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ABSTRACT: The telomere G-quadruplex-binding and telomerase-inhibiting capacity of two cationic (TMPyP4 and PIPER) and two anionic (phthalocyanine and Hemin) G-quadruplex-ligands were examined under conditions of molecular crowding (MC). Osmotic experiments showed that binding of the anionic ligands, which bind to G-quadruplex DNA via π-π stacking interactions, caused some water molecules to be released from the G-quadruplex/ligand complex; in contrast, a substantial number of water molecules were taken up upon electrostatic binding of the cationic ligands to G-quadruplex DNA. These behaviors of water molecules maintained or reduced the binding affinity of the anionic and the cationic ligands, respectively, under MC conditions. Consequently, the anionic ligands (phthalocyanine and Hemin) robustly inhibited telomerase activity even with MC; in contrast, the inhibition of telomerase caused by cationic TMPyP4 was drastically reduced by MC. These results allow us to conclude that the binding of G-quadruplex-ligands to G-quadruplex via non-electrostatic interactions is preferable for telomerase inhibition under physiological conditions.
Methods 04/2013; · 4.01 Impact Factor
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ABSTRACT: We systematically investigated the effects of loop length on the conformation, thermodynamic stability, and hydration of DNA G-quadruplexes under dilute and molecular crowding conditions in the presence of Na+. Structural analysis showed that molecular crowding induced conformational switches of oligonucleotides with the longer guanine stretch and the shorter thymine loop. Thermodynamic parameters further demonstrated that the thermodynamic stability of G-quadruplexes increased by increasing the loop length from two to four, whereas it decreased by increasing the loop length from four to six. Interestingly, we found by osmotic pressure analysis that the number of water molecules released from the G-quadruplex decreased with increasing thermodynamic stability. We assumed that base-stacking interactions within the loops not only stabilized the whole G-quadruplex structure, but also created hydration sites by accumulating nucleotide functional groups. The molecular crowding effects on the stability of G-quadruplexes composed of abasic sites, which reduce the stacking interactions at the loops, further demonstrated that G-quadruplexes with fewer stacking interactions within the loops released a larger number of water molecules upon folding. These results showed that the stacking interactions within the loops determined the thermodynamic stability and hydration of the whole G-quadruplex.
The Journal of Physical Chemistry B 11/2012; · 3.70 Impact Factor
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ABSTRACT: Incorporation of modified nucleotides into nucleic acid strands often produces conformational constraints and steric hindrances that may change the property of base pairing. In this study, we investigated a 2'-deoxycytidine derivative that tethers a phenyl moiety to the exocyclic amino group of cytosine linked through a ureido group. This derivative compound is structurally similar to the carbamoylated DNA base lesions produced in cells. The thermodynamic and structural studies showed that the modified dC formed the base pair with dG in the complementary strand, but the base-pairing selectivity toward dG was decreased under poly(ethylene glycol)-mediated osmotic stress. The phenyl group and the ureido linker attached to dC provided selectivity for the formation of base pairing exclusively with dG in a wide range of pH conditions, whereas unmodified dC stabilized the pairings with dA or dC in acidic solutions. Moreover, this modified base could not form self-pairing through intermolecular hydrogen bonds. We suggest that formation of weak pairing and protonation of the cytosine base are hindered due to the base modification. These data provide insights into the pairing selectivity of carbamoylated cytosine lesions produced in cells, and suggest applications of the 2'-deoxycytidine derivatives in medical technologies, molecular biology experiments, and synthesis of a supramolecular network of DNA strands.
