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Naoki Sugimoto

Publications and source records attributed to Naoki Sugimoto.

At least 19 recordsLinked to original sources
PubMed2006

Influence of cationic molecules on the hairpin to duplex equilibria of self-complementary DNA and RNA oligonucleotides.

A self-complementary nucleotide sequence can form both a unimolecular hairpin and a bimolecular duplex. In this study, the secondary structures of the self-complementary DNA and RNA oligonucleotides with different sequences and lengths were investigated under various solution conditions by gel electrophoresis, circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy and a ultraviolet (UV) melting analysis. The DNA sequences tended to adopt a hairpin conformation at low cation concentrations, but a bimolecular duplex was preferentially formed at an elevated cationic strength. On the other hand, fully matched RNA sequences adopted a bimolecular duplex regardless of the cation concentration. The thermal melting experiments indicated a greater change in the melting temperature of the bimolecular duplexes (by approximately 20 degrees C) than that of the hairpin (by approximately 10 degrees C) by increasing the NaCl concentration from 10 mM to 1 M. Hairpin formations were also observed for the palindrome DNA sequences derived from Escherichia coli, but association of the complementary palindrome sequences was observed when spermine, one of the major cationic molecules in a cell, existed at the physiological concentration. The results indicate the role of cations for shifting the structural equilibrium toward a nucleotide assembly and implicate nucleotide structures in cells.

Cations↗
PubMed2006

Characterization of structure and stability of long telomeric DNA G-quadruplexes.

In the current study, we used a combination of gel electrophoresis, circular dichroism, and UV melting analysis to investigate the structure and stability of G-quadruplexes formed by long telomeric DNAs from Oxytricha and human, where the length of the repeat (n)=4 to 12. We found that the Oxytricha telomeric DNAs, which have the sequence (TTTTGGGG)n, folded into intramolecular and intermolecular G-quadruplexes depending on the ionic conditions, whereas human telomeric DNAs, which have the sequence (TTAGGG)n, formed only intramolecular G-quadruplexes in all the tested conditions. We further estimated the thermodynamic parameters of the intramolecular G-quadruplex. We found that thermodynamic stabilities of G-quadruplex structures of long telomeric DNAs (n=5 to 12) are mostly independent of sequence length, although telomeric DNAs are more stable when n=4 than when n>or=5. Most importantly, when n is a multiple of four, the change in enthalpy and entropy for G-quadruplex formation increased gradually, demonstrating that the individual G-quadruplex units are composed of four repeats and that the individual units do not interact. Therefore, we propose that the G-quadruplexes formed by long telomeric DNAs (n>or=8) are bead-on-a-string structures in which the G-quadruplex units are connected by one TTTT (Oxytricha) or TTA (human) linker. These results should be useful for understanding the structure and function of telomeres and for developing improved therapeutic agents targeting telomeric DNAs.

Animals↗
PubMed2006

Hydration regulates thermodynamics of G-quadruplex formation under molecular crowding conditions.

The effect of molecular crowding on the structure and stability of biomolecules has become a subject of increasing interest because it can clarify how biomolecules behave under cell-mimicking conditions. Here, we quantitatively analyzed the effects of molecular crowding on the thermodynamics of antiparallel G-quadruplex formation via Hoogsteen base pairs and of antiparallel hairpin-looped duplex (HP duplex) formation via Watson-Crick base pairs. The free energy change at 25 degrees C for G-quadruplex formation decreased from -3.5 to -5.5 kcal mol(-1) when the concentration of poly(ethylene glycol) 200 was increased from 0 to 40 wt %, whereas that of duplex formation increased from -9.8 to -6.9 kcal mol(-1). These results showed that the antiparallel G-quadruplex is stabilized under molecular crowding conditions, but that the HP duplex is destabilized. Moreover, plots of stability (ln K(obs)) of the DNA structures versus water activity (ln a(w)) demonstrated that the ln K(obs) for G-quadruplex formation decreased linearly as the ln a(w) increased, whereas that for duplex formation increased linearly with the increase in ln a(w), suggesting that the slope approximately equals the number of water molecules released or taken up during the formation of these structures. Thus, molecular crowding affects the thermodynamics of DNA structure formation by altering the hydration of the DNA. The stabilization of the DNA structures with Hoogsteen base pairs and destabilization of DNA structures with Watson-Crick base pairs under molecular crowding conditions lead to structural polymorphism of DNA sequences regulated by the state of hydration.

