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Shu-ichi Nakano

Publications and source records attributed to Shu-ichi Nakano.

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

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

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

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↗
PubMed2005

DNA base flipping by a base pair-mimic nucleoside.

On the basis of non-covalent bond interactions in nucleic acids, we synthesized the deoxyadenosine derivatives tethering a phenyl group (X) and a naphthyl group (Z) by an amide linker, which mimic a Watson-Crick base pair. Circular dichroism spectra indicated that the duplexes containing X and Z formed a similar conformation regardless of the opposite nucleotide species (A, G, C, T and an abasic site analogue F), which was not observed for the natural duplexes. The values among the natural duplexes containing the A/A, A/G, A/C, A/T and A/F pairs differed by 5.2 kcal mol(-1) while that among the duplexes containing X or Z in place of the adenine differed by only 1.9 or 2.8 kcal mol(-1), respectively. Fluorescence quenching experiments confirmed that 2-amino purine opposite X adopted an unstacked conformation. The structural and thermodynamic analyses suggest that the aromatic hydrocarbon group of X and Z intercalates into a double helix, resulting in the opposite nucleotide base flipping into an unstacked position regardless of the nucleotide species. This observation implies that modifications at the aromatic hydrocarbon group and the amide linker may expand the application of the base pair-mimic nucleosides for molecular biology and biotechnology.

Base Pairing↗
PubMed2005

Site-selective RNA cleavage by DNA bearing a base pair-mimic nucleoside.

We have synthesized the deoxyadenosine derivative tethering a phenyl group (X), which mimics the Watson-Crick A/T base pair. The RNA/DNA hybrid duplexes containing X in the middle of the DNA sequence showed a similar thermal stability regardless of the ribonucleotide species (A, G, C, or U) opposite to X, probably because of the phenyl group stacking inside of the duplex accompanied by the opposite ribonucleotide base flipped in an extrahelical position. The RNA strand hybridized with the DNA strand bearing X was cleaved on the 3'-side of the ribonucleotide opposite to X in the presence of MgCl2, and the RNA sequence to be cleaved was not restricted. The site-specific RNA hydrolysis suggests that the DNA strand bearing X has the advantage of the site-selective base flipping in the target sequence and the development of a "universal deoxyribozyme" to exclusively cleave a target RNA sequence.

Adenosine↗
PubMed2005

Coformational switch of oligonucleotide induced by spermine.

Conformational change of nucleic acids induced by a small molecule is essential for many biological reactions and useful for biotechnology. To construct a spermine-responsive nucleic acid, we designed DNA sequences which can potentially form a unimolecular hairpin loop structure and a bimolecular duplex by itself. On the basis of UV melting and native PAGE experiments, we found four DNA oligomers of d16cg, d14cg, d12cg, and d14ta adopting different secondary structures depending on the spermine concentration. Their dissociation constants (Kd) with spermine were estimated in the order of 10(-6).

Electrophoresis, Polyacrylamide Gel↗
PubMed2004

The effect of molecular crowding with nucleotide length and cosolute structure on DNA duplex stability.

The thermodynamics of DNA duplex structures in the presence of high concentrations of cosolutes in solution were investigated to discern nucleic acid structures and functions in living cells. In the presence of ethylene glycol (EG) and poly(ethylene glycol) (PEG) (MW = 200-8000), the stability of the oligomer DNA duplexes with differing nucleotide length varied, depending on the nucleotide length as well as the size of PEG. It was also revealed that the decrease of water activity is the primary factor for destabilization of the short (8-mer) duplex by addition of high molecular weight PEGs as well as low molecular weight PEGs and other low molecular weight cosolutes. In addition, the number of water molecules taken up per base pair formation was the same for all the PEGs and for 1,2-dimethoxyethane, which was greater than in the cases of glycerol, EG, 1,3-propanediol, and 2-methoxyethanol, suggesting that the solvation of nucleotides may differ, depending on the cosolute structure. These findings are useful not only for understanding nucleic acid structures and functions in cells but also for the design of oligonucleotides applicable for cells, such as antisense nucleic acids, RNAi, and DNA chips.

DNA↗
PubMed2004

Influences of ribonucleotide on a duplex conformation and its thermal stability: study with the chimeric RNA-DNA strands.

