Inhibition of RNA Phosphodiester Backbone Cleavage in the Presence of Organic Cations of Different Sizes.

Living cells contain various types of organic cations that may interact with nucleic acids. In order to understand the nucleic acid-binding properties of organic cations of different sizes, we investigated the ability of simple organic cations to inhibit the RNA phosphodiester bond cleavage promoted by Mg2+, Pb2+, and RNA-cleaving serum proteins. Kinetic analysis using chimeric DNA-RNA oligonucleotides showed that the cleavage at ribonucleotide sites was inhibited in the presence of monovalent cations comprising alkyl chains or benzene rings. The comparison of the cleavage rates in the presence of quaternary ammonium and phosphonium ions indicated that the steric hindrance effect of organic cations on their binding to the RNA backbone is significant when the cation size is larger than the phosphate-phosphate distance of a single-stranded nucleic acid. The cleavage inhibition was also observed for ribonucleotides located in long loops but not in short loops of oligonucleotide structures, indicating less efficient binding of bulky cations to structurally constrained regions. These results reveal the unique nucleic acid-binding properties of bulky cations distinct from those of metal ions.

Evaluation of weak interactions of proteins and organic cations with DNA duplex structures.

Molecular interactions and reactions in living cells occur with high background concentrations of organic compounds including proteins. Uncharged water-soluble polymers are commonly used cosolutes in studies on molecular crowding, and most studies argue about the effects of intracellular crowding based on results obtained using polymer cosolutes. Further investigations using protein crowders and organic cations are important in understanding the effects of cellular environments on nucleic acids with negatively charged surfaces. We assessed the effects of using model globular proteins, serum proteins, histone proteins, structurally flexible polypeptides, di- and polyamines, and uncharged polymers. Thermal stability analysis of DNA oligonucleotide structures revealed that unlike conventional polymer cosolutes, basic globular proteins (lysozyme and cytochrome c) at high concentrations stabilized long internal and bulge loop structures but not fully matched duplexes. The selective stabilization of long loop structures suggests preferential binding to unpaired nucleotides in loops through weak electrostatic interactions. Furthermore, the ability of the proteins to stabilize the loop structures was enhanced under macromolecular crowding conditions. Remarkably, the effects of basic proteins on the stability of fully matched duplexes were dissimilar to those of basic amino-acid-rich polypeptides and polyamines. This study provides new insights into the interaction of nucleic acid structures with organic cations.

Enhancement of the Catalytic Activity of Hammerhead Ribozymes by Organic Cations.

Catalytic turnover is important for the application of ribozymes to biotechnology. However, the turnover is often impaired because of the intrinsic high stability of base pairs with cleaved RNA products. Here, organic cations were used as additives to improve the catalytic performance of hammerhead ribozyme constructs that exhibit different kinetic behaviors. Kinetic analysis of substrate cleavage demonstrated that bulky cations, specifically tetra-substituted ammonium ions containing pentyl groups or a benzyl group, have the ability to greatly increase the turnover rate of the ribozymes. Thermal stability analysis of RNA structures revealed that the bulky cations promote the dissociation of cleaved products and refolding of incorrectly folded structures with small disruption of the catalytic structure. The use of bulky cations is a convenient method for enhancing the catalytic activity of hammerhead ribozymes, and the approach may be useful for advancing ribozyme technologies.

Controlling Interfacial Ion-Transport Kinetics Using Polyelectrolyte Membranes for Additive- and Effluent-free, High-Performance Electrodeposition.

The development of high-performance, environmentally friendly electrodeposition processes is critical for emerging coating technologies because current technologies use highly complex baths containing metal salts, supporting electrolytes, and various kinds of organic additives, which are problematic from both environmental and cost perspectives. Here, we show that a 200 µm-thin polyelectrolyte membrane sandwiched between electrodes effectively concentrates metal ions through interfacial penetration, which increases the conductance between the electrodes to 0.30 S and realizes solid-state electrodeposition that produces no mist, sludge, or even waste effluent. Both, experimental results and theoretical calculations, reveal that electrodeposition is controlled by ion penetration at the solution/polyelectrolyte interface, providing an intrinsically different ion-transport mechanism to that of conventional diffusion-controlled electrodeposition. The setup, which includes 0.50 mol L-1 copper sulfate and no additives, delivers a maximum current density of 300 mA cm-2, which is nearly fivefold higher than that of a current commercial plating bath containing organic additives.