Base Pairing↗
PubMed2006

Effect of molecular crowding on DNA polymerase activity.

Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo; however, the molecular conditions within live cells are inherently crowded. The present study investigates the effect of molecular crowding on DNA polymerase activity using polyethylene glycol PEG of various molecular weights as a crowding agent. Polymerase activity assays under various conditions demonstrated that the activities of T7 and Taq DNA polymerases depend on the molecular weight and concentration of the crowding agent. Furthermore, equilibrium and kinetic analyses demonstrated that the binding affinity and catalytic activity of the polymerase increase and decrease, respectively, with increasing PEG concentrations. Based on quantitative parameters of the polymerase reactions, we improved the efficiency of PCR amplification under conditions of molecular crowding. These results suggest that quantitative measurements of biomolecular structure and function are useful for understanding the behavior of biomolecules in vivo and for biotechnology applications in vitro.

Colloids↗
PubMed2006

A DNA duplex with extremely enhanced thermal stability based on controlled immobilization on gold nanoparticles.

The effect of DNA loadings on the thermal stability of DNA duplex immobilized on gold nanoparticles has been investigated. The modestly loaded duplexes on the gold nanoparticles showed enhanced thermal stability, as compared to that of the free duplex (without gold nanoparticles). However, the highly loaded duplex showed stability similar to that of free duplex. The stability could be controlled over a wide temperature range simply by varying the salt concentration (over 50 degrees C). Additionally, the gold nanoparticles with modestly loaded oligonucleotides could be used as nanoprobes for effective and fast strand exchange reactions, based on the increased thermal stability of the immobilized duplex. These results indicate that the interaction between the duplex and the nanoparticle surface plays an important role in determining the stability of the duplex.

Base Sequence↗
PubMed2006

Application of liquid chromatography-nuclear magnetic resonance spectroscopy for the identification of ethyldimethylpyrazine, a food flavouring agent.

The application of liquid chromatography-nuclear magnetic resonance spectroscopy (LC-NMR) for the direct identification of ethyldimethylpyrazine, a food flavouring agent, has been studied. The commercial product is a mixture of two regio-isomers, 2-ethyl-3,5-dimethylpyrazine (1) and 2-ethyl-3,6-dimethylpyrazine (2); however, the exact composition of the mixture is unknown. Structural characterization by LC-MS and GC-MS was not possible because both regio-isomers yield the same molecular related ion and ion fragmentation. To rapidly identify the two regio-isomers, the product was analyzed by LC-NMR with on-flow and fraction loop modes. From the results, the structure elucidations of the two regio-isomers could be carried out without the need to isolate the isomers by the usual procedures.

Chromatography, Liquid↗
PubMed2006

A novel stable RNA pentaloop that interacts specifically with a motif peptide of lambda-N protein.

To achieve a novel specific peptide-nucleic acid binding model, we designed an in vitro selection procedure to decrease the energetic contribution of the electrostatic interaction in the total binding energy and to increase the contribution of hydrogen bonding and pi-pi stacking. After the selection of hairpin-loop RNAs that specifically bound to a model peptide of lambda N protein (N peptide), a new thermostable pentaloop RNA motif (N binding thermostable RNA hairpin: NTS RNA) was revealed. The obtained NTS RNA was able to bind to the N peptide with superior specificity to the boxB RNA, which is the naturally occurring partner of the lambda N protein.

Amino Acid Motifs↗
PubMed2006

The roles of cosolutes on the hammerhead ribozyme activity.

