ABSTRACT
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 â¼405 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.
Subject(s)
Coumarins/chemistry , Light , Protein Biosynthesis , RNA, Transfer, Amino Acyl/metabolism , Acylation , Animals , CHO Cells , Codon/genetics , Cricetinae , Cricetulus , Frameshift Mutation/genetics , Green Fluorescent Proteins/metabolism , Lasers , Liposomes/chemistry , Peptide Elongation Factor Tu/metabolism , Photolysis , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
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. This study showed that the caged aminoacyl-tRNA was not deacylated, did not bind to EF-Tu, and was activated by light. Photo-dependent translation of an mRNA containing the four-base codon was demonstrated using the caged aminoacyl-tRNA.
Subject(s)
Amino Acids/metabolism , RNA, Transfer, Amino Acyl/metabolism , Protein Biosynthesis , Protein EngineeringABSTRACT
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. The influence of arginine monomethylation in histone H3 on the acetylation of lysine residues by histone acetyltransferase hGCN5 was investigated, and the results demonstrated that K9 acetylation was suppressed by the methylation of R8 and R17 but not by R26 methylation. K18 acetylation was not affected by the methylation of R8, R17 and R26. This site-specific modification strategy provides a way to explore the roles of post-translational modifications in the absence of heterogeneity due to other modifications.
Subject(s)
Arginine-tRNA Ligase/chemistry , Arginine/analogs & derivatives , Protein Biosynthesis , Amino Acid Sequence , Aminoacylation , Arginine/genetics , Catalysis , Citrulline/chemistry , Citrulline/genetics , Homoarginine/chemistry , Homoarginine/genetics , Methylation , Molecular Sequence Data , omega-N-Methylarginine/chemistry , omega-N-Methylarginine/geneticsABSTRACT
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.
Subject(s)
Cysteine/chemistry , Peptides, Cyclic/chemical synthesis , Amino Acid Sequence , Cyclization , Cysteine/chemical synthesis , Molecular Sequence Data , Peptides, Cyclic/chemistryABSTRACT
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.
Subject(s)
Escherichia coli/drug effects , Oligonucleotides, Antisense/chemistry , Peptide Nucleic Acids/chemistry , Pyrrolidines/chemistry , Amino Acid Sequence , Oligonucleotides, Antisense/pharmacology , Peptide Nucleic Acids/chemical synthesis , Peptide Nucleic Acids/pharmacologyABSTRACT
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.
Subject(s)
Amino Acids/chemistry , ErbB Receptors/metabolism , Fluorescent Dyes/chemistry , Peptide Library , Peptides/metabolism , Amino Acid Sequence , Humans , Molecular Structure , Peptides/chemistry , Protein BindingABSTRACT
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.
Subject(s)
Peptide Elongation Factor Tu/genetics , RNA, Transfer, Amino Acyl/isolation & purification , Transfer RNA Aminoacylation/genetics , Peptide Elongation Factor Tu/metabolismABSTRACT
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.
Subject(s)
Caenorhabditis elegans/genetics , Lactobacillus/genetics , RNA Interference , Animals , Humans , Lactobacillus/metabolism , Nematoda/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Amino Acids/chemistry , Fluorescence , Fluorescent Dyes/chemistry , Peptides/chemistry , Proteins/chemistry , Binding Sites , Molecular Structure , Peptide LibraryABSTRACT
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). We designed a theophylline-aptamer-inserted RRE-RNA (Theo-RRE) to detect theophylline as a model target molecule. Theo-RRE formed an RNA-protein conjugate with EYFP-Rev-ECFP in the presence of theophylline. As a result, theophylline was specifically detected down to 10 microM by the FRET increase in distinction from theophylline analogue, caffeine, in cell lysates.
Subject(s)
Aptamers, Nucleotide/chemistry , Proteins/chemistry , RNA/chemistry , Theophylline/analysis , Fluorescence Resonance Energy Transfer , HeLa Cells , Humans , Molecular WeightABSTRACT
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. A nonnatural amino acid is once charged onto an E. coli tRNA(Phe) by a mutant ARS in situ, and successively transferred from the tRNA to a target protein, namely the NEXT-A reaction. Besides alphaA294G mutation on the ARS, alphaT251A, betaG318W, or betaA356W double-mutation were effective to increase the introduction efficiency through the NEXT-A reaction. Protein specific fluorescence labelling via the NEXT-A reaction followed by Huisgen cycloaddition was also demonstrated.
