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Biomedical subjects

Naoki Sugimoto

Publications and source records attributed to Naoki Sugimoto.

At least 73 records · Page 4Linked to original sources
PubMed2003

A novel approach of cell-free peptide synthesis and SNPs detection using nano-circular single-stranded DNA.

Development of peptide/protein synthesis is very important for investigating the functions of peptides and proteins. The advantages of cell-free synthesis, which has been extensively developed, include the ability to synthesize toxic proteins, aggregated proteins and proteins containing unnatural amino acids. However, this promising approach does not have a high reaction yield. In this study, we examine whether the rolling synchronization system, which produces long RNAs with a repeated sequence encoded by very small circular ssDNAs without the promoter sequence for RNA polymerase, can be used for cell-free peptide synthesis to improve the reaction yield. As a result, we have found that rolling synchronization is useful for large-scale cell-free peptide/protein synthesis.

Base Sequence↗
PubMed2003

Large stacking stability of a base pair-mimic nucleotide on the DNA duplex.

We synthesized novel adenosine derivatives tethering an aromatic hydrocarbon group by an amido linker. The single adenosine derivatives at 5' dangling end stabilized the DNA duplex of 5'-ATGCGCAT-3' more or equally than Watson-Crick base pair. When the number of the dangling residues increased from one to three, the duplex stability became larger by 1.8-3.6 kcal/mol. When the adenosine derivative was opposite to an abasic site in a DNA duplex, the destabilization by the abasic site was significantly reduced. These observations suggest that the adenosine derivatives developed in this study can stack with a DNA base pair by forming a base pair-mimic geometry.

Base Pairing↗
PubMed2003

Synthesis of glycosylcurcuminoids.

Condensation of glycosylated arylaldehyde with acetylacetone-B(2)O(3) complex gave a corresponding diglycosylcurcuminoid, and a similar reaction using a mixture of arylaldehyde and glycosylarylaldehyde gave an unsymmetrical monoglycosylcurcuminoid, both as boron-complexes. The boron was removed from the complexes by heating in methanol, thus achieving the synthesis of di- and mono-glycosylcurcuminoids.

Aldehydes↗
PubMed2003

[Analysis of constituents in Jamaica quassia extract, a natural bittering agent].

Jamaica quassia extract, a natural bittering agent, is described as "a substance extracted from bark of Jamaica quassia (Quassia excelsa Sw.)" in the List of Existing Food Additives in Japan. The constituents in Jamaica quassia extract product were investigated as a part of an ongoing study to evaluate its quality and safety as a food additive. The main constituents of the extract were identified as quassin and two isomers of neoquassin by using LC/MS. The main constituent, quassin, was isolated and the structure was determined by spectral means. The quantification of their main constituents was performed by HPLC using quassin as a standard, and the concentrations of quassin and total of neoquassin isomers were 21.4% and 55.5%. In addition, it was confirmed that Jamaica quassia extract was different from quassia extract, which is extracted from bark of Picrasma quassioides Benn. belonging to the same family as Q. excelsa, by comparing their HPLC profiles.

Chromatography, High Pressure Liquid↗
PubMed2002

Molecular crowding regulates the structural switch of the DNA G-quadruplex.

Almost all biochemical reactions in vitro have been investigated through numerous experiments conducted in dilute solutions containing low concentrations of solutes. However, biomacromolecules such as nucleic acids, proteins, and polysaccharides are designed to function and/or form their native structures in a living cell containing high concentrations of biomacromolecules, substrates, cofactors, salts, and so on. In the present study, we have demonstrated quantitatively the effect of molecular crowding on structures and stabilities of the G-quadruplex of d(G(4)T(4)G(4)). Molecular crowding with poly(ethylene glycol) (PEG) induced a structural transition from the antiparallel to the parallel G-quadruplex of d(G(4)T(4)G(4)), while molecular crowding with polycations did not alter the structure of the antiparallel G-quadruplex. The binding constants of putrescine, one of the polycations, for d(G(4)T(4)G(4)) in the absence and presence of Na(+) are calculated to be 277 and 2.5 M(-)(1), respectively. This indicates that the polycations coordinate to d(G(4)T(4)G(4)) with electrostatic interactions. The thermodynamic parameters of the antiparallel G-quadruplex formation under the crowding and noncrowding conditions induced by putrescine were also estimated. The stability of the antiparallel G-quadruplex decreased (-DeltaG degrees (25) decreased from 28 to 22 kcal mol(-)(1)) with molecular crowding by putrescine. Also, enthalpy and entropy changes in the structural formation under crowding and noncrowding conditions clearly showed that destabilization was entropy-driven. These quantitative parameters indicated that both the volume excluded by PEG and chemical interactions such as electrostatic interaction with solute polycations are critical for determining how molecular crowding affects the structure and stability of highly ordered DNA structures.

