Biomedical subjects

Shinsuke Sando

Publications and source records attributed to Shinsuke Sando.

30 records · Page 2Linked to original sources

PubMed2004

Quenched auto-ligating DNAs: multicolor identification of nucleic acids at single nucleotide resolution.

We describe the synthesis and study of multicolor quenched autoligating (QUAL) probes for identification and discrimination of closely related RNA and DNA sequences in solution and in bacteria. In these probes, a dabsyl quencher doubles as an activator in the oligonucleotide-joining reaction. The oligonucleotides remain dark until they bind at adjacent sites, and "light up" on nucleophilic displacement of the dabsyl probe by the phosphorothioate probe. Four fluorescent dye conjugates were prepared and tested with probes and targets that differ by one nucleotide. Experiments on polymer beads show clear color-based discrimination of DNAs added in solution. Two-color quenched probe pairs were then tested in the discrimination of 16S rRNA sequences in Escherichia coli. Single nucleotide resolution was achieved in the cells with green/red QUAL probes, allowing identification of a one-base sequencing error in the 16S rRNA database. Finally, QUAL probes were successfully applied in live bacterial cells. The method requires only incubation followed by fluorescence imaging, and requires no enzymes, added reagents, cross-linking, fixing, or washes. Because probes must bind side-by-side to generate signal, there is little or no interference from unintended protein binding, which can occur with other probe types. The results suggest that QUAL probes may be of general use in the detection and identification of sequences in solution, on microarrays, and in microorganisms.

DNA↗

PubMed2003

Amplified nucleic acid sensing using programmed self-cleaving DNAzyme.

A newly designed target-assisted self-cleavage (TASC) probe composed of a target-binding site and a DNAzyme domain undergoes TASC when activated via hybridization with a target DNA/RNA. This self-splicing or self-dissociation reaction occurs in a catalytic manner with the probe as a substrate and the target as a catalyst, since the fragmented products are automatically released from the target, thus amplifying the sequence information of the latter under non-PCR, i.e., isothermal and enzyme/reagent-free, conditions. A fluorescence-reporting TASC probe having a fluorescein/dabsyl FRET pair across the cleavage site allows a mix-and-read discrimination of single-nucleotide differences in the target.

Biosensing Techniques↗

PubMed2003

Facile preparation of DNA-tagged carbohydrates.

We report here that unprotected carbohydrates (maltose, lactose, cellobiose, and maltoheptaose) can be attached to the aminoalkylated oligonucleotides under mild reductive-amination conditions (aqueous borate buffer, pH 8.0, NaBH(3)CN, 60 degrees C) without notable side reactions. Quadruplex-forming G-rich oligonucleotide, 5'-aminoalkyl d(TGGGGT), is glycosylated with maltoheptaose to afford a novel DNA-assisted tetrasaccharide cluster motif.

Alkylation↗

PubMed2003

Remarkably size-regulated cell invasion by artificial viruses. Saccharide-dependent self-aggregation of glycoviruses and its consequences in glycoviral gene delivery.

We here report a novel example of artificial glycoviral vectors constructed via number- and size-controlled gene (pCMVluc, 7040 bp) coating with micellar glycocluster nanoparticles (GNPs) of calix[4]resorcarene-based macrocyclic glycocluster amphiphiles having eight or five saccharide moieties with terminal alpha-glucose (alpha-Glc), beta-glucose (beta-Glc), or beta-galactose (beta-Gal) residues. The resulting glycoviruses are compactly packed (approximately 50 nm) and well charge-shielded (zeta approximately equal 0 mV), undergo saccharide-dependent (alpha-Glc > beta-Gal >> beta-Glc) self-aggregation, and transfect cell (Hela and HepG2) cultures as triggered by the pinocytic form of endocytosis. The semilogarithmic linear size-activity correlation suggests that size-restricted pinocytosis (<100 nm) is effective only for monomeric viruses. The activities of oligomeric and otherwise poorly active beta-Gal-functionalized viruses toward hepatic HepG2 cells are approximately 10(2)-times higher than expected on the size basis, owing to the receptor-mediated specific pathway involving the asialoglycoprotein receptors on the hepatic cell surfaces. The scope and prospect of artificial glycoviruses are discussed.