The hammerhead ribozyme is often used for gene regulations in a cell. One of the major differences between in vitro and in cell conditions is the molecular crowding. However, the influence of crowding conditions on the ribozyme activity is still unclear. Here, we investigated the activity of the hammerhead ribozyme under molecular crowding condition by ethylene glycol (EG), poly (ethylene glycol) (PEG), dextran, and Ficoll. These cosolutes enhanced the hammerhead ribozyme activity by 2 to approximately 7-times, and larger-sized PEGs exhibited the greater activity. More importantly, the rate constant at 1 mM Mg(2+) and 37 degrees C increased about 1000-times and 500-times upon the addition of 20 wt% PEG8000 and PEG200, respectively. Additionally, the ribozyme still retained the cleavage activity even at 50 degrees C in the presence of PEG8000. Our results suggest that the ribozyme activity can be enhanced by the addition of the crowding reagent even at a low magnesium ion concentration and even at a high temperature.

Dextrans↗
PubMed2006

Recognition of the base pair-mimic nucleosides by DNA polymerases.

We previously reported that the deoxyadenosine derivative tethering the phenyl group at the N6 position of deoxyadenosine, A(phe) stacked efficiently with the adjacent nucleotide bases at a DNA duplex terminus and in the middle of a DNA duplex. In contrast with the observations for A(phe), this study revealed that the phenyl group of the deoxycytidine derivative, C(phe) located outside the helix and allowed the base pair formation with guanine in a DNA duplex, although its phenyl group could stack with the adjacent DNA bases as efficient as A(phe). Klenow fragment of DNA polymerase I and T7 DNA polymerase selectively incorporated dTTP and dGTP opposite A(phe) and C(phe) in the template DNA, respectively, implying that the conformation of A(phe) differs between in the DNA polymerase and in solution.

Adenosine↗
PubMed2006

Factors regulating thermodynamic stability of DNA structures under molecular crowding conditions.

The condition in a living cell is molecularly crowded with various biomolecules. The total concentration of the biomolecules inside Escherichia coli is in the range of 300-400 g/L. This is distinct from typical biomolecular concentrations of less than 1g/L, which is generally used for experiments in vitro. Here, we analyzed quantitatively the effects of molecular crowding on the thermodynamics of antiparallel G-quadruplex formation via Hoogsteen base pairs and of antiparallel hairpin-looped duplex (HP duplex) formation via Watson-Crick base pairs. The free energy changes for G-quadruplex and duplex formations decreased and increased when the concentration of poly(ethylene glycol) 200 was increased from 0 to 40 wt%, respectively. These results showed that the antiparallel G-quadruplex is stabilized under molecular crowding conditions but the HP duplex is destabilized.

Aptamers, Nucleotide↗
PubMed2006

The effect of the structure of cosolutes on the DNA duplex formation.

A living cell generally contains macromolecules occupying 20 approximately 40% of the total volume. In order to mimicking the crowded cellular environment, we chose different structure molecules, glycerol, ethylene glycol (EG) and poly(ethylene glycol) (PEG), as cosolutes. The thermodynamics and kinetics of DNA duplex formation in the presence of high concentration of glycerol, EG and PEG were investigated to discern nucleic acid behavior under molecular crowding condition. Comparing with the data obtained in the dilute solution, the melting temperature (T(m)) of a 10-mer DNA duplex (5'-TAGGTTATAA-3'/5'-TTATAACCTA-3') decreased by 4.8 degrees C, 5.6 degrees C, or 7.0 degrees C in the presence of 20 wt% glycerol, EG, or PEG200, respectively. The kinetic results revealed that the destabilization was not only caused by decreasing the association rate constant but also caused by great increasing the dissociation rate constant. These results are useful for understanding nucleic acid behavior in cell.

DNA↗
PubMed2006

Properties of long human telomeric DNAs under cell-mimicking conditions.

