PubMed Health⌕ Search

Biomedical subjects

Shinsuke Sando

Publications and source records attributed to Shinsuke Sando.

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

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↗
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↗
PubMed2004

Aptamer selection for the inhibition of cell adhesion with fibronectin as target.

An affinity column immobilizing a decapeptide H(2)N-RGDSPASSKP-CO(2)H was used to select RGD-binding aptamers from a pool of 86-mer single-strand oligodeoxynucleotides (ODNs) containing a random 40-mer sequence. The enriched library thus obtained was further selected against adsorbed fibronectin and individual aptamers were monocloned in E. coli and sequenced to give a couple of highly homologous ODNs, which indeed inhibited fibronectin-integrin mediated cell adhesion.

Amino Acid Sequence↗
PubMed2004

A facile route to dynamic glycopeptide libraries based on disulfide-linked sugar-peptide coupling.

We report here that disulfide-linked dynamic glycopeptide libraries can be constructed from 1-thiosugar and cysteine-rich oligopeptide building blocks upon gentle air oxidation of a slightly basic (pH 7.8) aqueous solution thereof. A mixture of 1-thiogalactose and two oligopeptides H2N-CysGlyCysGly-CO2H and H2N-GlyCycCysGlyGly-CO2H, for example, affords a poorly HPLC-resolved disulfide library composed of various sugar-peptide conjugates and cyclic peptides, at least 10 of which can be identified by ESI mass spectrometry. The building components of disulfide members are exchangeable with each other in the presence of dithiothreitol as an initiator to allow dynamic equilibration. A preliminary SPR examination shows that the thiogalactose-derived library indeed contains active divalent galactoside species capable of cross-linking peanut lectin molecules.

Combinatorial Chemistry Techniques↗
PubMed2004

A quantum dot conjugated sugar ball and its cellular uptake. On the size effects of endocytosis in the subviral region.

The lipophilic CdSe quantum dot (QD) coated with trioctylphosphine oxide (TOPOQD) can be extracted from chloroform into water upon interaction with macrocyclic glycocluster amphiphile 1. The QD-conjugated and highly fluorescent sugar ball of a size of 15 nm (TOPOQD1) thus solubilized in water readily invades Hela cells via endocytosis. The endocytic activity of TOPOQD1 (15 nm), in light of those of the micellar homoaggregate of 1 (5 nm) and the virus-like 1-DNA conjugate (50 nm) as references, reveals a dramatic size effect (50 > 15 > 5) in the subviral size region. The optimal size at approximately 50 nm indicates that size complementarity which governs molecular recognition in small host-guest systems also plays key roles in the encapsulation of nanometric guest particles by the endocytic vesicles (</=100 nm) as a macrobiomolecular host. The work thus suggests an utmost importance of size control at the viral size when designing molecular (gene, drug, probe, etc.) delivery machines.

Endocytosis↗
PubMed2004

Macrocyclic proteoglycan mimics. Potent inhibition of cell adhesion by a bundle of chondroitin sulfate chains assembled on the calix[4]resorcarene platform.

Tailed calix[4]resorcarene macrocycle (tail=undecyl) can be used as a platform to assemble four glycosaminoglycan polysaccharide chains to give a new type of proteoglycan mimics. A tetra(chondroitin sulfate) derivative thus obtained from the reaction of macrocyclic octaamine and chondroitin sulfate lactone is readily immobilized on a tissue culture plastic (polystyrene) plate and inhibits fibronectin-mediated adhesion of BHK (baby hamster kidney) cells thereon remarkably strongly with 50% inhibition occurring at a 10 ng/mL or 40 pM concentration range.

Animals↗