Calixarenes↗

PubMed2003

Artificial viruses and their application to gene delivery. Size-controlled gene coating with glycocluster nanoparticles.

Number- and size-controlled macromolecular associations are common in biology with viruses as a typical example. We report here a novel example of artificial viruses, in which the double-helical DNA is coated with 4-nm sized neutral glycocluster nanoparticles (GNPs) with a coating stoichiometry of approximately 2 GNPs per helical pitch (10 base pairs), where GNP arises from micellization of a cone-shaped, quadruple-chain glycocluster amphiphile having eight saccharide moieties with beta-glucoside termini on the calix[4]resorcarene macrocycle. The resulting "glycoviruses" are compactly packed (54 nm in the case of 7040 base-pair plasmid pCMVluc), are well charge-shielded (zeta congruent with approximately 0 mV), and effectively transfect cell cultures without notable cytotoxicity. The use of artificial viral vectors thus allows a new (nonamine/noncationic/nonpolymeric) access to gene delivery, a potential but still tough subject which has been studied extensively over the last 15 years by using viral or amine-based cationic vectors. The remarkable adhesion-manipulation ability of saccharide clusters also provides a strategy of bottom-up construction of nanometric or mesoscopic sizes.

DNA↗

PubMed2003

Induction of a remarkable conformational change in a human telomeric sequence by the binding of naphthyridine dimer: inhibition of the elongation of a telomeric repeat by telomerase.

The binding of a dimeric form of the 2-amino-1,8-naphthyridine derivative (naphthyridine dimer) to a human telomeric sequence, TTAGGG, was investigated by UV melting, CD spectra, and CSI-MS measurements. Both the 9-mer d(TTAGGGTTA) and the 15-mer d(TTAGGGTTAGGGTTA) showed apparent melting temperatures (T(m)) of 45.6 and 63.6 degrees C, respectively, in the presence of naphthyridine dimer (30 microM) in sodium cacodylate buffer (50 mM, pH 7.0) containing 100 mM NaCl. The CD spectra at 235 and 255 nm of the 9-mer increased in intensity accompanied with strong induced CDs at 285 and 340 nm upon complex formation with naphthyridine dimer. UV titration of the binding of naphthyridine dimer to the 9-mer at 320 nm showed a hypochromism of the spectra. A Scatchard plot of the data showed the presence of multiple binding sites with different association constants. Cold spray ionization mass spectrometry of the complex between naphthyridine dimer and the 9-mer clearly showed that one to three molecules of the ligand bound to the dimer duplex of the 9-mer. Telomeric repeat elongation assay showed that the binding of naphthyridine dimer to the telomeric sequence inhibits the elongation of the sequence by telomerase.

Base Pair Mismatch↗

PubMed2003

Macrocyclic glycoclusters. Self-aggregation and phosphate-induced agglutination behaviors of calix[4]resorcarene-based quadruple-chain amphiphiles with a huge oligosaccharide pool.

Macrocyclic glycocluster compounds 2n (n = 2-7) with four alkyl (undecyl) chains and eight oligosaccharide moieties on the opposite sides of the calix[4]resorcarene macrocycle are prepared from the reactions of the corresponding octaamine derivative with maltooligosaccharide lactones. Combined evidence from dynamic light scattering (DLS), gel permeation chromatography (GPC), and transmission electron microscopy (TEM) indicates that they form small micelle-like nanoparticles (d congruent with 3 nm) in water. In the presence of Na2HPO4/NaH2PO4, nanoparticles are agglutinated with phosphate ions as a glue to grow in size up to 60-100 nm, as revealed by DLS as well as microscopy (TEM and AFM). The phosphate-induced agglutination processes can be followed by surface plasmon resonance (SPR). Amphiphile 2n is readily immobilized on the hydrophobized sensor chip of SPR to give a closely packed monolayer with oligosaccharide moieties exposed to bulk water. While there is no further adsorption of 2n on the resulting monolayer, this does occur when the latter is pretreated with the phosphate salts, ultimately giving rise to a multilayer upon repeated treatment of the chip with 2n and Na2HPO4/NaH2PO4 in an alternate manner. Kinetic analyses show that the phosphate-mediated inter(saccharide) interactions in terms of rate and affinity are markedly dependent on the oligosaccharide chain lengths (n), becoming more favorable with increasing n's. The novel aggregation and agglutination behaviors observed are discussed in terms of immobilizable and irreversible micelles on the basis of the cone-shaped structure of quadruple-chain amphiphile 2n having a huge saccharide pool and the efficiency of multiple hydrogen bonding therein. The unique intermolecular binding properties of compound 22 and analogues so far reported are reviewed in light of the present finding.