Subject(s)
Amino Acids/chemistry , Phenylalanine-tRNA Ligase/metabolism , Proteins/chemistry , Transferases/metabolism , Amino Acids/metabolism , Fluorescent Dyes/chemistry , Mutation , Phenylalanine-tRNA Ligase/genetics , Proteins/metabolism , RNA, Transfer, PheABSTRACT
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.
Subject(s)
Peptide Nucleic Acids/chemistry , RNA Interference , RNA, Double-Stranded/administration & dosage , Animals , Base Sequence , CHO Cells , Cricetinae , Cricetulus , Peptide Nucleic Acids/metabolism , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/metabolism , Transition TemperatureABSTRACT
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. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening.
Subject(s)
Fluorescent Dyes/chemistry , Peptide Elongation Factors/chemistry , Peptide Hydrolases/metabolism , Protease Inhibitors/analysis , Spectrometry, Fluorescence/methods , Caspase 3/metabolism , Caspase Inhibitors , Dipeptides/analysis , Dipeptides/metabolism , Fluorescent Dyes/analysis , Hydroxamic Acids/analysis , Hydroxamic Acids/metabolism , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase Inhibitors , Peptide Elongation Factors/analysis , Protease Inhibitors/metabolism , Substrate SpecificityABSTRACT
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.
Subject(s)
Fluorescent Dyes/chemistry , Gene Products, tat/chemistry , Photochemistry , RNA Interference , RNA, Small Interfering/administration & dosage , Ribonucleoprotein, U1 Small Nuclear/chemistry , Animals , CHO Cells , Cricetinae , Cricetulus , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Fluorescent Dyes/administration & dosage , Gene Products, tat/administration & dosage , Lasers , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/chemistry , Ribonucleoprotein, U1 Small Nuclear/administration & dosageABSTRACT
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.
Subject(s)
RNA/isolation & purification , Animals , Base Sequence , Biotinylation , Cattle , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Escherichia coli/chemistry , Escherichia coli/genetics , Humans , Methods , Molecular Sequence Data , Nucleic Acid Conformation , Peptide Nucleic Acids/chemistry , RNA/chemistry , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , RNA, Transfer, Leu/chemistry , RNA, Transfer, Leu/genetics , RNA, Transfer, Leu/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. We refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.
Subject(s)
Drug Delivery Systems , RNA Interference , RNA, Small Interfering/metabolism , AIDS Vaccines/chemistry , AIDS Vaccines/genetics , AIDS Vaccines/metabolism , Animals , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Electrophoretic Mobility Shift Assay , Endosomes/drug effects , Endosomes/metabolism , Endosomes/radiation effects , ErbB Receptors/drug effects , ErbB Receptors/genetics , ErbB Receptors/metabolism , Gene Silencing/drug effects , Green Fluorescent Proteins/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Photochemistry , RNA Interference/drug effects , RNA Interference/radiation effects , RNA, Small Interfering/chemistry , RNA, Small Interfering/pharmacokinetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
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. Moreover, redistributed RNA induced gene silencing only within the photo irradiated area.
Subject(s)
RNA Interference , RNA, Untranslated/metabolism , tat Gene Products, Human Immunodeficiency Virus/metabolism , Animals , CHO Cells , Cell Membrane Permeability , Cricetinae , Cricetulus , Endosomes/metabolism , Fluorescent Dyes , Microscopy, Fluorescence , Peptides/chemistry , Peptides/metabolism , Protein Structure, Tertiary , Protein Transport/radiation effects , Quinolinium Compounds , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U1 Small Nuclear/chemistry , Ribonucleoprotein, U1 Small Nuclear/metabolism , tat Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
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. casei; one produces shRNA which works in mammalian cells, and the other produces long dsRNA which works in nematode. We confirmed the expression of shRNA and dsRNA in the transformed L. casei by Northern blotting and RT-PCR analyses.