To understand the influences of the ribonucleotide on a duplex conformation and its stability, we systematically studied the CD spectra and the thermodynamics of nucleic acid duplexes formed by the chimeric RNA-DNA strand in which ribonucleotides and deoxyribonucleotides were covalently attached. It was found that the duplex stability was context-dependent and independent of the number of ribonucleotides in the chimeric strand, whereas the CD spectra showed less overall structural perturbation by the chimeric junctions. Combining the results of the CD and the thermodynamic data revealed a stability-structure relationship for the duplexes. Importantly, DeltaG(o)37 values estimated for the chimeric junction formation in the RNA-DNA/DNA and the RNA-DNA/RNA duplexes were close to those of RNA/DNA and RNA/RNA interactions, respectively. Furthermore, DeltaG(o)37s of the DNA-RNA/DNA and DNA-RNA/DNA-RNA junctions were similar to those of the DNA duplex, and the values of DNA-RNA/RNA-DNA were similar to those of the DNA/RNA. The thermodynamic analyses suggest that the 5'-nucleotide may be the crucial factor that determines the stability at the chimeric junction. Our results not only suggest influences of the ribonucleotide on a duplex conformation and its stability but also are useful for the design of RNA-DNA chimeric strands applicable to biotechnology.

Base Pairing↗
PubMed2004

Catalytic roles for proton transfer and protonation in ribozymes.

Utilization of proton transfer in catalysis, which is well known in the mechanisms of protein enzymes, has been described only relatively recently for RNA enzymes. In this article, we present a current understanding of proton transfer by nucleic acids. Rate enhancement and specificity conferred by general acid-base catalysis are discussed. We also present possibilities for electrostatic catalysis from general acids and bases as well as cationic base pairs. The microenvironments of a large RNA provide the possibility of histidine-like pK(a)s for proton transfer, as well as lysine- and arginine-like pK(a)s for electrostatic catalysis. Discussion on proton transfer focuses on the hepatitis delta virus (HDV) and hairpin ribozymes, with select examples drawn from the protein literature. Discussion on electrostatic catalysis also draws on these two ribozymes, and a postulate for electrostatic catalysis by a cationic base pair in the mechanism of peptidyl transfer in the ribosome is presented. We also provide a perspective on possibilities for phosphoryl transfer mechanisms involving phosphorane intermediates and unusual tautomeric forms of the bases. Lastly, a distinction is made between ground state and "transition state" pK(a)s. We favor a model in which changes in pH lead to changes in the distribution of reactive and nonreactive ionizations of the ribozyme molecules in the ground state, and therefore suggest that "pK(a) changes in the transition state" do not provide an acceptable explanation for observed pH-rate profiles.

Catalysis↗
PubMed2004

Stabilization of a DNA duplex under molecular crowding conditions of PEG.

A living cell generally contains macromolecules occupying 20-40% of the total volume. To mimic the crowded cellular condition, we prepared solutions including poly(ethylene glycol) (PEG) as a cosolute and investigated the influence of the cosolute on the DNA duplex stability. In the presence of PEG 200 or PEG 8000, the Tm (melting temperature) of a self-complementary duplex of 5'-dATGCGCAT-3' decreased by 11.8 degrees C in the presence of 20 wt% PEG200 and by 1.5 degrees C in the presence of 20 wt% PEG 8000. The dln K(obs) vs. dln a(w) plots for PEG 200 and PEG 8000 were linear with a negative slope, suggesting the association of water molecules upon the duplex formation. Interestingly, when the NaCl concentration decreased from 1 M to 400 mM, the Tm increased in the presence of PEGs. Our results imply that the nucleic acid stabilities in a living cell may be different from those in in vitro conditions.

Base Pairing↗
PubMed2004

Secondary structure change of oligonucleotide induced by salts.

Formation of a unimolecular structure is essential for many biological processes such as transcription and translation, and the (deoxy)ribozyme activities. Since such nucleic acids consist of self-complementary sequence, they also have a potential to form a bimolecular structure as well. In this study, we designed 13 RNA and DNA sequences which can potentially form a hairpin loop structure by one strand and a duplex by two strands. On the basis of the UV melting and CD spectra measurements, we found that an RNA oligomer of rGCAAGCAAGCUUGC adopted different secondary structures depending on NaCl concentration, for example, a duplex at 1 M NaCl and a hairpin loop structure at 10 mM NaCl. On the other hand, the structure of DNA oligomer with the same sequence was unchanged. This observation indicates that the structural transition by the change of the NaCl concentration is not due to the nature of the sequence.