Bridged-Ring Compounds↗

PubMed2003

Encapsulation of DNA with neutral glycocluster nanoparticles. A step toward artificial viruses.

Number and size-controlled macromolecular associations are common in biology with viruses as a typical example. We report here a novel example of artificial viruses, in which the double-helical DNA is coated with 4-nm sized neutral glycocluster nanoparticles (GNPs). The resulting "glycoviruses" are compactly-packed, well charge-shielded (zeta approximately = 0 mV), and effectively transfect cell cultures without notable cytotoxicity.

DNA↗

PubMed2002

Imaging of RNA in bacteria with self-ligating quenched probes.

We report on the application of a new class of oligonucleotide reporter probes, QUAL probes, that "light up" when a nucleophilic phosphorothioate probe binds adjacent to a dabsyl-quenched probe. These self-ligating DNA probes were used for sequence-specific detection of 16S rRNA in Escherichia coli cells. Strong fluorescence was observed only when the phosphorothioate and quenched dabsyl probes bind side-by-side on a 16S rRNA target. The results demonstrate the use of QUAL probes to detect specific RNA sequences in bacterial cells without enzymes and without washing steps.

DNA Probes↗

PubMed2002

Quencher as leaving group: efficient detection of DNA-joining reactions.

We describe a new fluorescence reporting strategy in which dabsyl, a well-known quencher, activates a hydroxyl group in a probe to convert it to a leaving group. When a nucleophilic phosphorothioate probe binds adjacent to a dabsyl quenched probe, autoligation occurs, releasing the quencher, and lighting up the probes, This signal change can be used to detect single nucleotide differences in DNA without enzymes or reagents.

Base Sequence↗

PubMed2002

Nonenzymatic DNA ligation in Escherichia coli cells.

We report on a new fluorescence reporting strategy in which dabsyl, a well-known quencher, activates a hydroxyl group in a probe to convert it to a leaving group. When a nucleophilic phosphorothioate probe binds adjacent to a dabsyl quenched probe, autoligation occurs, releasing the quencher, and lighting up the probes. These self-ligating DNA probes were used for sequence-specific detection of 16S rRNA in E. coli cells. Strong fluorescence was observed only when the phosphorothioate and quenched dabsyl probes bind side-by-side on a 16S rRNA target. The results demonstrate the use of QUAL probes to detect specific RNA sequences in bacterial cells without enzymes and without washing steps.

DNA Probes↗

PubMedDate not supplied

RNAi silencing of exogenous and endogenous reporter genes using a macrocyclic octaamine as a "compact" siRNA carrier. Studies on the nonsilenced residual activity.

A macrocyclic octaamine 1 having a covalently linked lipid-bundle structure was introduced as a new type of siRNA carrier. Gel electrophoresis, DLS, and SPR results indicate that it strongly binds to a luciferase-targeting 21-mer (42P) siRNA with a ratio of 1/P congruent with 0.3 (1/N congruent with 2.4) to give remarkably compact 1-siRNA complexes with an average size of approximately 10 nm. The 1-mediated siRNA silencing of the exogenous luciferase gene occurs with a 90-95% efficiency. The overall suppression-[siRNA] profile with a 5-10% residual activity in the saturation region is commonly observed irrespective of the cell type (HeLa, HepG2, or HEK293), the order, or timing (stepwise or simultaneous) of supply of the siRNA and that of the luciferase-encoding plasmid, the level of mRNA transcribed, or the type of carriers (1 vs lipofectamine 2000). The silencing of the endogenous DsRed2 gene stably incorporated in the genome of HeLa cells also has a similar overall profile. These results suggest that (1) the cellular uptake of the plasmid and that of the siRNA are basically independent of each other and (2) the incomplete silencing is not due to insufficient siRNA delivery. Implication of item 2 is briefly discussed.

Cell Line↗