Effects of Molecular Crowding on the Structures, Interactions, and

Dec 23, 2013 - (226) Mel'niov, S. M.; Khan, M. O.; Lindman, B.; Jönsson, B. J. Am. Chem. Soc. 1999 .... (280) Blount, K. F.; Breaker, R. R. Nat. Biot...
0 downloads 0 Views 11MB Size
Download PDF
Review pubs.acs.org/CR

Effects of Molecular Crowding on the Structures, Interactions, and Functions of Nucleic Acids Shu-ichi Nakano,* Daisuke Miyoshi,* and Naoki Sugimoto* Department of Nanobiochemistry, Faculty of Frontiers of Innovative Research in Science and Technology (FIRST) and Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan 5.1. Noncanonical Nucleic Acid Structures in a Cell 5.2. Left-Handed Double Helix Structures 5.3. Branched Structures 5.4. Triple Helix Structures 5.5. Quadruplex Structures 5.6. Higher-Order RNA Structures 5.7. Enzymatic Activity of the Ribozymes 6. Perspectives Author Information Corresponding Authors Notes Biographies Acknowledgments References

CONTENTS 1. Introduction 2. Nucleic Acids under the Molecular Crowding Condition 2.1. Molecular Environment of a Living Cell 2.2. Nucleic Acids in the Cytoplasm 2.3. Nucleic Acids in the Nucleoplasm 2.4. Nucleic Acids Immobilized on Solid Surfaces 2.5. Computer Simulations of Intracellular Environments 3. Theoretical Background of the Molecular Crowding Effects on Nucleic Acid Interactions 3.1. Water-Soluble Cosolutes for Creating the Molecular Crowding Conditions 3.2. Excluded Volume Effect by Polymer Cosolutes 3.3. Influences of Polymer Cosolutes on DNA− Protein Interactions 3.4. Effect of Osmotic Pressure Created by Cosolutes 3.5. Cosolute Studies for DNA−Protein Interactions 3.6. Cosolute Studies for DNA−Ligand Interactions 4. Cosolute Effects on the Structures and Interactions of Nucleic Acid Duplexes 4.1. DNA Condensation 4.2. Stability of the Polynucleotide Duplexes 4.3. Structure, Thermodynamics, and Kinetics of the Short Duplexes 4.4. Ion Binding and Dielectric Constant Effects 4.5. Osmotic Pressure Studies of Oligonucleotides 5. Cosolute Effects on the Noncanonical Structures and Their Functions © 2013 American Chemical Society

2733 2735 2735 2736 2736 2737

2748 2748 2748 2748 2749 2750 2751 2752 2753 2753 2753 2753 2753 2754

1. INTRODUCTION Nucleic acids are excellent at recognizing complementary sequences through the formation of Watson−Crick base pairs. Since the base pairing enables the highly selective hybridization with a target sequence, synthetic DNA and RNA oligonucleotides are regarded as some of the most promising materials for therapeutic and diagnostic purposes, including human gene regulation,1−5 gene expression analysis,6−9 and target molecule sensing.10−13 In particular, clinical applications have provided many successes in target-specific gene regulations for the treatments of cancers,14−16 cardiovascular disease,17 and inflammatory diseases.18 Quantitative data of nucleic acid interactions are very useful for improving a number of oligonucleotide technologies. The strengths of the interbase hydrogen bonding and base stacking determine the hybridization efficiency and stability of nucleic acid structures; however, considerations of the hydration and counterion condensation are also important because water and cations associate with the polar purine and pyrimidine bases and the negatively charged sugar−phosphate backbone (Figure 1). Since the base pair formation is accompanied by the association or dissociation of ions and water molecules, nucleic acid interaction energies are significantly influenced by the solution composition.19−22 The stability of the Watson−Crick base pairing has been extensively studied using aqueous dilute solutions containing salts.23−27 These thermodynamic data allow the accurate predictions of the hybridization energy and secondary structures of a given sequence,28−30 and the

