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Naoki Sugimoto - One of the best experts on this subject based on the ideXlab platform.
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nearest neighbor parameters for predicting dna duplex stability in diverse Molecular Crowding conditions
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Saptarshi Ghosh, Shuntaro Takahashi, Tamaki Endoh, Tatsuya Ohyama, Hisae Tateishikarimata, Naoki SugimotoAbstract:The intracellular environment is crowded and heterogeneous. Although the thermodynamic stability of nucleic acid duplexes is predictable in dilute solutions, methods of predicting such stability under specific intracellular conditions are not yet available. We recently showed that the nearest-neighbor model for self-complementary DNA is valid under Molecular Crowding condition of 40% polyethylene glycol with an average Molecular weight of 200 (PEG 200) in 100 mM NaCl. Here, we determined nearest-neighbor parameters for DNA duplex formation under the same Crowding condition to predict the thermodynamics of DNA duplexes in the intracellular environment. Preferential hydration of the nucleotides was found to be the key factor for nearest-neighbor parameters in the Crowding condition. The determined parameters were shown to predict the thermodynamic parameters (∆H°, ∆S°, and ∆G°37) and melting temperatures (Tm) of the DNA duplexes in the Crowding condition with significant accuracy. Moreover, we proposed a general method for predicting the stability of short DNA duplexes in different cosolutes based on the relationship between duplex stability and the water activity of the cosolute solution. The method described herein would be valuable for investigating biological processes that occur under specific intracellular crowded conditions and for the application of DNA-based biotechnologies in crowded environments.
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2020Co-Authors: Takeshi Fujimoto, Daisuke Miyoshi, Shuichi Nakano, Naoki SugimotoAbstract: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 Gquadruplex 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
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Validation of the nearest-neighbor model for Watson-Crick self-complementary DNA duplexes in Molecular Crowding condition.
Nucleic Acids Research, 2019Co-Authors: Saptarshi Ghosh, Shuntaro Takahashi, Tamaki Endoh, Hisae Tateishi-karimata, Soumitra Hazra, Naoki SugimotoAbstract:Recent advancement in nucleic acid techniques inside cells demands the knowledge of the stability of nucleic acid structures in Molecular Crowding. The nearest-neighbor model has been successfully used to predict thermodynamic parameters for the formation of nucleic acid duplexes, with significant accuracy in a dilute solution. However, knowledge about the applicability of the model in Molecular Crowding is still limited. To determine and predict the stabilities of DNA duplexes in a cell-like crowded environment, we systematically investigated the validity of the nearest-neighbor model for Watson-Crick self-complementary DNA duplexes in Molecular Crowding. The thermodynamic parameters for the duplex formation were measured in the presence of 40 wt% poly(ethylene glycol)200 for different self-complementary DNA oligonucleotides consisting of identical nearest-neighbors in a physiological buffer containing 0.1 M NaCl. The thermodynamic parameters as well as the melting temperatures (Tm) obtained from the UV melting studies revealed similar values for the oligonucleotides having identical nearest-neighbors, suggesting the validity of the nearest-neighbor model in the Crowding condition. Linear relationships between the measured ΔG°37 and Tm in Crowding condition and those predicted in dilute solutions allowed us to predict ΔG°37, Tm and nearest-neighbor parameters in Molecular Crowding using existing parameters in the dilute condition, which provides useful information about the thermostability of the self-complementary DNA duplexes in Molecular Crowding.
