Detection of L-DNA-tagged PCR products by surface plasmon resonance imaging.
Explore the source record for details and available documents.
Base Sequence↗
Biomedical subjects
Akio Kobori
Publications and source records attributed to Akio Kobori.
Evaluation of mismatch-binding ligands as inhibitors for Rev-RRE interaction.
Drugs targeting the stem-loop IIB of Rev responsible element (RRE) of HIV-1 mRNA are potential therapeutic agents for HIV-1 infection. The stem loop is characterized by an internal loop consist of consecutive G-G and G-A mismatches, which is the single binding site for Rev protein for nuclear export of viral mRNA. We report here that ligands binding to G-G and G-A mismatches in duplex DNA also bind to the internal loop in competition with Rev peptide and lead to the dissociation of pre-formed Rev-RRE complex in a model system.
Base Pair Mismatch↗
Synthesis and cross-linking activities of oligodeoxynucleotides containing a 4-oxoalkenal group.
The 4-oxoalkenal group, which has been characterized as part of a novel lipid-peroxidation-derived genotoxin, reacts with dG, dC, and dA, yielding etheno adducts. To develop a new interstrand cross-linking system based on the covalent bond formed between the 4-oxoalkenal group and DNA bases, we prepared 4-oxoalkenal-derivatized oligodeoxynucleotides. The protected 4-oxoalkenal derivative with a primary amino group was synthesized and incorporated to the 5' end of the oligodeoxynucleotide. In the presence of the complementary strand, cross-linking products were observed by HPLC and MALDI-MS analysis.
Chromatography, High Pressure Liquid↗
Solid-phase detection of RNA using bispyrene-modified RNA probe.
Bispyrene modified 2'-O-methyl oligoRNA (OMUpy2), which is a useful fluorescent probe for specific RNA detection, was immobilized on a glass substrate via various linker molecules to develop a more convenient RNA detection chip for gene expression study. As the fluorescence intensity of OMUpy2 enhanced greatly when it hybridized with its complementary RNA, it was expected that RNA detection by OMUpy2-immobilized chip (RNA-chip) required neither fluorescent labels to target nucleic acid nor washing protocol after the hybridization. As the linker molecules, oligo(dT), agarose, and poly(ethylene glycol) (PEG) were chosen, and the fluorescence from the chip was measured by a fluorescence microscope. Results indicated that the RNA-chip was able to detect the complementary oligoribonucleotide (cORN) without target labeling and washing protocols. Furthermore, the background emission was reduced as the length of the linker increased. In the case of PEG linker, 25-fold enhancement of the fluorescence intensity of the OMUpy2 was observed upon the addition of cORN.
Fluorescent Dyes↗
Properties of novel antisense oligonucleotides containing 2'-O-modified adenosine with a photo-reactive group.
In order to enhance the efficiency of antisense molecules for target RNA regulation, a novel photo-reactive antisense oligonucleotide was developed. We designed and synthesized a photo-reactive antisense oligonucleotide containing an adenosine in which 2'-OH was modified with 4,5',8-trimethylpsoralen (Ps) via an ethoxymethylene linkage (2'-Ps-eom). We evaluated the photo-cross-linking efficiency and sequence specificity toward complementary RNA (match-RNA). 2'-Ps-eom was selectively photo-cross-linked to the match-RNA. The photo-cross-linking efficiency was about 75% upon UVA-irradiation (365 nm) for 10 min. Previously, we reported oligonucleotides that had an adenosine anchoring Ps at 2'-O-position via a methylene linkage (2'-Ps-met). The photo-cross-linking efficiency of 2'-Ps-met and match-RNA was about 35% upon UVA-irradiation for 120 min. The photo-cross-linking efficiency of 2'-Ps-eom was dramatically enhanced in comparison with the one of 2'-Ps-met.
Adenosine↗
Various properties of polymeric carriers improved the transfection efficiency.