2737 2738 2738 2739 2739 2740 2742 2743 2744 2744 2744 2745 2745 2745

Received: February 22, 2013 Published: December 23, 2013

2748 2733

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

derivatives, methylamines, methylsulfonium compounds, and urea inside cells of marine animals and mammalian kidneys are estimated to be at or above 600 mM,36 and an extreme example in mammals is the kidney medulla cell that contains urea concentrations up to 5.4 M, corresponding to 30% by mass.37,38 Under the condition regarded as “small-molecule crowding”, the molecules bind with water molecules and restrict their mobility, resulting in the physicochemical properties of intracellular water being quite different from those of a dilute solution. As a consequence of the crowded environments, intracellular nucleic acids are supposed to have thermodynamic and kinetic properties different from those in a dilute solution. Recent progresses in microscopy and spectroscopy technologies for intracellular measurements, quantitative studies using experimental model systems, and computer simulations have led to an improved understanding of the interactions and reactions of nucleic acids under the macromolecular crowding and small-molecule crowding conditions, which are the central theme of this review. There is increasing evidence that the molecular environment has significant effects on the diffusion rate, hybridization efficiency, folded structure, and structural stability and interactions of nucleic acids, and the effects are not always the same as in the case of proteins.39−43 It is also known that molecular crowding is highly relevant to drug delivery systems,44 electrochemical DNA sensing,45 and the manipulations of nanomaterials or single DNA molecules.46−50 In this review, we introduce the features of the behavior of nucleic acids inside living cells and those immobilized on material surfaces where molecules are highly crowded. We also describe nucleic acid structures, DNA−protein interactions, and DNA− small ligand interactions, studied under the molecular crowding conditions created by cosolute molecules. The organization of this review is as follows: Section 2 describes the biological implications of the condition of molecular crowding. Section 3 presents the effects of molecular crowding on the nucleic acid interactions with proteins and small ligands. Sections 4 and 5

Figure 1. (a) Interbase hydrogen bonding and base stacking of the Watson−Crick base pairs. (b) Binding sites of water and metal ions in a deoxyadenosine unit.

predictions for long RNA sequences can be further improved by combining the chemical modification data.31 The energetic aspects are important for the design of oligonucleotide sequences. As an example of the mRNA targeting therapeutics using antisense oligonucleotides, the prediction parameters determined for RNA•DNA hybrid duplexes23,32,33 can be used as a guideline for designing the antisense DNA sequences. Moreover, the thermodynamic properties of the base pair formations of mRNA•DNA and DNA•DNA were found to have a strong correlation with the level of the mRNA transcription in yeast.34 Since most thermodynamic studies have been conducted using dilute solutions, it is unclear whether these data are capable of predicting nucleic acids inside living cells. One of the most distinguishing features of the intracellular environment is the condition of being crowded with macromolecules and cell organelles.35 The environment crowded with macromolecules is referred to as “macromolecular crowding”. In addition to the large components, small hydrophilic molecules, such as metabolites and osmolytes, are highly concentrated in a cell. The total amounts of polyols and sugars, amino acids and

Table 1. Glossary of Keywords and Terms Used in This and Other Papers Molecular crowding: The term, crowding, is used for a crowded environment. In cells, no single molecular species is present at an extremely high concentration, but a significant proportion of the volume is physically occupied by various macromolecules and cell organelles. Some papers seem to use the term to refer to macromolecular crowding.40,59 The present review uses the term when a crowded condition is caused either by high-molecular-weight molecules or low-molecular-weight molecules. Macromolecular crowding: An environment crowded with large molecules. The term describes the fact that the environment inside a living cell is crowded with macromolecules such as proteins and nucleic acids.55 A significant proportion of the cellular volume is physically occupied and unavailable to other molecules, causing repulsive interactions and structural obstacles to the free motions of biomolecules. Small-molecule crowding: An environment in which small molecules are present in a large amount. The excluded volume per molecule is relatively small, but the solvent properties, such as the water activity, may change because a large number of water molecules are involved in the hydration of the molecules. The small molecules may also have influence through favorable or unfavorable interactions with biomolecules. Excluded volume: The space excluded by molecules due to the physical nonspecific effect originating from steric repulsion. The excluded volume effect, also called the volume exclusion effect, can be caused by macromolecular crowding or the spatial confinement of molecules in a small volume.42 Many of the effects can be predicted using a simple statistical thermodynamic model.40,61 Preferential interaction: The nature of interactions of solvent components (water and cosolutes) with a biomolecule. The preferential binding between a cosolute and a biomolecule leads to an accumulation of cosolutes in the vicinity of the biomolecule. The biomolecule−solvent interactions can be expressed by mutual perturbations of the chemical potentials in the three-component system of water, a biomolecule, and a cosolute.19,39,51,54,56 Cosolute: Polymer molecules used for creating macromolecular crowding behave like solutes rather than solvents of biomolecules. Conversely, small hydrophilic molecules used for creating small-molecule crowding are solvated by water, behaving like solutes, and also solvate biomolecules as water does, behaving like solvents. These properties are not mutually exclusive,54 and some papers refer to the solvent additives as cosolvents,52,53 solutes,60 or cosolutes.58 Specifically, the term cosolute is used when a hard sphere particle is assumed in the theory of the excluded volume effect.57 In the context of this review, it refers to both polymers and small molecules as cosolutes in order to emphasize the osmotic properties that play a significant role in the effects on nucleic acids. Dilute solution: An aqueous solution that does not create the molecular crowding environment. The term is used as the opposite of solutions crowded with cosolutes. 2734

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

summarize the effects of molecular crowding on the canonical and noncanonical nucleic acid structures, respectively, and section 5 includes results of studies on the tertiary folding and functions of nucleic acids. Section 6 discusses the perspectives for future studies. The definitions for the keywords and terms used in this and other papers19,40,42,51−60 are presented in Table 1.