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Bisubstrate Function of RNA Polymerases Triggered by Molecular Crowding Conditions
2019Co-Authors: Shuntaro Takahashi, Hiromichi Okura, Naoki SugimotoAbstract:Since the origin of life on Earth, the role of carrying genetic information has been presumably transferred from RNA to DNA. At present, cellular environments are extremely dense, packed with cosolutes and macromolecules. Hence, the preference between RNA-dependent RNA and DNA polymerization may be affected by Molecular Crowding. In this study, we investigated both RNA-dependent RNA and DNA polymerizations by tC9Y polymerase ribozyme, T7 RNA polymerase (T7 RNAP), and Klenow fragment DNA polymerase (KF) under different Molecular Crowding conditions. Poly(ethylene glycol) (PEG) of various Molecular weights was used as a Crowding agent and found to promote both RNA and DNA ribozyme-catalyzed polymerizations. In contrast, PEG with an average Molecular weight of 200 (PEG200) reduced the level of RNA polymerization by proteinaceous T7 RNAP but simultaneously promoted DNA polymerization, without affecting the activity of KF. Thus, proteinaceous RNA polymerase might potentially display bisubstrate specificity, which can be switched in response to changes in the dielectric constant and excluded volume in crowded environments. Our findings validate the bisubstrate activity of RNA polymerase from an evolutionary perspective for the development of non-natural materials
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Characterization of Intracellular Crowding Environments with Topology-Based DNA Quadruplex Sensors
2019Co-Authors: Shuntaro Takahashi, Johtaro Yamamoto, Akira Kitamura, Masataka Kinjo, Naoki SugimotoAbstract:Molecular Crowding creates a unique environment in cells and imposes physical constraints such as the excluded volume effect, water activity, and dielectric constant that can affect the structure and function of biomolecules. It is therefore important to develop a method for quantifying the effects of Molecular Crowding in cells. In this study, we developed a Förster resonance energy transfer (FRET) probe based on a guanine-quadruplex (G4) DNA motif that shows distinct FRET signals in response to Crowding conditions in the presence of salt and poly(ethylene glycol). FRET efficiencies varied in different solutions, reflecting the dependence of G4 stability and topology on salt concentration and water activity. In living cells, FRET signals in the nucleus were higher than those in the cytosol; the signals in membraneless nuclear compartments (i.e., nucleolus) were especially high, suggesting that a decrease in water activity is important for the Crowding effect in the nucleus. Thus, the use of DNA sensors with variable structures can elucidate the local effects of Molecular Crowding in cells
Daisuke Miyoshi - One of the best experts on this subject based on the ideXlab platform.
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2020Co-Authors: Takeshi Fujimoto, Daisuke Miyoshi, Shuichi Nakano, Naoki SugimotoAbstract: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 Gquadruplex 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
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study on effects of Molecular Crowding on g quadruplex ligand binding and ligand mediated telomerase inhibition
Methods, 2013Co-Authors: Hidenobu Yaku, Shuichi Nakano, Daisuke Miyoshi, Hisae Tateishikarimata, Takashi Murashima, Naoki SugimotoAbstract: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.
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beads on a string structure of long telomeric dnas under Molecular Crowding conditions
Journal of the American Chemical Society, 2012Co-Authors: Shuichi Nakano, Daisuke Miyoshi, Naoki SugimotoAbstract:The structure and stability of long telomeric DNAs, (T2AG3)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 impli...
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Molecular Crowding and hydration regulating of g quadruplex formation
Topics in Current Chemistry, 2012Co-Authors: Daisuke Miyoshi, Takeshi Fujimoto, Naoki SugimotoAbstract:Intracellular space is highly crowded with soluble and insoluble biomolecules that range from large polymers, such as proteins and nucleic acids, to small molecules, including metabolites and metal ions. It is therefore of interest to understand the effects of Molecular Crowding on the structure, stability, and function of biomolecules. Moreover, Molecular Crowding is observed not only intracellularly but also in the extracellular matrix and under the conditions used in in vitro biotechnological and nanotechnological processes. However, most biochemical studies of biomolecules are performed under dilute conditions. Recent studies have demonstrated critical effects of Molecular Crowding on nucleic acids. In the present study we discuss how Molecular Crowding affects the properties of G-quadruplexes as well as other non-canonical nucleic acid structures.
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stabilization of three way junctions of dna under Molecular Crowding conditions
Journal of the American Chemical Society, 2009Co-Authors: Sanjukta Muhuri, Daisuke Miyoshi, Kenta Mimura, Naoki SugimotoAbstract:We examined the effects of Molecular Crowding conditions on the structures and thermodynamics of three-way junctions (TWJs) of DNA. Structural analysis utilizing gel electrophoresis and circular dichroism spectroscopy showed that the designed DNAs folded into TWJ structures in the presence of Na+ and Mg2+ under both dilute and Molecular Crowding conditions with polyethylene glycol 200 (PEG 200). From the thermodynamic parameters evaluated by UV melting techniques in the absence and presence of 5 mM Mg2+ under dilute and Molecular Crowding conditions, it was clear that Mg2+ stabilized all TWJs under the dilute condition, although the extent of stabilization depended on the stacking partners of TWJs. For example, thermodynamic stability (−ΔG°37) of A/B-stacked TWJs (A, B, and C are the three helices of TWJ, and among these helices, A and B are stacked together) increased from 3.7 to 5.6 kcal/mol by the addition of 5 mM Mg2+, while that of A/C-stacked TWJs (A and C are stacked together) increased only from 3...