We investigated the stability and structure of long telomeric DNAs derived from human, (TTAGGG)(n) (n=4-12) in the presence of 100 mM K(+) at 0 wt% or 20 wt% poly(ethylene glycol) 200 (PEG200) utilizing circular dichroism and UV melting analysis. The results showed that the values of enthalpy and entropy changes for the G-quadruplex formation of the telomeric DNAs whose repeat number was multiple of four, such as n=4, n=8, and n=12, increased gradually under the dilute condition (100 mM K(+)), demonstrating no interaction existed between the individual G-quadruplex units composing of four repeats. Therefore, the reasonable arrangement of the intramolecular G-quadruplexes formed by long telomeric DNAs (n> or =8) was proposed to be a bead-string structure in which the G-quadruplex units were connected each other by one TTA linker. Furthermore, the results of melting experiments demonstrated that thermodynamic stabilities of G-quadruplex structures of the long telomeric DNAs (n=5-12) are mostly independent of sequence length, although telomeric DNA including four repeats (n=4) is more stable than the longer ones. Moreover, the melting temperatures of the G-quadruplexes under the crowding condition (100 mM K(+) and 20 wt% PEG200) are higher than those under the dilute condition, indicating the crowding condition can increase the stability of G-quadruplex. These information are useful for researches of the telomere biology and a better development of therapeutic agents targeting telomeric DNAs.

DNA↗
PubMed2006

Functional role of the cofactor on activation process of L-histidine dependent ribozyme.

The activity of an L-histidine dependent ribozyme was analyzed with many pseudo-cofactors to investigate the activation process of the ribozyme. As a result, only L-histidine was able to induce the ribozyme activity among all chemicals used in this study. On the other hand, many L-histidine analogues inhibited the activation of the ribozyme by L-histidine, indicating the binding site permitted the analogues' access. These results suggest that the ribozyme recognizes L-histidine and express its activity highly specifically via an activation process followed by the histidine binding.

Coenzymes↗
PubMed2006

Development of molecular logic gates using the structural switch of telomere DNAs.

Telomere DNAs consisting of double-stranded G-rich and C-rich sequences are particularly promising as scaffolds for molecular devices because they form high-ordered structures and have a highly polymorphic nature depending on surrounding factors. Based on the structural polymorphism of telomere DNAs, excellent molecular devices such as molecular motors and switches have been reported. Here we found that the dynamic structural conversion of telomere DNAs can be controlled by both monovalent cations (M(+)) and pH (H(+)). Based on this conversion, we propose a new concept of molecular logic gates in response to the surrounding conditions (M(+) and H(+)) with fluorescence intensity changes as the output signal.

Cations, Monovalent↗
PubMed2006

[Analysis of constituents in urushi wax, a natural food additive].

Urushi wax is a natural gum base used as a food additive. In order to evaluate the quality of urushi wax as a food additive and to obtain information useful for setting official standards, we investigated the constituents and their concentrations in urushi wax, using the same sample as scheduled for toxicity testing. After methanolysis of urushi wax, the composition of fatty acids was analyzed by GC/MS. The results indicated that the main fatty acids were palmitic acid, oleic acid and stearic acid. LC/MS analysis of urushi wax provided molecular-related ions of the main constituents. The main constituents were identified as triglycerides, namely glyceryl tripalmitate (30.7%), glyceryl dipalmitate monooleate (21.2%), glyceryl dioleate monopalmitate (2.1%), glyceryl monooleate monopalmitate monostearate (2.6%), glyceryl dipalmitate monostearate (5.6%), glyceryl distearate monopalmitate (1.4%). Glyceryl dipalmitate monooleate isomers differing in the binding sites of each constituent fatty acid could be separately determined by LC/MS/MS.

Chromatography, Liquid↗
PubMed2006

Identification of the main constituents in sandarac resin, a natural gum base.

Sandarac resin, a natural gum base, is described as "a substance composed mainly of sandaracopimaric acid obtained from the secretion of sandarac trees" in the List of Existing Food Additives in Japan. To evaluate its quality as a food additive, the main constituents in a sandarac resin product were investigated. Three constituents were isolated and identified as sandaracopimaric acid, sandaracopimarinol and 4-epidehydroabietic acid by MS and 2D-NMR. Quantification of the main constituent, sandaracopimaric acid, was performed by HPLC and its content in the product was determined to be 11.6%.

Chromatography, High Pressure Liquid↗