In order to develop novel efficient gene carriers, we have been focusing on the transcription of transgene in the nucleus among various steps in the gene transfer system. Optimal carrier properties for improving the transgene recognition by transcription factors have not been clarified so far. In the present study, we established a novel evaluation system for the intranuclear transcription efficiency of the transgene using microinjection technique. Polyplexes composed of polypeptides with different molecular weights were microinjected into the cytoplasm or nucleus of COS-1 cells, and the relationship between the carrier properties, such as molecular weight, and the intranuclear transcription efficiency was evaluated. The molecular weight (Mw) dependency of the transgene transcription in the living cells was successfully quantified, and the low Mw polymers were found not to suppress the transcription but high Mw polymers allowed almost no transcription. Interestingly, the transcription efficiencies of poly(arginine) (PR) and poly(lysine) (PK) were almost same although the PR is widely reported to be more efficient gene carrier than PK. The difference in the various properties and intracellular trafficking of PR/DNA and PK/DNA polyplexes will be discussed.
Animals↗
N,N'-Bis(3-aminopropyl)-2,7-diamino-1,8-naphthyridine stabilized a single pyrimidine bulge in duplex DNA.
We here show the first identified ligand 2,7-diamino-1,8-naphthyridine (DANP) that strongly and specifically binds to the single cytosine and thymine bulges with exclusively 1:1 stoichiometry.
DNA↗
Small-molecule ligand induces nucleotide flipping in (CAG)n trinucleotide repeats.
DNA trinucleotide repeats, particularly CXG, are common within the human genome. However, expansion of trinucleotide repeats is associated with a number of disorders, including Huntington disease, spinobulbar muscular atrophy and spinocerebellar ataxia. In these cases, the repeat length is known to correlate with decreased age of onset and disease severity. Repeat expansion of (CAG)n, (CTG)n and (CGG)n trinucleotides may be related to the increased stability of alternative DNA hairpin structures consisting of CXG-CXG triads with X-X mismatches. Small-molecule ligands that selectively bound to CAG repeats could provide an important probe for determining repeat length and an important tool for investigating the in vivo repeat extension mechanism. Here we report that napthyridine-azaquinolone (NA, 1) is a ligand for CAG repeats and can be used as a diagnostic tool for determining repeat length. We show by NMR spectroscopy that binding of NA to CAG repeats induces the extrusion of a cytidine nucleotide from the DNA helix.
DNA↗
A new ligand binding to G-G mismatch having improved thermal and alkaline stability.
Naphthyridine dimer (ND) specially binds to guanine-guanine (G-G) mismatch in duplex DNA. In order to improve the thermal and alkaline stability and binding ability of the ligand, we have examined structural modification of the linker. A new ligand (NNC) possessing 2-amino-1,8-naphthyridines and a carbamate linker is much more thermally stable than ND. The half-life of NNC is 2.5 times longer than that of ND at 80 degrees C. NNC is also much more stable than ND under alkaline conditions. In addition, NNC binds to G-G mismatch more strongly than ND. The improved stability and the binding of NNC to the G-G mismatch would be suitable for the practical use of NNC-immobilized sensor.
Base Pair Mismatch↗
Synthesis of antisense oligonucleotides containing a photocleavable protecting group on a guanine base and their photoinduced duplex formation.
An oligonucleotide containing a photocleavable protecting group at a guanine base was synthesized to induce the duplex formation by photo-irradiation. Alpha-methyl-2-nitropiperonyl (MeNP) group was used for the photocleavable protecting group at O6 position of deoxyguanosine. The oligonucleotide containing MeNP group (MeNP-ODN:5'-dTTCTG(MeNP)TCTGT-3') was synthesized by phosphoramidite method. The MeNP group was found to be removable by UV irradiation at wavelength of 365 nm for 5 min in 98% yield. UV-melting temperature (Tm value) analysis indicated that the duplex of MeNP-ODN with the complementary RNA was significantly unstable compared with the unmodified DNA/RNA duplex (deltaTm = -25 degrees C). After UV irradiation at 365 nm, the Tm value of the mixture increased to the same as that of the unmodified duplex. These results suggest that the RNA binding ability of the MeNP-ODN can be induced by photocleavage of the MeNP group.
Benzodioxoles↗
Synthesis and properties of photo-reactive antisense oligonucleotides containing 2'-O-psoralen-conjugated adenosine.