Stabilization of DNA Loop Structures by Large Cations.

The DNA-binding properties of large cations differ from those of metal ions due to steric exclusion from base-paired regions. In this study, the thermal stability of DNA secondary structures, including duplexes, internal loops, bulge loops, hairpin loops, dangling ends, and G-quadruplexes, was investigated in the presence of cations of different sizes. Large cations, such as tetrabutylammonium and tetrapentylammonium ions, reduced the stability of fully matched duplexes but increased the stability of duplexes with a long loop. The cations also increased the stability of G-quadruplexes with a long loop, and the degree of stabilization was greater for low-stability G-quadruplexes. Analysis of the salt concentration dependence indicates that large cations bind to the loop nucleotides, leading to counteracting the destabilization effect on base pairing. It is likely that binding occurs when loop nucleotides are sufficiently flexible to allow for greater accessibility for large cations. These results provide insight into nucleic acid interactions with large cationic molecules and suggest a potential method for stabilizing noncanonical DNA structures under intracellular conditions.

Bulky cations greatly increase the turnover of a native hammerhead ribozyme.

Methods to facilitate the catalytic turnover of ribozymes are required for advancing oligonucleotide-based technologies. This study examined tetraalkylammonium ions for their ability to increase the efficiency of catalytic turnover of a native hammerhead ribozyme. Kinetic analysis showed that large tetraalkylammonium ions significantly increased the turnover rate of the ribozyme and was much more effective than poly(ethylene glycol) (PEG) and urea. The magnitude of the rate increase depended on the concentrations of Mg2+ and tetrapentylammonium ions, and the rate was enhanced by more than 180-fold at the optimal concentrations of these salts. The results provide physical insights into interactions of ribozymes with large cationic molecules through electrostatic forces and steric hindrance.

Thermal stability and conformation of DNA and proteins under the confined condition in the matrix of hydrogels.

Spatially confined environments are seen in biological systems and in the fields of biotechnology and nanotechnology. The confinement restricts the conformational space of polymeric molecules and increasing the degree of molecular crowding. Here, we developed preparation methods for agarose and polyacrylamide gels applicable to UV spectroscopy that can evaluate the confinement effects on DNA and protein structures. Measurements of UV absorbance and CD spectra showed no significant effect of the confinement in the porous media of agarose gels on the base-pair stability of DNA polynucleotides [poly(dA)/poly(dT)] and oligonucleotides (hairpin, duplex, and triplex structures). On the other hand, a highly confined environment created by polyacrylamide gels at high concentrations increased the stability of polynucleotides while leaving that of oligonucleotides unaffected. The changes in the base-pair stability of the polynucleotides were accompanied by the perturbation of the helical conformation. The polyacrylamide gels prepared in this study were also used for the studies on proteins (lysozyme, bovine serum albumin, and myoglobin). The effects on the proteins were different from the effects on DNA structures, suggesting different nature of interactions within the gel. The experimental methods and results are useful to understand the physical properties of nucleic acids and proteins under confined conditions.

DNA G-Wire Formation Using an Artificial Peptide is Controlled by Protease Activity.

The development of a switching system for guanine nanowire (G-wire) formation by external signals is important for nanobiotechnological applications. Here, we demonstrate a DNA nanostructural switch (G-wire <--> particles) using a designed peptide and a protease. The peptide consists of a PNA sequence for inducing DNA to form DNA-PNA hybrid G-quadruplex structures, and a protease substrate sequence acting as a switching module that is dependent on the activity of a particular protease. Micro-scale analyses via TEM and AFM showed that G-rich DNA alone forms G-wires in the presence of Ca2+, and that the peptide disrupted this formation, resulting in the formation of particles. The addition of the protease and digestion of the peptide regenerated the G-wires. Macro-scale analyses by DLS, zeta potential, CD, and gel filtration were in agreement with the microscopic observations. These results imply that the secondary structure change (DNA G-quadruplex <--> DNA/PNA hybrid structure) induces a change in the well-formed nanostructure (G-wire <--> particles). Our findings demonstrate a control system for forming DNA G-wire structures dependent on protease activity using designed peptides. Such systems hold promise for regulating the formation of nanowire for various applications, including electronic circuits for use in nanobiotechnologies.