Table 2. Physical Parameters Representing the Intracellular Environmenta parameter

features

concentration

protein in E. coli: 200−300 mg mL−1 RNA in E. coli: 75−150 mg mL−1 mammalian cell: 4 × 10−12 L organelle (of 50 nm diameter): 6 × 10−20 L bacterial virus: 2 × 10−16 L hepatocyte internal membrane: 1 × 105 μm2 hepatocyte plasma membrane: 2 × 103 μm2 mammalian cell cytoskeleton: 3 × 103 μm2 mitochondria: 102−103 μm2

volume

2. NUCLEIC ACIDS UNDER THE MOLECULAR CROWDING CONDITION

surface area

2.1. Molecular Environment of a Living Cell

Living cells consist of a large number of macromolecules of proteins, nucleic acids, carbohydrates, etc. (Figure 2). These

a

See the references cited in the papers by Luby-Phelps35 and by Ellis.55

in a small volume also provides the excluded volume effect. These steric effects change the thermodynamic activities of dissolved molecules and, consequently, affect the thermodynamics and kinetics of cellular reactions. It is mentioned that weak interactions that have no significant role in vitro could be more pronounced under the molecular crowding conditions. The large cellular components also cause interference with the movements and dynamics of molecules,41,80 which affect the rate of molecular diffusion and the efficiency of molecular collisions (Figure 3b). It is possible to observe the mobility of macromolecules and their spatial organization in living cells using the fluorescence microscopy-based methods.81 The fluorescence measurements of a probe molecule have proved the significance of the intracellular viscosity on the diffusiondependent kinetics of cellular reactions.82 The diffusion rate of macromolecules is reduced in cytoplasm, but anomalous diffusion of certain proteins observed in cells may not be as a consequence of macromolecular crowding but of specific interactions with cellular components.41,83 It was also suggested that the anomalous diffusion property only slightly varies among the different cell types, and the degree of macromolecular crowding in the cytoplasm and nucleoplasm seems to be conserved.84 The solvent properties of intracellular water are different from those of a dilute solution. The cellular components tightly associate with water molecules and form layers of ordered water (Figure 3c), and the bound water becomes osmotically inactive.35,69,85,86 The amount of intracellular free water changes depending on the status of a cell: the volume fraction of free water changes during the cell cycle,87 and there exists a lower number of free water molecules in tumor cells than in normal cells, which might have some relevance to their different cellular activities.88 It was reported that the amount of cytoplasmic water is the primary determinant of the growth rate of an osmotically stressed E. coli, presumably by perturbing the cytoplasmic molecular interactions at the increased osmolality; however, their DNA−protein interactions are relatively insensitive to the change in the salt concentration in vivo, whereas they are sensitive in vitro.89,90 The intracellular dielectric properties are also different from a dilute solution. The dielectric constant of a yeast cell is estimated to be around 50 or even less,91,92 which is much lower than the value of pure water at about 80. A computer simulation has also predicted values in the range of 30−50 under the molecular crowding conditions.93 It is mentioned that the dielectric constant within protein crystals, in which the molecular density is similar to that of the cellular macromolecules, is about 55.94 A decrease in the dielectric constant results in increased electrostatic attractions

Figure 2. Illustration of the molecular environment inside a cell.

components can be observed using high performance microscopes: a high-resolution electron microscope at nanometer resolution visualizes the distinct shapes of ribosomes, proteasomes, and cytoskeleton without perturbation of the cellular environment,62−65 and fluorescence microscopy allows for the probing of single molecules to measure the subcellular distributions, stability, and dynamics of individual proteins inside a cell.65−67 The total amounts of biomolecules inside E. coli. are estimated to be 300−400 mg mL−1, including 200 mg mL−1 protein, 75 mg mL−1 RNA, and 10−20 mg mL−1 DNA. Eukaryotic cells contain 50−400 mg mL−1 biomolecules in the cytoplasm, 100−400 mg mL−1 in the nuclei, 100−200 mg mL−1 in nuclear organelles, and 270−560 mg mL−1 in the mitochondrial matrix, although the amounts will change depending on the cell type, differentiation stage, and cell volume.55,68−74 These amounts of biomolecules are much higher than those used for the in vitro experiments, typically below 1−10 mg mL−1. In addition, the conditions of a small reaction volume and substantial surface area of the cytoplasmic organelles and cell membranes (Table 2) are fundamentally different from those in a dilute solution. It is proposed that the intracellular environment plays a key role in many cellular processes, including intracellular phase separation, molecular compartmentation, glucose metabolism, tumor generation, and susceptibility to diseases with aging.75−77 It is also known that regulation of the cell volume plays an essential role in the cell growth and proliferation by modulating the extent of the intracellular crowding and osmolality.78,79 The highly concentrated and confined environment changes the properties of the media of cellular reactions. The space excluded by macromolecules and organelles is inaccessible to other components, generating an excluded volume due to steric hindrance (Figure 3a).40 The spatial confinement of molecules 2735