Organic & Biomolecular Chemistry 11/2012; · 3.70 Impact Factor
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ABSTRACT: The structure and stability of long telomeric DNAs, (T(2)AG(3))(n) (n = 4-20), were studied under dilute and molecular crowding conditions in the presence of Na(+) and K(+). Structural analysis showed that the long telomeric DNAs formed intramolecular G-quadruplexes under all conditions. In the presence of Na(+), the telomeric DNAs formed an antiparallel G-quadruplex under both dilute and molecular crowding conditions. However, in the presence of K(+), molecular crowding induced a conformational change from mixed to parallel. These results are consistent with numerous structural studies for G-quadruplex units under molecular crowding conditions. Thermodynamic analysis showed that G-quadruplexes under the molecular crowding conditions were obviously more stable than under dilute condition. Interestingly, this stabilization effect of molecular crowding was reduced for the longer telomeric DNAs, indicating that the G-quadruplex structure of long telomeric DNAs is not as stable under molecular crowding conditions, as implied from the large stabilization of isolated G-quadruplex units as previously reported. Moreover, a hydration study revealed that upon structure folding, the interior of a G-quadruplex unit was dehydrated, whereas the linker between two units was more hydrated. It is thus possible to propose that the linkers between G-quadruplex units are ordered structures but not random coils, which could have an important influence on the stability of the entire structure of long telomeric DNAs. These results are significant to elucidate the biological characteristics of telomeres, and can aid in the rational design of ligands and drugs targeting the telomere and related proteins.
Journal of the American Chemical Society 08/2012; · 9.91 Impact Factor
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ABSTRACT: The thermal stability of nucleic acid structures is perturbed under the conditions that mimic the intracellular environment, typically rich in inert components and under osmotic stress. We now describe the thermodynamic stability of DNA oligonucleotide structures in the presence of high background concentrations of neutral cosolutes. Small cosolutes destabilize the basepair structures, and the DNA structures consisting of the same nearest-neighbor composition show similar thermodynamic parameters in the presence of various types of cosolutes. The osmotic stress experiments reveal that water binding to flexible loops, unstable mismatches, and an abasic site upon DNA folding are almost negligible, whereas the binding to stable mismatch pairs is significant. The studies using the basepair-mimic nucleosides and the peptide nucleic acid suggest that the sugar-phosphate backbone and the integrity of the basepair conformation make important contributions to the binding of water molecules to the DNA bases and helical grooves. The study of the DNA hydration provides the basis for understanding and predicting nucleic acid structures in nonaqueous solvent systems.
Biophysical Journal 06/2012; 102(12):2808-17. · 3.65 Impact Factor
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ABSTRACT: Characterization of metal ion binding to RNA and DNA base pairs is important for understanding their energy contribution to the folding and conformational changes of nucleic acid structures. In this study, we examine the equilibrium shift from the hairpin toward the dimer formation, induced by nonspecifically bound metal ions. The hairpin dimerization is markedly enhanced in the presence of high background concentrations of poly(ethylene glycol) (PEG) and several small organic molecules. The simple volume exclusion effect and the base pair stability cannot entirely account for this increase. We find that the dielectric constant correlates well with the dimerization efficiency in the conditions caused by small alcohol molecules and amide compounds as well as PEG. The hairpin dimerization experiments reveal the potential of PEG for enhancing the binding affinity between nucleic acids and metal ions, by reducing the solution dielectric constant without decreasing the thermodynamic stability of nucleic acid structures. The results presented here contribute to the understanding of nucleic acid folding and its ability to switch between alternative conformations under the condition of limited cation availability and cellular physiology.
The Journal of Physical Chemistry B 06/2012; 116(25):7406-15. · 3.70 Impact Factor
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ABSTRACT: We studied the kinetic and thermodynamic effects of locked nucleic acid (LNA) modifications on parallel and antiparallel DNA duplexes. The LNA modifications were introduced at cytosine bases of the pyrimidine strand. Kinetic parameters evaluated from melting and annealing curves showed that the association and dissociation rate constants for the formation of the LNA-modified parallel duplex at 25.0 °C were 3 orders of magnitude larger and 6 orders of magnitude smaller, respectively, than that of the unmodified parallel duplex. The activation energy evaluated from the temperature-dependent rate constants was largely altered by the LNA modifications, suggesting that the LNA modifications affected a prenucleation event in the folding process. Moreover, thermodynamic parameters showed that the extent of stabilization by the LNA modification for parallel duplexes (3.6 kcal mol(-1) per one modification) was much more significant than that of antiparallel duplexes (1.6 kcal mol(-1)). This large stabilization was due to the decrease in ΔH° that was more favorable than the decrease in TΔS°. These quantitative parameters demonstrated that LNA modification specifically stabilized the noncanonical parallel duplex. On the basis of these observations, we succeeded to stabilize the parallel duplex by LNA modification at the physiological pH. These results can be useful in the rational design of functional molecules such as more effective antisense and antigene strands, more sensitive strands for detection of target DNA and RNA strands, and molecular switches responding to solution pH.
