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Biomedical subjects

Yasuhiro Aoyama

Publications and source records attributed to Yasuhiro Aoyama.

At least 19 recordsLinked to original sources
PubMed2006

In vitro selection of RNA aptamer against Escherichia coli release factor 1.

A pool of 84-nt RNAs containing a randomized sequence of 50 nt was selected against gel-immobilized Escherichia coli release factor 1 (RF-1) responsible for translation termination at amber (UAG) stop codon. The strongest aptamer (class II-1) obtained from 43 clones bound to RF-1, but not to UAA/UGA-targeting RF-2, with Kd = 30+/-6 nM (SPR). A couple of unpaired hairpin domains in the aptamer were suggested as the sites of attachment of RF-1. By binding to and hence inhibiting the action of RF-1 specifically or bio-orthogonally, aptamer class II-1 enhanced the amber suppression efficiency in the presence of an anticodon-adjusted (CUA) suppressor tRNA without practically damaging the protein translation machinery of the cell-free extract of E. coli, as confirmed by the translation of amber-mutated (gfp(amber141) or gfp(amber178)) and wild-type (gfp(wild)) genes of GFP.

Aptamers, Nucleotide↗
PubMed2006

Systematic lactose-functionalization of amphiphilic octaamine macrocycle as a gene carrier. Optimization of the charge, size, toxicity, and receptor factors for hepatocyte targeting.

We investigated the transfection properties of singly and triply lactose-functionalized derivatives, Lac(1) and Lac(3), and non-glycosylated one, Lac(0), of calix[4]resorcarene-based amphiphilic octaamine 1 in light of those previously reported for more extensively glycosylated compounds Lac(5) and Lac(8). They all strongly bind to the luciferase-encoding plasmid DNA pCMVluc (7040 base-pairs) with a saturation stoichiometry of Lac(n)/P approximately =0.5 (n=0, 1, or 3) or 0.7 (n=5 or 8), where P stands for a phosphate moiety of the plasmid DNA. The resulting carrier-DNA conjugates are positively charged and monomeric (monomolecular with respect to DNA) as such when n=0 or 1, neutral and monomeric when n=3, or neutral and aggregated when n=5 or 8. Transfection of HeLa (uterine) and HepG2 (hepatic) cell lines shows a general trend of decreasing luciferase expression efficiencies (E) in lively cells as well as cytotoxicities with increasing n's. The cell selectivities for HepG2 over HeLa sharply increase with increasing n's; E(HepG2)/E(HeLa)=0.3, 0.6, 7, 14, and 120 for Lac(0), Lac(1), Lac(3), Lac(5), and Lac(8), respectively, as a result of specific receptor pathway involving the asialoglycoprotein receptors on the hepatic (HepG2) cell surfaces and clustering lactose moieties of the carrier-DNA conjugates. The toxicity-corrected, overall efficiency of gene delivery to hepatocytes is optimized at Lac(3), which forms compactly packed (approximately 40 nm), charge-masked (xi approximately = 0 mV), and less toxic virus-like particles capable of receptor-mediated hepatocyte targeting.

Asialoglycoprotein Receptor↗
PubMed2006

Cerasome as an infusible, cell-friendly, and serum-compatible transfection agent in a viral size.

Alanine-based cationic lipid 1 having a (EtO)3SiCH2CH2CH2 group on the quaternized ammonium nitrogen forms a liposome which self-rigidifies via in situ sol-gel processes (Si-OEt + H2O --> Si-OH + EtOH followed by 2Si-OH --> Si-O-Si + H2O) on the surface. The resulting cerasome (partially ceramic- or silica-coated liposome) (60-70 nm) retains the integrity of such in the complexation with lucifarase-encoding plasmid DNA pGL3. The resultant pGL3 complex of infusible or monomeric cerasome in a viral size ( approximately 70 nm) exhibits a remarkable transfection performance toward HeLa and HepG2 cells with a 102-3-fold higher efficiency (relative to that of the nonsilylated reference lipid 2), minimized cytotoxicity, and serum compatibility. Reference lipid 2, i.e., alanine-based lipid having a simple quaternized ammonium headgroup, forms liposome (60-70 nm) which is less self-confined and more mobile undergoes DNA-induced fusion to give endocytosis-irrelevant and more toxic bigger (100-300 nm) particles. The silicon strategy thus provides a simple and widely applicable tool to overcome general problems associated with current technology of artificial gene delivery.

Cell Line, Tumor↗
PubMed2006

Efforts toward codon table engineering: expansion of unnatural substrates acceptable by the E. coli ribosome.

The ribosome catalyzes oligo/polymerization of amino acids. We designed an allowable modification of amino acid backbone, based on the hypothesized mechanism of peptidyl transfer reaction. Nonsense suppression method was used to investigate the acceptability of these substrates in the prokaryotic ribosomal system. The E.coli ribosome showed a restricted tolerance to the backbone modification, especially for main-chain elongation. However, our designed homologous beta-hydroxyalkanoic acid with elongated methylene (backbone) chain-length was revealed to be a possible substrate for the E.coli ribosome.