Animals↗
PubMed2002

Long RNA dangling end has large energetic contribution to duplex stability.

Long terminal unpaired nucleotides known as dangling ends play interesting roles in biological systems. Previous studies, however, only dealt with the energy contributions of single dangling bases. The energy contributions of long dangling ends on the stability of duplexes have not been systematically studied. We now report a quantitative increase in stability of RNA-RNA and DNA-DNA duplexes containing a long dangling end. We found a larger enhancement of the stability by the long RNA dangling end of the RNA-RNA duplex than has been observed for the DNA duplexes. It is also found that structural stabilizations by long dangling ends seem to originate from the single-stranded stacking interactions of nucleotides. These results indicate that RNA stability can be achieved by increasing the length of the dangling end. The thermodynamic parameters of the long dangling ends are useful for designing ribozymes and antisense oligonucleotides, and for the prediction of the RNA secondary structure like the pseudoknot.

DNA↗
PubMed2002

Characterization and thermodynamic properties of quadruplex/duplex competition.

Structural characteristics and thermodynamic properties of dG(3)(T(2)AG(3))(3), d(C(3)TA(2))(3)C(3) and dG(3)(T(2)AG(3))(3)/d(C(3)TA(2))(3)C(3) were intensively investigated. It was indicated that metal ions greatly affected the conformation and stability of the G-quadruplex. A competition of a structure transition among the G-quadruplex, I-motif, and the duplex was confirmed to be dependent on both cation species and pH values. The structural competitive mechanism is discussed for the first time. This study shows an intriguing potential in modulating DNA structures in vivo, which is of great importance in drug design and cancer chemotherapy.

Calorimetry↗
PubMed2002

Effect of divalent cations and cytosine protonation on thermodynamic properties of intermolecular DNA double and triple helices.

The contribution of divalent cations and cytosine protonation to conformation and stability of duplex and triplex formation were intensively investigated and characterized by ultraviolet (UV), circular dichroism (CD), differential scanning calorimetry (DSC), and electrophoresis mobility shift assay (EMSA). CD spectra showed that the divalent cations investigated would not significantly distort nucleotide geometry, while UV and DSC melting experiments revealed that the cation binding abilities to duplexes and triplexes were clearly dependent on the types of cations under near physiological conditions. The calorimetric enthalpies were generally underestimated relative to the corresponding van't Hoff enthalpies for Hoogsteen and Watson-Crick transitions, but free energy changes derived from the DSC measurements were in good agreement with those derived from the UV measurements. The adjacent placing of the C(+) x G.C triplets in triplexes lowered the stabilities of not only Hoogsteen base-pairing but also Watson-Crick base-pairing. The protonation contribution of the given cytosine residues might depend on the local and global structure of the protonated cytosine complex. A rigid structural targeted-strand would favor the protonation of cytosine residues. The apparent pK(a) values for parallel duplex and triplex investigated were determined to be 6.4 and 7.6, respectively, which are considerably heightened by 2.1 and 3.3 pH unit as compared to the intrinsic pK(a) value of the free cytosine residues.

Calorimetry, Differential Scanning↗
PubMed2002

Immobilized small deoxyribozyme to distinguish RNA secondary structures.

The RNA folding variation due to one or more mutations leads to different RNA splicing, RNA processing, and translational controls as a result of differences in the primary and higher-ordered structures that interact with other cellular molecules. Thus, distinguishing RNA folding is one of the guides to detect the gene functions related to disease and drug responses. We found, previously, a small Ca(2+)-dependent deoxyribozyme with its site-specific RNA cleavage [Sugimoto, N., Okumoto, Y., and Ohmichi, T. (1999) J. Chem. Soc., Perkin Trans. 2, 1382-1388]. In this study, we report the potential of this deoxyribozyme as a useful tool to distinguish RNA foldings. It is found that the immobilized deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The systematic design for the target RNA hairpin loops shows that the immobilized deoxyribozyme is able to cleave them with a > or =17 nucleotide loop size at only one site under single-turnover conditions. Furthermore, an RNA cleavage reaction is detected using the immobilized deoxyribozyme on a surface plasmon resonance (SPR) sensor chip. These results show that the immobilized deoxyribozymes on a column and on an SPR sensor chip become a novel and useful tool to distinguish the RNA foldings.

Base Sequence↗
PubMed2002

Temperature dependence of thermodynamic properties for DNA/DNA and RNA/DNA duplex formation.