In order to selectively regulate mRNA having a point mutation, the photo-reactive antisense oligonucleotides were developed. Two types of photo-reactive oligonucleotides containing adenosine whose 2'-OH was modified with 4,5',8-trimethylpsoralen (psoralen) were synthesized (2'-Ps-oligo). One contains psoralen via a methylene linkage (2'-Ps-met), and the other via an amidomethylene linkage (2'-Ps-amd). 2'-Ps-oligos were then subjected to the photo-cross-linking reaction. 2'-Ps-met cross-linked to the complementary RNA and scarcely did to the RNA having a single mismatch base. Contrarily, 2'-Ps-amd did not cross-link to both RNA strands. These results suggest the structure of the linkage might affect the efficiency of the photo-cross-linking.
Adenosine↗
The destabilization of polyplexes facilitates intranuclear transcription efficiency.
We have been focusing on the last step of transfection, that is, the intranuclear recognition of polyplexes by transcription factors. In this work, we studied about the relationship between the intracellular destabilization of polyplexes and the transcription efficiency. Polypeptides containing specific sequences which are digested by intracellular proprotein convertase (PC), furin (Fur-polypeptide) were synthesized as novel carriers. When Fur-polypeptide/plasmid DNA polyplexes were incubated with furin in the cell-free transcription/translation system, the sequence specific expression was observed as time. Additionally, Fur-polypeptides/plasmid DNA polyplexes led to site-specific effective transgene expression even in COS-1 cells. These results indicate that shortened carriers lose their abilities to form polyplexes in response to furin activity in the cells, and it seems to enhance the destabilization of polyplexes, the recognition of transgene in polyplexes by transcription factors, and the expression efficiency.
Animals↗
Solution structure of a small-molecular ligand complexed with CAG trinucleotide repeat DNA.
NMR structure of the first identified ligand, naphthyridine-azaquinolone (NA), complexed with the CAG-CAG triad is reported. The determined structure revealed the invasive ligands binding to the A-A mismatch and flanking G-C base pairs, causing the widowed cytosines to flip out from pi-stack. Hydrogen-bond pairs between NA and DNA, naphthyridine-guanine and azaquinolone-adenine, are well stacked in the right-handed DNA helix, showing structural mimicry of Watson-Crick base pairing. This is the first observation that the small molecular ligand induced the base flipping of the nucleotide base in the Watson-Crick base pair.
Base Pairing↗
2-Ureidoquinoline: a useful molecular element for stabilizing single cytosine and thymine bulges.
We have demonstrated that aromatic heterocycles having hydrogen-bonding surfaces complementary to those of nucleotide bases are effective molecular elements for the binding to single nucleotide bulges and base mismatches. We here report that a new molecule, 2-ureidoquinoline having an alignment of hydrogen-bonding groups in the order of acceptor-donor-donor stabilizes single cytosine and thymine bulges in duplex DNAs. Furthermore, a dimeric form of 2-ureidoquinoline stabilizes cytosine-cytosine and cytosine-thymine mismatches.
Base Pair Mismatch↗
The binding of guanine-guanine mismatched DNA to naphthyridine dimer immobilized sensor surfaces: kinetic aspects.
Naphthyridine dimer composed of two naphthyridine chromophores and a linker connecting them strongly, and selectively, binds to the guanine-guanine mismatch in duplex DNA. The kinetics for the binding of the G-G mismatch to the naphthyridine dimer was investigated by surface plasmon resonance assay. The sensor surface was prepared by immobilizing naphthyridine dimer through a long poly(ethylene oxide) linker with the ligand density of 9.1 x 10(-12) fmolnm(-2). The kinetic analyses revealed that the binding of the G-G mismatch was sequence dependent on the flanking base pairs, and the G-G mismatches flanking at least one G-C base pair bound to the surface via a two-step process with a 1:1 DNA-ligand stoichiometry. The first association rate constant for the binding of the G-G mismatch in the 5'-CGG-3'/3'-GGC-5' sequence to the naphthyridine dimer-immobilized sensor surface was 3.2 x 10(3)M(-1)s(-1) and the first dissociation rate constant was 1.4 x 10(-2)s(-1). The association and dissociation rate constants for the second step were insensitive to the flanking sequences, and were almost of the same order of magnitude as the first dissociation rate constant. This indicates that the second step had only a small energetic contribution to the binding. The association constant calculated from kinetic parameters was 2.7 x 10(5)M(-1), which is significantly smaller than the apparent association constants obtained from experiments in solution. Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry on the complex produced from the G-G mismatch and naphthyridine dimer showed the formation of the 1:1 complex and a 1:2 DNA-ligand complex in solution. The latter complex became the dominant complex when a six-fold excess of naphthyridine dimer was added to DNA.