Modulation of Ribozyme and Deoxyribozyme Activities Using Tetraalkylammonium Ions.

Nucleic acid enzymes require specific metal ions to be catalytically active. The functions of the metal ions having structural and catalytic roles are affected by competing cations. Large-sized tetraalkylammonium ions have a propensity to preferentially bind to single strands of RNA and DNA. Here, the large cations are used in the reactions of lead-dependent ribozyme and 17E deoxyribozyme that require divalent metal ions to cleave a nucleic acid substrate. Kinetic analysis shows that tetraalkylammonium ions influence the rate of substrate cleavage, and the effects are different depending on the nucleic acid enzymes and metal ions used. Importantly, the large cations used here increase the dependence of cleavage rates on metal ion concentration and enhance the ability of the enzyme to monitor changes in metal ion concentrations. The same effect is also observed for the metal ion concentration dependence of the thermal stability of RNA and DNA structures, indicating that the large cations affect the binding of structural metal ions. The use of large tetraalkylammonium ions provides new ways to study the importance of metal ions to nucleic acid enzymes, and also to modulate the functionality of nucleic acid enzymes.

Newly characterized interaction stabilizes DNA structure: oligoethylene glycols stabilize G-quadruplexes CH-π interactions.

Oligoethylene glycols are used as crowding agents in experiments that aim to understand the effects of intracellular environments on DNAs. Moreover, DNAs with covalently attached oligoethylene glycols are used as cargo carriers for drug delivery systems. To investigate how oligoethylene glycols interact with DNAs, we incorporated deoxythymidine modified with oligoethylene glycols of different lengths, such as tetraethylene glycol (TEG), into DNAs that form antiparallel G-quadruplex or hairpin structures such that the modified residues were incorporated into loop regions. Thermodynamic analysis showed that because of enthalpic differences, the modified G-quadruplexes were stable and the hairpin structures were slightly unstable relative to unmodified DNA. The stability of G-quadruplexes increased with increasing length of the ethylene oxides and the number of deoxythymidines modified with ethylene glycols in the G-quadruplex. Nuclear magnetic resonance analyses and molecular dynamics calculations suggest that TEG interacts with bases in the G-quartet and loop via CH-π and lone pair-π interactions, although it was previously assumed that oligoethylene glycols do not directly interact with DNAs. The results suggest that numerous cellular co-solutes likely affect DNA function through these CH-π and lone pair-π interactions.

Effects of trimethylamine N-oxide and urea on DNA duplex and G-quadruplex.

We systematically investigated effects of molecular crowding with trimethylamine N-oxide (TMAO) as a zwitterionic and protective osmolyte and urea as a nonionic denaturing osmolyte on conformation and thermodynamics of the canonical DNA duplex and the non-canonical DNA G-quadruplex. It was found that TMAO and urea stabilized and destabilized, respectively, the G-quadruplex. On the other hand, these osmolytes generally destabilize the duplex; however, it was observed that osmolytes having the trimethylamine group stabilized the duplex at the lower concentrations because of a direct binding to a groove of the duplex. These results are useful not only to predict DNA structures and their thermodynamics under physiological environments in living cells, but also design of polymers and materials to regulate structure and stability of DNA sequences.

Model studies of the effects of intracellular crowding on nucleic acid interactions.