Biochemistry 08/2011; 50(34):7414-25. · 3.42 Impact Factor
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ABSTRACT: The DNA chip that immobilizes DNA oligonucleotides on a solid plate surface is used for many diagnostic applications. For maximizing the detection sensitivity and accuracy, it is important to control the DNA density on a chip surface and establish a convenient method for optimizing the density. Here, the binding of DNA mismatch-binding protein MutS to the DNA substrate on the chip was investigated, which can be applied for high-throughput single-nucleotide polymorphism analysis in a genome. We prepared the DNA chips where the DNA substrate density was changed simply by using a mixed DNA solution. The binding of MutS was significantly influenced by the amount of DNA substrate on the chip as a consequence of steric crowding, and the moderate density that gave the distance between the DNA substrates greater than the size of the protein was appropriate to obtain accurate kinetic parameters. The substrate density-controlled DNA chip prepared using the mixed DNA solution has distinctive advantages for maximizing the detection capability and kinetic analysis of the binding of MutS and probably also other large proteins.
Analytical Chemistry 08/2011; 83(16):6368-72. · 5.86 Impact Factor
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ABSTRACT: We found that Hoogsteen base pairs were stabilized by molecular crowding and a histone H3-mimicking peptide, which was not observed for Watson-Crick base pairs. Our findings demonstrate that the type of DNA base pair is critical for the interaction between DNA and histones.
Chemical Communications 03/2011; 47(10):2790-2. · 6.17 Impact Factor
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ABSTRACT: Both cellular environmental factors and chemical modifications critically affect the properties of nucleic acids. However, the structure and stability of DNA containing abasic sites under cell-mimicking molecular crowding conditions remain unclear. Here, we investigated the molecular crowding effects on the structure and stability of the G-quadruplexes including a single abasic site. Structural analysis by circular dichroism showed that molecular crowding by PEG200 did not affect the topology of the G-quadruplex structure with or without an abasic site. Thermodynamic analysis further demonstrated that the degree of stabilization of the G-quadruplex by molecular crowding decreased with substitution of an abasic site for a single guanine. Notably, we found that the molecular crowding effects on the enthalpy change for G-quadruplex formation had a linear relationship with the abasic site effects depending on its position. These results are useful for predicting the structure and stability of G-quadruplexes with abasic sites in the cell-mimicking conditions.
Journal of nucleic acids 01/2011; 2011:857149.
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ABSTRACT: Unnatural nucleosides have been explored to expand the properties and the applications of oligonucleotides. This paper briefly summarizes nucleic acid analogs in which the base is modified or replaced by an unnatural stacking group for the study of nucleic acid interactions. We also describe the nucleoside analogs of a base pair-mimic structure that we have examined. Although the base pair-mimic nucleosides possess a simplified stacking moiety of a phenyl or naphthyl group, they can be used as a structural analog of Watson-Crick base pairs. Remarkably, they can adopt two different conformations responding to their interaction energies, and one of them is the stacking conformation of the nonpolar aromatic group causing the site-selective flipping of the opposite base in a DNA double helix. The base pair-mimic nucleosides can be used to study the mechanism responsible for the base stacking and the flipping of bases out of a nucleic acid duplex.
Journal of nucleic acids 01/2011; 2011:967098.
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ABSTRACT: Base stacking is important for the base pair interaction of a DNA duplex, DNA replication by polymerases, and single-stranded nucleotide overhangs. To study the mechanisms responsible for DNA stacking interactions, we measured the thermal stability of DNA duplexes containing a non-natural nucleotide tethered to a simple aromatic hydrocarbon group devoid of dipole moments and hydrogen bonding sites. The duplexes containing tetrahydrofuran were paired with a deoxyadenosine derivative (A/T base pair analog) or a deoxycytidine derivative (C/G base pair analog) and showed a lower stability than Watson-Crick base pairing, partly due to the loss of interbase hydrogen bonds. Conversely, non-natural nucleotides present at a dangling end yielded an interaction energy as high as that observed with base pairing. Importantly, the non-natural nucleotides yielded an interaction energy with a linear correlation similar to that of the analogous Watson-Crick base pairs both in the middle and at the end of a DNA duplex, although a different stacking mechanism between the middle and the end was suggested. Moreover, a positive cooperativity was observed in dangling end stacking of the nucleotide base moiety and aromatic hydrocarbon group. These observations are useful to understand nucleic acid interactions and to design new non-natural nucleotides.
