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Biomedical subjects

Daisuke Miyoshi

Publications and source records attributed to Daisuke Miyoshi.

At least 19 recordsLinked to original sources
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

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

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

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↗
PubMed2005

SPR sensor chip for detection of small molecules using molecularly imprinted polymer with embedded gold nanoparticles.

Molecularly imprinted polymer gel with embedded gold nanoparticle was prepared on a gold substrate of a chip for a surface plasmon resonance (SPR) sensor for fabricating an SPR sensor sensitive to a low molecular weight analyte. The sensing is based on swelling of the imprinted polymer gel that is triggered by an analyte binding event within the polymer gel. The swelling causes greater distance between the gold nanoparticles and substrate, shifting a dip of an SPR curve to a higher SPR angle. The polymer synthesis was conducted by radical polymerization of a mixture of acrylic acid, N-isopropylacrylamide, N,N'-methylenebisacrylamide, and gold nanoparticles in the presence of dopamine as model template species on a sensor chip coated with allyl mercaptan. The modified sensor chip showed an increasing SPR angle in response to dopamine concentration, which agrees with the expected sensing mechanism. Furthermore, the gold nanoparticles were shown to be effective for enhancing the signal intensity (the change of SPR angle) by comparison with a sensor chip immobilizing no gold nanoparticles. The analyte binding process and the consequent swelling appeared to be reversible, allowing one the repeated use of the presented sensor chip.

Gold↗
PubMed2005

DNA-based biosensor for monitoring pH in vitro and in living cells.

DNA is a promising material for the construction of a biosensor or bioindicator because its structure is sensitive to the binding of cofactors. In the current studies, we found that a combination of two DNA oligonucleotides, 5'-TCTTTCTCTTCT-3' and 5'-AGAAAGAGAAGA-3', exhibit a novel structural transition from a Watson-Crick antiparallel duplex to a parallel Hoogsteen duplex as the pH changes from pH 7.0 to 5.0. By labeling this DNA for fluorescence resonance energy transfer, we were able to develop a sensitive pH indicator that can detect changes between pH 7.0 and 5.0. Moreover, using DNA-based hairpin parallel-stranded duplex in conjunction with fluorescence microscopy, we were able to observe the pH changes in living cells during apoptosis as an easily detected change in color. These results indicate that the DNA-based pH indicator should be useful for detecting pH changes between pH 7.0 and 5.0 in living cells.

Base Pairing↗
PubMed2005

Roles of Mg2+ in TPP-dependent riboswitch.

We quantified the effect of Mg(2+) on thiamine pyrophosphate (TPP) binding to TPP-dependent thiA riboswitch RNA. The association constant of TPP binding to the riboswitch at 20 degrees C increased from 1.2 x 10(6) to 50 x 10(6) M(-1) as the Mg(2+) concentration increased from 0 to 1 mM. Furthermore, circular dichroic spectra under various conditions showed that 1 mM Mg(2+) induced a local structural change of the riboswitch, which might be pivotal for TPP binding. These results indicate that a physiological concentration of Mg(2+) can regulate TPP binding to the thiA riboswitch.

5' Untranslated Regions↗
PubMed2005

Structure and stability of DNA quadruplexes under molecular crowding conditions.

A living cell contains generally macromolecules occupying 20-40% of the total volume. To mimic the crowded cellular condition, we prepared solutions including various cosolutes and investigated the influence of the cosolute on the structure and stability of DNA G-quadruplex. As a result, Tetrahymena telomere sequences form well-ordered G-wires in the presence of cosolutes, whereas human telomere sequences remain as compact G-quadruplexes. Since these sequence motif differ by only one base, these results demonstrate that a single base difference in telomere sequences leads to drastically different structures under the molecular crowding conditions. The findings are useful for understanding G-quadruplex structures in cell-like conditions and for design of DNA nanomaterials.

Animals↗
PubMed2005

Structural switch of telomere DNA by pH and monovalent cation.