Zhaosheng Liu - One of the best experts on this subject based on the ideXlab platform.
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improvement of imprinting effect of ionic liquid Molecularly imprinted polymers by use of a Molecular Crowding agent
Analytical and Bioanalytical Chemistry, 2018Co-Authors: Man Jia, Zhaosheng Liu, Jian Yang, Ya Kun Sun, Xi Bai, Haji Akber AisaAbstract:We aimed to improve the imprinting effect of ionic liquid Molecularly imprinted polymers (MIPs) by use of a Molecular Crowding agent. The ionic liquid 1-vinyl-3-ethylimidazolium tetrafluoroborate ([VEIm][BF4]) was used as the functional monomer and aesculetin was used as the template molecule in a Crowding environment, which was made up of a tetrahydrofuran solution of polystyrene. The ionic liquid MIPs that were prepared in the Crowding environment displayed an enhanced imprinting effect. NMR peak shifts of active hydrogen of aesculetin suggested that interaction between the functional monomer and the template could be increased by the use of a Crowding agent in the self-assembly process. The retention and selectivity of aesculetin were affected greatly by high Molecular Crowding, the amount of high Molecular weight Crowding agent, and the ratio of [VEIm][BF4] to aesculetin. The optimal MIPs were used as solid-phase extraction sorbents to extract aesculetin from Cichorium glandulosum. A calibration curve was obtained with aesculetin concentrations from 0.0005 to 0.05 mg mL−1 (correlation coefficient R 2 of 0.9999, y = 1519x + 0.0923). The limit of quantification was 0.12 μg mL−1, and the limit of detection was 0.05 μg mL−1. The absolute recovery of aesculetin was (80 ± 2)% (n = 3), and the purity of aesculetin was (92 ± 0.5)% (n = 5). As a conclusion, Molecular Crowding is an effective approach to obtain ionic liquid MIPs with high selectivity even in a polar solvent environment.
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effect of polyethylene glycol as a Molecular Crowding agent on reducing template consumption for preparation of Molecularly imprinted polymers
Analytical Methods, 2016Co-Authors: Man Jia, Zhaosheng Liu, Guangyin Sun, Yong Xin Zhao, Haji Akber AisaAbstract:The efforts in the present work focus on the topic of reducing template consumption in the preparation of Molecularly imprinted polymers (MIPs). For this purpose, the effect of polyethylene glycol (PEG) as a Molecular Crowding agent on reducing template consumption in the preparation of MIPs was investigated. To prepare MIP monoliths with punicalagin, acrylamide was used as the functional monomer, and ethylene glycol dimethacrylate was the cross-linking monomer, with a mixture of PEG–THF–toluene as a porogen. It was found that the Molecular weight of PEG, the ratio of the template to functional monomer, amount of PEG, level of DMF and the composition of the mobile phase greatly affected the retention of the template and the performance of Molecular recognition. The PEG-based imprinted monolith still showed a specific imprinting effect even at the ratio of functional monomer to template of 1353 : 1. The optimal MIPs can be used as solid-phase extraction sorbents for the purification of punicalagin with a recovery of 85% and a purity of 97.7% from the crude extract of pomegranate-rind.