Base Pair Mismatch↗
Highly sensitive detection of GG mismatched DNA by surfaces immobilized naphthyridine dimer through poly(ethylene oxide) linkers.
Naphthyridine dimer is a unique molecule that strongly, and selectively, binds to the guanine-guanine mismatch in duplex DNA. We have synthesized naphthyridine dimers possessing a different length of poly(ethylene oxide) (PEO) linker, and immobilized them to CM5 sensor chip to carry out a surface plasmon resonance (SPR) assay of DNA duplexes containing a single base mismatch. The sensitivity of the sensor remarkably increased with increasing numbers of PEO units incorporated into the linker. With the sensor surface immobilized naphthyridine dimer for 1.5 x 10(3) response unit (RU) through three PEO units, the distinct SPR signal was observed at a concentration of 1 nM of the 27-mer G-G mismatch.
Base Pair Mismatch↗
The SPR sensor detecting cytosine[bond]cytosine mismatches.
We have synthesized the first surface plasmon resonance (SPR) sensor that detects cytosine-cytosine (C[bond]C) mismatches in duplex DNA by immobilizing aminonaphthyridine dimer on the gold surface. The ligand consisting of two 2-aminonaphthyridine chromophores and an alkyl linker connecting them strongly stabilized the C[bond]C mismatches regardless of the flanking sequences. The fully matched duplexes were not stabilized at all under the same conditions. The C[bond]T, C[bond]A, and T[bond]T mismatches were also stabilized with a reduced efficiency. SPR analyses of mismatch-containing 27-mer duplexes were performed with the sensor surface on which the aminonaphthyridine dimer was immobilized. The response for the C[bond]C mismatch in 5'-GCC-3'/3'-CCG-5' was about 83 times stronger than that obtained for the fully matched duplex. The sensor successfully detects the C[bond]C mismatch at the concentration of 10 nM. SPR responses are proportional to the concentration of the C[bond]C mismatch in a range up to 200 nM. Aminonaphthyridine dimer could bind strongly to the C[bond]C mismatches having 10 possible flanking sequences with association constants in the order of 10(6) M(-1). The facile protonation of 2-aminonaphthyridine chromophore at pH 7 producing the hydrogen-bonding surface complementary to that of cytosine was most likely due to the remarkably high selectivity of 1 to the C[bond]C mismatch.
Base Pair Mismatch↗
Detection of guanine-adenine mismatches by surface plasmon resonance sensor carrying naphthyridine-azaquinolone hybrid on the surface.
We have discovered a new molecule naphthyridine-azaquinolone hybrid (Npt-Azq) that strongly stabilized the guanine-adenine (G-A) mismatch in duplex DNA. In the presence of Npt-Azq, the melting temperature (T(m)) of 5'-d(CTA ACG GAA TG)-3'/3'-d(GAT TGA CTT AC)-5' containing a single G-A mismatch increased by 15.4 degrees C, whereas fully matched duplex increased its T(m) only by 2.2 degrees C. Npt-Azq was immobilized on the sensor surface for the surface plasmon resonance (SPR) assay to examine SPR detection of duplexes containing a G-A mismatch. Distinct SPR signals were observed when 27mer DNA containing a G-A mismatch was analyzed by the Npt-Azq immobilized sensor surfaces, whereas the signal of the fully matched duplex was approximately 6-fold weaker in intensity. The SPR signals for the G-A mismatch were proportional to the concentration of DNA in a range up to 1 microM, confirming that the SPR signal is in fact due to the binding of the G-A mismatch to Npt-Azq immobilized on the surface. Examination of all 16 G-A mismatches regarding the flanking sequence revealed that the sensor surface reported here is applicable to eight flanking sequences, covering 50% of all possible G-A mismatches.
Adenine↗