Molecular interactions and reactions in living cells occur with high concentrations of background molecules and ions. Many research studies have shown that intracellular molecules have characteristics different from those obtained using simple aqueous solutions. To better understand the behavior of biomolecules in intracellular environments, biophysical experiments were conducted under cell-mimicking conditions in a test tube. It has been shown that the molecular environments at the physiological level of macromolecular crowding, spatial confinement, water activity and dielectric constant, have significant effects on the interactions of DNA and RNA for hybridization, higher-order folding, and catalytic activity. The experimental approaches using in vitro model systems are useful to reveal the origin of the environmental effects and to bridge the gap between the behaviors of nucleic acids in vitro and in vivo. This paper highlights the model experiments used to evaluate the influences of intracellular environment on nucleic acid interactions.

Thermal Stability of RNA Structures with Bulky Cations in Mixed Aqueous Solutions.

Bulky cations are used to develop nucleic-acid-based technologies for medical and technological applications in which nucleic acids function under nonaqueous conditions. In this study, the thermal stability of RNA structures was measured in the presence of various bulky cations in aqueous mixtures with organic solvents or polymer additives. The stability of oligonucleotide, transfer RNA, and polynucleotide structures was decreased in the presence of salts of tetrabutylammonium and tetrapentylammonium ions, and the stability and salt concentration dependences were dependent on cation sizes. The degree to which stability was dependent on salt concentration was correlated with reciprocals of the dielectric constants of mixed solutions, regardless of interactions between the cosolutes and RNA. Our results show that organic solvents affect the strength of electrostatic interactions between RNA and cations. Analysis of ion binding to RNA indicated greater enhancement of cation binding to RNA single strands than to duplexes in media with low dielectric constants. Furthermore, background bulky ions changed the dependence of RNA duplex stability on the concentration of metal ion salts. These unique properties of large tetraalkylammonium ions are useful for controlling the stability of RNA structures and its sensitivity to metal ion salts.

A reversible B-A transition of DNA duplexes induced by synthetic cationic copolymers.

Although the B-form duplex is the canonical DNA structure, the A-form duplex plays critical roles in controlling gene expression. Here, reversible B-A transitions of DNA duplexes were induced by synthetic cationic and anionic polymers. Thermodynamic analysis demonstrated that the B-A transition was regulated by the dehydration of the DNA duplex caused by polymer binding.

Use of a Ureido-Substituted Deoxycytidine Module for DNA Assemblies.

Ureido-substituted cytosine derivatives are promising for constructing self-assembly structures that can be applied to nanotechnology research. However, conventional cytosine modules achieve assembly in organic solvents. In this study, an N-phenylcarbamoyl deoxycytidine nucleoside was incorporated into a C-rich oligonucleotide to achieve self-assembly in aqueous solution. The results show that the capability of the module to form DNA assemblies varied depending on the number of modules incorporated. The deoxycytidine derivative has a potential application in the development of smart materials based on DNA assembly.

The structural stability and catalytic activity of DNA and RNA oligonucleotides in the presence of organic solvents.

Organic solvents and apolar media are used in the studies of nucleic acids to modify the conformation and function of nucleic acids, to improve solubility of hydrophobic ligands, to construct molecular scaffolds for organic synthesis, and to study molecular crowding effects. Understanding how organic solvents affect nucleic acid interactions and identifying the factors that dominate solvent effects are important for the creation of oligonucleotide-based technologies. This review describes the structural and catalytic properties of DNA and RNA oligonucleotides in organic solutions and in aqueous solutions with organic cosolvents. There are several possible mechanisms underlying the effects of organic solvents on nucleic acid interactions. The reported results emphasize the significance of the osmotic pressure effect and the dielectric constant effect in addition to specific interactions with nucleic acid strands. This review will serve as a guide for the selection of solvent systems based on the purpose of the nucleic acid-based experiments.

Thermodynamic properties of water molecules in the presence of cosolute depend on DNA structure: a study using grid inhomogeneous solvation theory.