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low cross linked Molecularly imprinted monolithic column prepared in Molecular Crowding conditions
Journal of Chromatography A, 2011Co-Authors: Xianhua Wang, Yanping Huang, Liang Zhao, Zhaosheng LiuAbstract:Molecular Crowding is a new approach to promoting Molecular imprinting more efficiently. In this work, this concept was applied to the preparation of low cross-linked imprinted polymers in the presence of an immobilised template for stabilizing binding sites and improving Molecular recognition. An imprinted monolithic column was synthesized using a mixture of 2,4-diamino-6-methyl-1,3,5-triazine (template), 2,4-diamino-6-(methacryloyloxy) ethyl-1,3,5-triazine (polymerisable template), methacrylic acid, ethylene glycol dimethacrylate, and polystyrene (Molecular Crowding agent). Some polymerization factors, such as template-monomer molar ratio, the composition of the porogen and crosslinking density, on the imprinting effect of resulting MIP monolith were systematically investigated. The results indicated that the imprinted monolithic columns prepared in the presence of Molecular Crowding agent retained affinity and specificity for template even when prepared with a level of cross-linker as low as 9%. Moreover, a stoichiometric displacement model for retention was successfully applied to evaluate the interaction between the solute and the stationary phase. Compared with the low cross-linked MIP prepared by conventional polymerization, the Molecular Crowding-based low cross-linked monolithic MIPs showed higher selectivity. The results suggested that Molecular Crowding is a powerful strategy to increase the effect of Molecular imprinting at a low level of crosslinker.
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preparation of imprinted monolithic column under Molecular Crowding conditions
Chinese Chemical Letters, 2011Co-Authors: Xin Liu, Li Hong Bai, Hongquan Duan, Yanping Huang, Zhaosheng LiuAbstract:Abstract Molecular Crowding is a new concept to obtain Molecularly imprinted polymers (MIPs) with greater capacity and selectivity. In this work, Molecular Crowding agent was firstly applied to the preparation of MIPs monolithic column. A new polymerization system based on Molecular Crowding surrounding was developed to prepare enrofloxacin-imprinted monolith, which was composed of polystyrene and tetrahydrofuran. The result showed that the monolithic MIPs under Molecular Crowding conditions presented good Molecular recognition for enrofloxacin with an imprinting factor of 3.03.
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cec separation of ofloxacin enantiomers using imprinted microparticles prepared in Molecular Crowding conditions
Electrophoresis, 2011Co-Authors: Xiaoxu Shi, Hongquan Duan, Yanping Huang, Zhaosheng LiuAbstract:Molecular Crowding is a new concept to obtain Molecularly imprinted polymers (MIPs) with greater capacity and selectivity, which could shift the equilibrium of a print molecule reacting with functional monomers in the direction of complex formation side. In this work, Molecular Crowding agent was first applied to the preparation of MIPs microparticles by precipitation polymerization. A new system of Molecular Crowding surrounding was developed, composed of polystyrene and tetrahydrofuran, in the presence of the template (S)-ofloxacin. Partial filling capillary electrochromatography (CEC) was utilized to evaluate imprinting effect of the resulting microparticles by chiral separations of ofloxacin. Some important parameters in the preparation, i.e. template to monomer ratio, influence of cross-linking monomers and functional monomer composition on the CEC separation of MIP microparticles were investigated. Baseline separation of ofloxacin (R(s) =1.53) was obtained under optimized conditions and the highest theory plate of the later eluent (S)-ofloxacin was 5400. The textural and morphological parameters for imprinted particles, such as Brunauer-Emmett-Teller surface areas, pore volumes and pore size distributions have also been determined. Compared to the MIP microparticle prepared by conventional precipitation polymerization, the (S)-ofloxacin-imprinted particles formed under Molecular Crowding conditions showed higher selectivity (α=1.09) and separation efficiency (<25 min) in the CEC mode.
Randy M Wadkins - One of the best experts on this subject based on the ideXlab platform.