Molecular BioSystems 10/2010; 6(10):2023-9. · 3.53 Impact Factor
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ABSTRACT: We now present the first example in which triplet repeat DNAs adopt the i-motif structure at neutral pH by molecular crowding. Crowding stabilized the i-motif and the pK(a) of N3 of cytosine was raised in such a microenvironment. Molecular crowding is known to accelerate the formation of the multi-stranded i-motif while the triplet repeats adopt the single-strand structure.
Chemical Communications 02/2010; 46(8):1299-301. · 6.17 Impact Factor
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ABSTRACT: Short RNA sequences exhibiting the activity of a target RNA cleavage are promising for cellular gene regulation and biosensor research, but the reaction media different from an aqueous solution may cause unanticipated molecular interactions and properties. In this study, we investigated the molecular crowding effects arising from steric crowding and altered solvent properties on the hammerhead ribozyme activity using water-soluble neutral cosolutes. Poly(ethylene glycol) (PEG) and other cosolutes at 20 wt % increased the RNA hydrolysis rate by a factor of 2.0-6.6 at 10 mM MgCl(2) and much more at lower MgCl(2) concentrations. Remarkably, although the cosolutes decreased the stability of the ribozyme stem helices, the thermal inactivation temperature of the ribozyme was significantly raised, resulting in a higher reaction rate, up to 270 times at 50 degrees C. More significantly, PEG decreased the metal ion concentration to perform the reaction even with a limiting Mg(2+) or Na(+) concentration, facilitated the catalytic turnover, and activated a catalytically less active ribozyme sequence. These observations agreed that the cosolutes acted as an osmolyte stabilizing the water-release reaction of the RNA tertiary folding but destabilizing the water-uptake reaction of Watson-Crick base pairing. The opposite cosolute effect on the stabilities of RNA secondary and tertiary structures, which is fundamentally different from a protein folding, suggests how RNA stabilizes a tertiary structure and enhances the catalytic activity in molecular crowding media.
Journal of the American Chemical Society 10/2009; 131(46):16881-8. · 9.91 Impact Factor
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ABSTRACT: A base flipping conformation is found in many biological processes, including DNA repair and DNA and RNA modification processes. To investigate the dynamics and energetics of this unusual conformation in a double helix, base flipping induced by the base pair analogues of deoxyadenosine and deoxycytidine derivatives tethering a phenyl or naphthyl group was investigated. DNA strands bearing the base pair analogues stabilized the base flipping conformation of a complementary RNA, resulting in a site-specific hydrolysis by specific base catalysis. Measurements of the hydrolysis rate and the thermal stability of DNA/RNA duplexes suggested an unconstrained flexibility of the flipped-out ribonucleotide. As established in the base flipping by DNA repair and DNA and RNA modification enzymes, the results suggested that base flipping occurred in competition with base pair formation. In addition, the deoxycytidine derivatives discriminated G from I (inosine), with respect to the base pair interaction energy, as observed for a damaged base or a weakened base pair search by DNA repair proteins. The base pair mimic nucleosides would be useful for investigating the base flipping conformation under the equilibrium with base pairing.
Biochemistry 10/2009; 48(47):11304-11. · 3.42 Impact Factor
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ABSTRACT: Nucleotide folding accompanies cation binding that shields the electronegative potential of phosphate groups, and metal ions in the condensation layer predominantly associate diffusely with base-paired nucleotides. Although metal ions bound at specific sites have been well studied, information of diffusely bound cations, that usually have a weak binding affinity than those associating at specific sites, have not been thoroughly studied. We explored a convenient experimental system using a self-complementary nucleotide sequence for analyzing cationic ligands diffusely bound to DNA or RNA base pairs. To study the metal ion-nucleotide interaction under a non-homologous aqueous condition, solutions containing a large amount of PEG (polyethylene glycol) were examined. We found that PEG (e.g., 20 wt%) substantially influenced the metal ion binding to nucleotides, suggesting significances of the molecular environment on nucleotide-cation interactions.