Codon↗
PubMed2006

Visible sensing of nucleic acid sequences using a genetically encodable unmodified mRNA probe.

We previously reported a molecular beacon-mRNA (MB-mRNA) strategy for nucleic acid detection/sensing in a cell-free translation system using unmodified RNA as a probe. Here in this presentation, we report that a combination with RNase H activity, which induces an additional process of irreversible cleavage of MB-domain, achieves an improved sequence selectivity (one nucleotide selectivity) and an enhanced sensitivity. This improved system finally enabled visible sensing of target nucleic acid sequence at a single nucleotide resolution under isothermal conditions.

Base Sequence↗
PubMed2006

Regulation of cancer-related growth factor expression by artificial zinc-finger proteins.

One attractive approach to anticancer therapy is repression of expression of vascular endothelial growth factor (VEGF) gene, which is a potent target for prevention of tumor growth. To achieve this, artificial transcription factors (ATF) designed for VEGF gene regulation were fused to cell-penetrating peptides (CPPs). We demonstrated ATFs fused to CPPs, designated CPP-ATFs or designed regulatory proteins (DRPs), could penetrate into mammalian cells and transiently repress expression of a reporter gene, which was under control of the VEGF promoter/5'-UTR. We discuss gene-regulatory properties of CPP-ATFs in detail.

Angiogenesis Inhibitors↗
PubMed2006

Application of artificial zinc-finger proteins to inhibition of DNA replication of human papillomavirus.

Recently, we have demonstrated that plant DNA virus replication could be inhibited in Arabidopsis thaliana by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic A. thaliana resistant to DNA virus infection. Here we apply the AZP technology to inhibition of replication of a mammalian DNA virus, human papillomavirus (HPV) type 18. Two AZPs, designated AZP(HPV)-1 and AZP(HPV)-2, were designed to block binding of the HPV-18 E2 replication protein to the replication origin. Both the designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited the viral DNA replication: AZP(HPV)-2, especially, reduced the replication level to approximately 10%. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses, but also to mammalian DNA viruses.

Binding Sites↗
PubMed2006

Inhibition of DNA replication of human papillomavirus by artificial zinc finger proteins.

Recently, we demonstrated that plant DNA virus replication was inhibited in planta by using an artificial zinc finger protein (AZP) and created AZP-based transgenic plants resistant to DNA virus infection. Here we apply the AZP technology to the inhibition of replication of a mammalian DNA virus, human papillomavirus type 18 (HPV-18). Two AZPs, designated AZP(HPV)-1 and AZP(HPV)-2, were designed by using our nondegenerate recognition code table and were constructed to block binding of the HPV-18 E2 replication protein to the replication origin. Both of the newly designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and they efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited viral DNA replication, especially AZP(HPV)-2, which reduced the replication level to approximately 10%. We also demonstrated in transient replication assays, using plasmids with mutant replication origins, that AZP(HPV)-2 could precisely recognize the replication origin in mammalian cells. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses but also to mammalian DNA viruses.

Cell Line↗
PubMed2005

A small-molecule-based approach to sense codon-templated natural-unnatural hybrid peptides. Selective silencing and reassignment of the sense codon by orthogonal reacylation stalling at the single-codon level.

In the presence of the stable sulfamoyl analogue of phenylalanyl adenylate (Phe-SA), the UUU/UUC sense codon for phenylalanine (Phe) can be silenced and reassigned to a naphthylalanine (Nap) conjugated to tRNAPhe. We have demonstrated the efficiency and selectivity or orthogonality of the Phe-to-Nap reassignment induced by an "orthogonal reacylation stalling" strategy at the single-codon level in the translation of mRNAs of dihydrofolate reductase and a 24-mer oligopeptide. We used a prokaryotic translation system with an essential preincubation, during which the endogenous precharged phenylalanyl-tRNAPhe undergoes deacylation and the reacylation of the resulting tRNAPhe is stalled by the action of Phe-SA to inhibit the phenylalanyl-tRNA synthetase activity. We discuss the significance of the present small-molecule-based approach to sense-codon templated natural-unnatural peptides.

Acylation↗
PubMed2005

Doubly catalytic sensing of HIV-1-related CCR5 sequence in prokaryotic cell-free translation system using riboregulator-controlled luciferase activity.

A molecular-beacon-type riboregulator (mRNA) was applied to multiply catalytic gene sensing. It consists of a reporter gene for firefly protein luciferase and, upstream thereof, a regulator hairpin domain composed of an RBS/anti-RBS stem (RBS = ribosome binding site) and a loop which is complementary to the target. The hairpin and, hence, the RBS are rendered open upon binding of a target oligonucleotide of the human CC chemokine receptor 5 sequence in a prokaryotic cell-free translation system (10 muL) to ignite ribosomal catalytic translation, or transcription/translation when using a DNA form of the probe, to produce luciferase, which is assayed by a catalytic chemiluminescence reaction. The sensing, using an unmodified RNA or even dsDNA as a probe with a chemiluminescence output, is thus doubly catalytic or amplifiable with a sensitivity at </=50 fmol in respect to the target with 4.5 fmol (1 ng/muL) of probe and a single nucleotide resolution.