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epigenetic modification dehydration and Molecular Crowding effects on the thermodynamics of i motif structure formation from c rich dna
Biochemistry, 2014Co-Authors: Yogini P Bhavsarjog, Eric Van Dornshuld, Tracy A Brooks, Gregory S Tschumper, Randy M WadkinsAbstract:DNA sequences with the potential to form secondary structures such as i-motifs (iMs) and G-quadruplexes (G4s) are abundant in the promoters of several oncogenes and, in some instances, are known to regulate gene expression. Recently, iM-forming DNA strands have also been employed as functional units in nanodevices, ranging from drug delivery systems to nanocircuitry. To understand both the mechanism of gene regulation by iMs and how to use them more efficiently in nanotechnological applications, it is essential to have a thorough knowledge of factors that govern their conformational states and stabilities. Most of the prior work to characterize the conformational dynamics of iMs have been done with iM-forming synthetic constructs like tandem (CCT)n repeats and in standard dilute buffer systems. Here, we present a systematic study on the consequences of epigenetic modifications, Molecular Crowding, and degree of hydration on the stabilities of an iM-forming sequence from the promoter of the c-myc gene. Our results indicate that 5-hydroxymethylation of cytosines destabilized the iMs against thermal and pH-dependent melting; contrarily, 5-methylcytosine modification stabilized the iMs. Under Molecular Crowding conditions (PEG-300, 40% w/v), the thermal stability of iMs increased by ∼10 °C, and the pKa was raised from 6.1 ± 0.1 to 7.0 ± 0.1. Lastly, the iM's stability at varying degrees of hydration in 1,2-dimethoxyethane, 2-methoxyethanol, ethylene glycol, 1,3-propanediol, and glycerol cosolvents indicated that the iMs are stabilized by dehydration because of the release of water molecules when folded. Our results highlight the importance of considering the effects of epigenetic modifications, Molecular Crowding, and the degree of hydration on iM structural dynamics. For example, the incorporation of 5-methylycytosines and 5-hydroxymethlycytosines in iMs could be useful for fine-tuning the pH- or temperature-dependent folding/unfolding of an iM. Variations in the degree of hydration of iMs may also provide an additional control of the folded/unfolded state of iMs without having to change the pH of the surrounding matrix.
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epigenetic modification dehydration and Molecular Crowding effects on the thermodynamics of i motif structure formation from c rich dna
Biochemistry, 2014Co-Authors: Yogini P Bhavsarjog, Eric Van Dornshuld, Tracy A Brooks, Gregory S Tschumper, Randy M WadkinsAbstract:DNA sequences with the potential to form secondary structures such as i-motifs (iMs) and G-quadruplexes (G4s) are abundant in the promoters of several oncogenes and, in some instances, are known to regulate gene expression. Recently, iM-forming DNA strands have also been employed as functional units in nanodevices, ranging from drug delivery systems to nanocircuitry. To understand both the mechanism of gene regulation by iMs and how to use them more efficiently in nanotechnological applications, it is essential to have a thorough knowledge of factors that govern their conformational states and stabilities. Most of the prior work to characterize the conformational dynamics of iMs have been done with iM-forming synthetic constructs like tandem (CCT)n repeats and in standard dilute buffer systems. Here, we present a systematic study on the consequences of epigenetic modifications, Molecular Crowding, and degree of hydration on the stabilities of an iM-forming sequence from the promoter of the c-myc gene. Our...
Shuichi Nakano - One of the best experts on this subject based on the ideXlab platform.
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2020Co-Authors: Takeshi Fujimoto, Daisuke Miyoshi, Shuichi Nakano, Naoki SugimotoAbstract: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 Gquadruplex 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
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study on effects of Molecular Crowding on g quadruplex ligand binding and ligand mediated telomerase inhibition
Methods, 2013Co-Authors: Hidenobu Yaku, Shuichi Nakano, Daisuke Miyoshi, Hisae Tateishikarimata, Takashi Murashima, Naoki SugimotoAbstract: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.
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beads on a string structure of long telomeric dnas under Molecular Crowding conditions
Journal of the American Chemical Society, 2012Co-Authors: Shuichi Nakano, Daisuke Miyoshi, Naoki SugimotoAbstract:The structure and stability of long telomeric DNAs, (T2AG3)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 impli...
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Molecular Crowding of the cosolutes induces an intraMolecular i motif structure of triplet repeat dna oligomers at neutral ph
Chemical Communications, 2010Co-Authors: Arivazhagan Rajendran, Shuichi Nakano, Naoki SugimotoAbstract: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 pKa 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.
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facilitation of rna enzyme activity in the Molecular Crowding media of cosolutes
Journal of the American Chemical Society, 2009Co-Authors: Shuichi Nakano, Hisae Karimata, Yuichi Kitagawa, Naoki SugimotoAbstract: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.