A clear difference in the enthalpy changes derived from spectroscopic and calorimetric measurements has recently been shown. The exact interpretation of this deviation varied from study to study, but it was generally attributed to the non-two-state transition and heat capacity change. Although the temperature-dependent thermodynamics of the duplex formation was often implied, systemic and extensive studies have been lacking in universally assigning the appropriate thermodynamic parameter sets. In the present study, the 24 DNA/DNA and 41 RNA/DNA oligonucleotide duplexes, designed to avoid the formation of hairpin or slipped duplex structures and to limit the base pair length less than 12 bp, were selected to evaluate the heat capacity changes and temperature-dependent thermodynamic properties of duplex formation. Direct comparison reveals that the temperature-independent thermodynamic parameters could provide a reasonable approximation only when the temperature of interest has a small deviation from the mean melting temperature over the experimental range. The heat capacity changes depend on the base composition and sequences and are generally limited in the range of -160 to approximately -40 cal.mol-1.K-1 per base pair. In contrast to the enthalpy and entropy changes, the free energy change and melting temperature are relatively insensitive to the heat capacity change. Finally, the 16 NN-model free energy parameters and one helix initiation at physiological temperature were extracted from the temperature-dependent thermodynamic data of the 41 RNA/DNA hybrids.

Base Pairing↗
PubMed2002

Role of 2'-OH on thermodynamic stability and structure of chimeric oligonucleotides.

Thermodynamic parameters of RNA/DNA chimeric duplexes were obtained systematically. Stability of the duplexes including chimeric junctions was context dependent, although CD spectra showed less structural perturbation by the junctions. Nearest-neighbor delta G degree 37 values for these chimeric RNA-DNA junctions suggested that 2'-OH on the 5'-side nucleotide at the junction may be the determinant for the nearest-neighbor stability of the chimeric junction.

Base Sequence↗
PubMed2002

Effect of secondary structure of short double-stranded RNA on RNAi efficiency.

The dangling nucleotides are very important for the RNA interference (RNAi). We investigate the effect of the dangling end on both the thermodynamic property of the RNA helix and efficiency of gene regulation in RNAi pathway. The result indicates that the four dangling adenines, which are more stable than two or three dangling residues, is not suitable for the RNAi efficiency.

Gene Expression Regulation↗
PubMed2002

Structure of acid-stable carmine.

Acid-stable carmine has recently been distributed in the U.S. market because of its good acid stability, but it is not permitted in Japan. We analyzed and determined the structure of the major pigment in acid-stable carmine, in order to establish an analytical method for it. Carminic acid was transformed into a different type of pigment, named acid-stable carmine, through amination when heated in ammonia solution. The features of the structure were clarified using a model compound, purpurin, in which the orientation of hydroxyl groups on the A ring of the anthraquinone skeleton is the same as that of carminic acid. By spectroscopic means and the synthesis of acid-stable carmine and purpurin derivatives, the structure of the major pigment in acid-stable carmine was established as 4-aminocarminic acid, a novel compound.

Acids↗
PubMed2002

[Identification of acid-stable carmine in imported apple syrup product].

An unknown red pigment was purified from an apple syrup product imported from Canada, using a DIAION HP-20 column with methanol as the eluent. By spectroscopic means and chemical synthesis, the isolated pigment was identified as 4-aminocarminic acid, which is the major pigment of acid-stable carmine (a red colorant illegal in Japan). In addition, HPLC and TLC methods were proposed to detect this illegal colorant. While the color of carminic acid changed from yellow to red in the pH range of McIlvaine buffer (3.0-7.0), the color of 4-aminocarminic acid was always red, and also the ultraviolet/visible (UV/Vis) spectra did not change. These characteristics are useful to distinguish 4-aminocarminic acid from carminic acid.

Carmine↗
PubMed2002

Analysis of rubusoside and related compounds in tenryocha extract sweetener.

The main and minor constituents of tenryocha extract product, a natural sweetener, were investigated as a part of an ongoing study to evaluate its quality and safety as a food additive. Four constituents, namely rubusoside, steviolmonoside, panicloside IV, and ent-16 alpha, 17-dihydroxykauran-19-oic acid, were isolated. The concentration of rubusoside, the main sweet constituent, was 8.65% in the tenryocha extract product. In addition, it was confirmed that the origin of the extract was the leaves of Rubus suavissimus S. Lee (Rosaseae), as determined by comparing TLC and HPLC profiles of the product and hot-water extract prepared from the leaves of R. suavissimus.

Chromatography, High Pressure Liquid↗