Base Sequence↗
PubMed2005

Locked TASC probes for homogeneous sensing of nucleic acids and imaging of fixed E. coli cells.

We have designed a second-generation TASC (target-assisted self-cleavage) probe. It is based on the switching-on of incorporated cis-acting DNAzyme activity upon the target-induced conformational change of the otherwise inactive off-target probes locked in an intrastrand base-paired hairpin geometry. With E. coli 16S ribosomal RNA-relevant oligonucleotides as targets, the locked TASC probe exhibits an allosteric factor of k(on)/k(off) = 65 and the sequence selectivity is high, in terms of single nucleotide difference, when particular sequence and length of targets are chosen. Preliminary experiments with fixed E. coli cells show that the locked TASC probe with a FRET pair can be used to image fixed E. coli cells.

Allosteric Regulation↗
PubMed2005

A simple approach to sense codon-templated synthesis of natural/unnatural hybrid peptides.

In the presence of Phe-SA, the stable sulfamoyl analogue of phenylalanyl adenylate, the codon (UUU/UUC) for phenylalanine (Phe) can be reassigned to naphthylalanine (Nap) bound to tRNA(Phe). The efficiency and selectivity of this Phe-to-Nap reassignment induced by the "orthogonal reacylation stalling" method was demonstrated at the single-codon level in the translation of mRNAs of dihydrofolate reductase (DHFR) and a 24-mer oligopeptide. In the prokaryotic translation system with essential preincubation, the endogenous precharged phenylalanyl-tRNA(Phe) undergoes deacylation and reacylation of the resulting tRNA(Phe) is inhibited by the action of Phe-SA to kill the phenylalanyl-tRNA synthetase activity. The significance of the present small-molecule-based approach to sense-codon templated natural-unnatural peptides is discussed.

Adenosine↗
PubMed2005

In vitro read-through polysome/ribosome display of full-length protein ORF and it's applications.

Combination of nonsense suppression and protein-ribosome-mRNA (PRM) complexation techniques leads to a new strategy "read-through polysome/ribosome display", which is designed to display full-length open reading frame (ORF) domain of the protein on the natural mRNA templates. The optimised conditions are to use the anticodon-adjusted tRNA for Leu as a nonsense suppressor in a reconstituted translation system containing diminished amounts of release factors (RFs). When applied to pseudo-natural mRNAs of Escherichia coli dihydrofolate reductase (E. coli DHFR), the input mRNA was recovered as a polysome complex displaying full-length DHFR.

3' Untranslated Regions↗
PubMed2005

In vitro selection of RNA aptamers for the Escherichia coli release factor 1.

We carried out an in vitro selection of RNA aptamers that bind to Escherichia coli release factor 1 (E. coli RF1). The selected aptamer (class II) showed an apparent dissociation constant of nM range. The binding of the class II aptamer with E. coli RF1 is highly specific (orthogonal), allowing selective inhibition of RF1 activity in the E. coli translation system.

Aptamers, Nucleotide↗
PubMed2005

Highly sensitive genotyping using artificial riboregulator system.

A molecular-beacon-type riboregulator (mRNA) was applied to multiply catalytic gene sensing. It consists of a reporter gene for firefly protein luciferase and, upstream thereof, a regulator hairpin domain composed of an RBS/anti-RBS stem (RBS = ribosome binding site) and a loop which is complementary to the target. The sensing of target gene, using an unmodified RNA or even dsDNA as a probe with a chemiluminescence output, was demonstrated with a sensitivity at < or = 50 fmol of the target and a single nucleotide resolution.

Bacterial Proteins↗
PubMed2005

Ribosome-catalyzed synthesis of protein/oligopeptides with unnatural backbone.

Nonsense suppression method was used to probe the allowable modification of substrate (amino acid) backbone in the prokaryotic ribosomal system. Dihydrofolate reductase (DHFR) with an amber mutation was translated in the RF1-diminished prokaryotic cell free translation system in the presence of chemically-misacylated yeast tRNA(Phe)CUA. The prokaryotic ribosome showed a restricted tolerance to the backbone modification. Although natural-type alpha-amino acid was accepted as a good substrate for the ribosome, incorporation of beta-aminopropionic acid was not detected under our experimental conditions. Interestingly, we found that the homologous beta-hydroxyalkanoic acid with elongated methylene (backbone) chain-length can be a substrate for the ribosome, giving an important implication for the chemical mechanism of the ribosome-catalyzed peptide bond forming reaction.

Base Sequence↗