Nucleic Acids Symposium Series 01/2009;
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Shu-Ichi Nakano,
Lei Wu,
Hirohito Oka,
Hisae Tateishi Karimata,
Toshimasa Kirihata,
Yuichi Sato,
Satoshi Fujii,
Hiroshi Sakai,
Masayasu Kuwahara,
Masayuki Kuwahara,
Hiroaki Sawai,
Naoki Sugimoto
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ABSTRACT: Water-soluble neutral cosolutes can be used to quantify biomolecular properties in the particular molecular environment occurring in a cell. We studied the conformation and the thermal stability of DNA and RNA structures in the presence of PEG [poly(ethylene glycol)] and smaller cosolutes of glycerol, ethylene glycol, 1,3-propanediol, 2-methoxyethanol, and 1,2-dimethoxyethane. Although the neutral cosolutes destabilized the oligonucleotide duplex and the hairpin structures, the left-handed Z-form duplex was more energetically favored in the cosolute-containing solutions. These observations were due to the contribution of water molecule on the nucleotide structure formations because the cosolutes act as an osmolyte to reduce the water activity of a solution. Moreover, the sodium ion condensation for the duplex and the hairpin formations was reduced in the presence of PEG, while that for the transition from the B-form to the Z-form was unaltered. The CD (circular dichroism) and EPR (electron paramagnetic resonance) spectra demonstrated that the cosolutes changed the helical conformation of the unstructured oligonucleotides, but not those of the ordered structures. The results of the favorable formation of the noncanonical nucleotide structures, and minimized conformational and thermal perturbations of the ordered nucleotide structures in the cosolute-containing solutions implicate the significance of the intracellular environment on DNA and RNA structures in a cell.
Molecular BioSystems 07/2008; 4(6):579-88. · 3.53 Impact Factor
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ABSTRACT: We obtained the intrinsic binding affinity for metal ions, polyamines, and oligolysine peptides diffusely bound to base-paired sites in DNA by monitoring the shift of the hairpin-duplex equilibrium of the self-complementary DNA sequences, which can be widely used for capturing cationic ligands bound diffusely to nucleotide base pairs.
Chemical Communications 03/2008; · 6.17 Impact Factor
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ABSTRACT: Although metal ion is essential for the DNA and RNA folding, there are a limited number of reports describing the influence of molecular environments on the metal ion binding. Here, we investigated the binding properties of Mg(2+) and Na(+) toward the hammerhead ribozyme in the presence of PEG [poly(ethylene glycol)] as a cosolute that changes the solvent property of the reaction. PEG8000 (PEG with the average molecular weight of 8000) increased the reaction rate at lower Mg(2+) concentrations but not at higher concentrations. We also found that PEG8000 unchanged the number of Mg(2+) bound while the binding cooperativity of Na(+) in the reaction without divalent metal ion was altered. The change of Na(+) condensation by cosolute has also been reported for diffusely bound Na(+) to Watson-Crick base pairs. It is thus supposed that diffusely bound Na(+) stabilizes the ribozyme active form in the absence of divalent metal ion. This study provides insights into the RNA-metal ion interaction and the hammerhead ribozyme activity under the molecular crowding condition occurred in a cell and on biosensor devices.
Nucleic Acids Symposium Series 02/2008;
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ABSTRACT: The kinetics of DNA duplex formation was affected by the addition of PEGs with different masses (MW = 200-8000) to an aqueous solution; for each condition, two duplexes (5'-TAGGTTATAA-3'/5'-TTATAACCTA-3' and 5'-CAGGTCACAG-3'/5'-CTGTGACCTG-3') with different stabilities were formed after overcoming the same association activation energy barrier, suggesting that the formation of consecutive GC base pairs in the helices rather than the helix terminus is the initiation nucleus for DNA duplex formation not only in the absence, but also in the presence of PEGs.
Chemical Communications 08/2007; · 6.17 Impact Factor