In conditions that mimic those of the living cell, where various biomolecules and other components are present, DNA strands can adopt many structures in addition to the canonical B-form duplex. Previous studies in the presence of cosolutes that induce molecular crowding showed that thermal stabilities of DNA structures are associated with the properties of the water molecules around the DNAs. To understand how cosolutes, such as ethylene glycol, affect the thermal stability of DNA structures, we investigated the thermodynamic properties of water molecules around a hairpin duplex and a G-quadruplex using grid inhomogeneous solvation theory (GIST) with or without cosolutes. Our analysis indicated that (i) cosolutes increased the free energy of water molecules around DNA by disrupting water-water interactions, (ii) ethylene glycol more effectively disrupted water-water interactions around Watson-Crick base pairs than those around G-quartets or non-paired bases, (iii) due to the negative electrostatic potential there was a thicker hydration shell around G-quartets than around Watson-Crick-paired bases. Our findings suggest that the thermal stability of the hydration shell around DNAs is one factor that affects the thermal stabilities of DNA structures under the crowding conditions.

Effects of Cosolvents on the Folding and Catalytic Activities of the Hammerhead Ribozyme.

The RNA cleavage activity of the hammerhead ribozyme has been compared in various mixed aqueous solutions containing cosolvents. Kinetic analysis revealed that the tested cosolvents enhanced the ribozyme activity, particularly at low MgCl2 concentrations. These enhancements, in some cases of more than tenfold, resulted from a reduction in the Mg(2+) concentration required for substrate cleavage. An inverse correlation was found between the MgCl2 concentration essential for efficient catalysis and the dielectric constant values. In contrast, FRET measurements showed no substantial influence of cosolvents on the Mg(2+) -induced structural transitions. The results suggest that the solution environment has various effects on the Mg(2+) interactions involved in the catalysis and global folding of the ribozyme.

Effects of background anionic compounds on the activity of the hammerhead ribozyme in Mg(2+)-unsaturated solutions.

Cellular ribozymes exhibit catalytic activity in media containing large numbers of anionic compounds and macromolecules. In this study, the RNA cleavage activity of the hammerhead ribozyme induced by Mg(2+) was investigated using the solutions containing background nucleic acids, small phosphate and carboxylic acid compounds, and neutral polymers. Analysis of the substrate cleavage kinetics showed that the anionic compounds do not affect the ribozyme activity in Mg(2+)-saturated solutions and there is almost no effect of the anion-Mg(2+) complexes formed. On the other hand, the rate of substrate cleavage in Mg(2+)-unsaturated solutions was reduced under conditions of a high background of anionic compounds found in cells. The extent of the reduction was more with a greater net negative charge, caused by decreased amounts of Mg(2+) that could be used for the ribozyme reaction. It was remarkable that background DNA and RNA molecules having phosphodiester bonds reduced the cleavage rate as much as adenosine monophosphates having a charge of -2 when the effects of the same amount of phosphate groups were compared. Greater reductions in rates than those expected from the molecular charge were also observed in the background of fatty acids that form micelles. An addition of poly(ethylene glycol) to the solutions partially restored the ribozyme activity, suggesting a possible role of macromolecular crowding in counteracting the inhibitory effects of background anions on the ribozyme reaction. The results have biological and practical implications with respect to the effects of molecular environment on the efficiency of ion binding to RNA.

Roles of the amino group of purine bases in the thermodynamic stability of DNA base pairing.

The energetic aspects of hydrogen-bonded base-pair interactions are important for the design of functional nucleotide analogs and for practical applications of oligonucleotides. The present study investigated the contribution of the 2-amino group of DNA purine bases to the thermodynamic stability of oligonucleotide duplexes under different salt and solvent conditions, using 2'-deoxyriboinosine (I) and 2'-deoxyribo-2,6-diaminopurine (D) as non-canonical nucleotides. The stability of DNA duplexes was changed by substitution of a single base pair in the following order: G • C > D • T ≈ I • C > A • T > G • T > I • T. The apparent stabilization energy due to the presence of the 2-amino group of G and D varied depending on the salt concentration, and decreased in the water-ethanol mixed solvent. The effects of salt concentration on the thermodynamics of DNA duplexes were found to be partially sequence-dependent, and the 2-amino group of the purine bases might have an influence on the binding of ions to DNA through the formation of a stable base-paired structure. Our results also showed that physiological salt conditions were energetically favorable for complementary base recognition, and conversely, low salt concentration media and ethanol-containing solvents were effective for low stringency oligonucleotide hybridization, in the context of conditions employed in this study.