acid may be introduced by suppressing an amber codon that may appear from a (NNK)(n) nucleotide sequence on the mRNA. The mRNA display was expressed in an Escherichia coli in vitro translation system in the presence of three types of tRNAs carrying different four-base anticodons and a tRNA carrying an amber anticodon, the tRNAs being chemically aminoacylated with different nonnatural amino acids. The complexity of the starting mRNA-displayed peptide library was estimated to be 1.1 × 10(12) molecules. The effectiveness of the four-base codon mediated mRNA display method was demonstrated in the selection of biocytin-containing peptides on streptavidin-coated beads. Moreover, a novel streptavidin-binding nonnatural peptide containing benzoylphenylalanine was obtained from the nonnatural peptide library. The nonnatural peptide library from the four-base codon mediated mRNA display provides much wider functional and structural diversity than conventional peptide libraries that are constituted from 20 naturally occurring amino acids

Carrier PNA for shRNA delivery into cells

A peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (carrier PNA) was used as 'bridgebuilder' 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

Cell Adhe sio n Inhibitio n by R GD Peptide s Linke d with a Photoiso meriza ble No nnatural Amino Acid

Abstract : RGD peptides linked with a nonnatural amino acid , phenylazophenyl alaninine (azoAla) , were synthesized and applied to cell adhesion inhibitors. The RGD peptides linked with azoAla at C 2terminal showed potent binding to the integrin on the surface of HeLa cells. Photoisomerization effect of the azobenzene side chain of synthesized peptides on the cell adhesion inhibition was further investigated. It was demonstrated that the cis 2form of azoAla2linked RGD peptides revealed a little weak cell adhesion inhibition effect as compared with trans2form of azoAla2linked RGD peptide

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. On irradiation at ∼405 nm at 125 mW cm(-2), DEACM-aa-tRNA is converted into active aa-tRNA with a half-life of 19 s. Notably, this rapid uncaging induced by visible light does not impair the translation system. Translation is photoinduced when DEACM-aa-tRNA carrying a CCCG or a CUA anticodon is uncaged in the presence of mRNAs harbouring a CGGG four-base codon or a UAG amber codon, respectively. Protein synthesis is phototriggered in several model systems, including an in vitro translation system, an agarose gel, in liposomes and in mammalian cells

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

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