Because of the importance of telomere DNAs, the structures of these DNAs in vivo and in vitro are currently of great research interest in the medical, pharmaceutical, chemical, and industrial fields. In this study, we investigated the structure and thermodynamic properties of the telomere DNAs in the presence of monovalent cations, K+ or Na+. The results demonstrate that the duplex-quadruplex conversion of the 1:1 mixture of telomere G-rich and C-rich sequences can be induced depending on cation species and its concentration. Furthermore, slightly acidic condition stabilizes the i-motif and forces the mixture to dissociate. These results suggest that the structure and stability of telomere DNAs can be controlled by monovalent cation and pH.

Cations, Monovalent↗
PubMed2005

DNA nanowire sensitive to the surrounding condition.

We designed and synthesized an artificial DNA that undergoes a structural transition induced by metal ions. A 2, 2'-bipyridine unit was incorporated into the main chain of d(G4T4G4) instead of thymine. Structural analyses of the artificial DNA with and without metal ions demonstrated that micromolar concentrations of metal ions can induce a structural transition of antiparallel G-quadruplex to a G-wire, which shows the DNA is promising as a controllable and switchable supramolecular device.

2,2'-Dipyridyl↗
PubMed2004

Composite of Au nanoparticles and molecularly imprinted polymer as a sensing material.

A molecularly imprinted polymer with immobilized Au nanoparticles (Au-MIP) is reported as a novel type of sensing material. The sensing mechanism is based upon the variable proximity of the Au nanoparticles immobilized in the imprinted polymer, which exhibits selective binding of a given analyte accompanied by swelling that causes a blue-shift in the plasmon absorption band of the immobilized Au nanoparticles. Using adrenaline as the model analyte, it was shown that molecular imprinting effectively enhanced the sensitivity and selectivity, and accordingly, Au-MIP selectively detects the analyte at 5 microM. The combination of molecular imprinting and the Au nanoparticle-based sensing system was shown to be a general strategy for constructing sensing materials in a tailor-made fashion due to wide applicability of the imprinting technique and the independence of the sensing mechanism from the analyte recognition system.

Chemistry Techniques, Analytical↗
PubMed2004

Duplex dissociation of telomere DNAs induced by molecular crowding.

Because of the importance of telomere DNAs, the structures of these DNAs in vivo are currently of great research interest in the medical, pharmaceutical, chemical, and industrial fields. To understand the structure of biomolecules in vivo, their properties studied in vitro are extrapolated to the in vivo condition, while the condition in a living cell is inherently molecularly crowded and a nonideal solution contains various biomolecules. We investigated the effect of molecular crowding, which is one of the most important cellular environmental conditions, on the structure and stability of the telomere and G-rich and C-rich DNAs using circular dichroism (CD) spectra, CD melting curves, and isothermal titration calorimetry (ITC). The CD spectra and CD melting curves of G-rich DNA, C-rich DNA, and the 1:1 mixture of G-rich and C-rich DNAs showed that each G-rich DNA, C-rich DNA, and the 1:1 mixture form the antiparallel G-quadruplex, I-motif, and duplex, respectively, in the noncrowding condition as previously considered. On the contrary, the G-rich and C-rich DNAs individually form the parallel G-quadruplex and I-motif, respectively, in the molecular crowding condition, and the 1:1 mixture folds into the parallel G-quadruplex and I-motif but does not form a duplex. The ITC measurements indicated that the thermodynamic stability (DeltaG degrees (20)) of the duplex formation between the G-rich and C-rich DNAs in the noncrowding condition was -10.2 kcal mol(-)(1), while only a small heat change was observed in the ITC measurements in the molecular crowding condition. These ITC results also demonstrated that the molecular crowding condition prevents any duplex formation between G-rich and C-rich DNAs. These results indicate that a structural polymorphism of the telomere DNAs is induced by molecular crowding in vivo.

Calorimetry↗
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↗