Buffers↗
PubMed2004

Conformational change of a nucleotide by a base-pair mimic nucleoside in the complementary DNA strand.

On the basis of the non-covalent bond interactions in nucleic acids, we have synthesized a novel adenosine derivative tethering a phenyl group. Structure and thermal stability of the DNA duplexes bearing the adenosine derivative in the middle of a strand were investigated by CD spectra and the UV melting curves. We found that the conformation and the interactions of the duplexes were similar regardless of the nucleotide facing with the adenosine derivative in a duplex. This observation suggests the base pair-mimic geometry of the adenosine derivative accompanied with a conformational change of the nucleotide in the complementary strand.

Adenosine↗
PubMed2003

Structural competition involving G-quadruplex DNA and its complement.

Structural competition between the G-quadruplex, the I-motif, and the Watson-Crick duplex has been implicated for repetitive DNA sequences, but the competitive mechanism of these multistranded structures still needs to be elucidated. We investigated the effects of sequence context, cation species, and pH on duplex formation by the G-quadruplex of dG(3)(T(2)AG(3))(3) and its complement the I-motif of d(C(3)TA(2))(3)C(3), using ITC, DSC, PAGE, CD, UV, and CD stopped-flow kinetic techniques. ITC and PAGE experiments confirmed Watson-Crick duplex formation by the complementary strands. The binding constant of the two DNA strands in the presence of 10 mM Mg(2+) at pH 7.0 was shown to be 5.28 x 10(7) M(-1) at 20 degrees C, about 400 times larger than that in the presence of 100 mM Na(+) at pH 5.5. The dynamic transition traces of the duplex formation from the equimolar mixture of G-/C-rich complementary sequences were obtained at both pH 7.0 and pH 5.5. Fitting to a single-exponential function gave an observed rate of 8.06 x 10(-3) s(-1) at 20 degrees C in 10 mM Mg(2+) buffer at pH 7.0, which was about 10 times the observed rate at pH 5.5 under the same conditions. Both of the observed rates increased as temperature rose, implying that the dissociation of the single-stranded structured DNAs is the rate-limiting step for the WC duplex formation. The difference between the apparent activation energy at pH 7.0 and that at pH 5.5 reflects the fact that pH significantly influences the structural competition between the G-quadruplex, the I-motif, and the Watson-Crick duplex, which also implies a possible biological role for I-motifs in biological regulation.

Animals↗
PubMed2003

Mechanistic characterization of the HDV genomic ribozyme: classifying the catalytic and structural metal ion sites within a multichannel reaction mechanism.

Prior studies of the metal ion dependence of the self-cleavage reaction of the HDV genomic ribozyme led to a mechanistic framework in which the ribozyme can self-cleave by multiple Mg2+ ion-independent and -dependent channels [Nakano et al. (2001) Biochemistry 40, 12022]. In particular, channel 2 involves cleavage in the presence of a structural Mg2+ ion without participation of a catalytic divalent metal ion, while channel 3 involves both structural and catalytic Mg2+ ions. In the present study, experiments were performed to probe the nature of the various divalent ion sites and any specificity for Mg2+. A series of alkaline earth metal ions was tested for the ability to catalyze self-cleavage of the ribozyme under conditions that favor either channel 2 or channel 3. Under conditions that populate primarily channel 3, nearly identical K(d)s were obtained for Mg2+, Ca2+, Ba2+, and Sr2+, with a slight discrimination against Ca2+. In contrast, under conditions that populate primarily channel 2, tighter binding was observed as ion size decreases. Moreover, [Co(NH3)6]3+ was found to be a strong competitive inhibitor of Mg2+ for channel 3 but not for channel 2. The thermal unfolding of the cleaved ribozyme was also examined, and two transitions were found. Urea-dependent studies gave m-values that allowed the lower temperature transition to be assigned to tertiary structure unfolding. The effects of high concentrations of Na+ on the melting temperature for RNA unfolding and the reaction rate revealed ion binding to the folded RNA, with significant competition of Na+ (Hill coefficient of 1.5-1.7) for a structural Mg2+ ion and an unusually high intrinsic affinity of the structural ion for the RNA. Taken together, these data support the existence of two different classes of metal ion sites on the ribozyme: a structural site that is inner sphere with a major electrostatic component and a preference for Mg2+, and a weak catalytic site that is outer sphere with little preference for a particular divalent ion.

Barium↗
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↗