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

Figure 3. (a) Inaccessible and accessible areas between hard spherical molecules. The inaccessible area represents the volume excluded to the center of other molecules. (b) The diffusion model in the presence of cosolute molecules acting as an obstacle having an exclusion volume. (c) Illustration of hydrated cosolutes. The mobility of the bound water molecules is restricted, leading to a decreasing amount of free water and changes in the solution properties including the water activity and dielectric constant. (d) The equilibrium constant for a biomolecular association in the presence of cosolutes, Kca becomes different from that in a dilute solution, Ka, when the cosolute effects on the biomolecules before and after the association (Ku and Kn, respectively) are different.

other hand, the 18S rRNA in soybean seedling leaves showed alternate structures different from those determined in vitro.103 The study using an RNA self-cleavage reaction that probed the intracellular conformation showed that the RNA secondary structure is sequentially folded in yeast, and the rate of exchange between alternative secondary structures interacting with adjacent upstream or downstream regions is much faster than the exchange in vitro.104 Another outstanding approach to identify the intracellular RNA structures is in-cell spectroscopy. The NMR (nuclear magnetic resonance) and EPR (electron paramagnetic resonance) techniques allow for the observation of three-dimensional structures, dynamics, and interactions of a labeled RNA molecule in a living cell;105−107 however, there are some difficulties with the in-cell measurements due to instability of RNA molecules in the cellular milieu. These methods are very useful to determine the structures and interactions of intracellular RNA molecules, and it is currently challenging to systematically investigate the individual structures of intracellular nucleic acids.

and repulsions. Furthermore, the coexisting molecules may preferentially interact with nucleic acid strands. Consequently, the molecular environment of living cells is supposed to heavily influence the equilibria and rates of nucleic acid reactions (Figure 3d). 2.2. Nucleic Acids in the Cytoplasm

How does the molecular environment of the cytoplasm affect nucleic acids? It was reported that intracellular organelles, cytoskeleton, and macromolecules hinder the diffusion of nucleic acids and cause an anomalous rate of diffusion-mediated searching for their interaction partners, such as transcription factors and mRNA molecules.95,96 The diffusion rates of DNA fragments characterized by measuring the rates of the fluorescence recovery after photobleaching showed that the diffusion in the HeLa cell cytoplasm is much slower than that in an aqueous solution, and the diffusion coefficients relative to that in water are 0.19 for a 100-base pair (bp), 0.06 for a 250bp, and less than 0.01 for >2 × 103-bp DNA fragments.97 The study using FRET (fluorescence resonance energy transfer)labeled 16-mer and 12-mer DNA duplexes showed several-fold accelerated and decelerated hybridization kinetics, respectively, in a single HeLa cell, in comparison to those obtained in vitro.98 The size-dependent diffusion rates suggest substantial obstructions by or interactions with cytoplasmic components. The efficiency and process of intracellular RNA folding are sometimes different from those determined in vitro. RNA structures inside cells can be investigated using dimethyl sulfate, providing the RNA methylation patterns that reflect the folded structure and the interactions with other molecules. The chemical modification experiments showed that the rRNA intron in Tetrahymena thermophila, the U3A small nuclear RNA in Saccharomyce cerevisiae, the thrS sequence in Bacillus subtilis, and the MFA2 mRNA in a yeast strain adopt the structures nearly equivalent to those determined in vitro.99−102 On the

2.3. Nucleic Acids in the Nucleoplasm

Eukaryotic nuclei are highly heterogeneous and crowded with a variety of subnuclear structures and protein complexes, including nucleolus, Cajal bodies, promyelocytic leukemia bodies, splicing-factor compartments, and replication and transcription factors.108,109 It is proposed that the molecular crowding condition promotes the organization of the nuclear architecture.110,111 The experiments, in which penetrating polymers of PEG (poly(ethylene glycol) of an average molecular weight, MW, of 8 × 103) and dextran (MW = 10.5 × 103) were injected into the nuclei of a K562 cell, showed that the degree of macromolecular crowding plays an essential role in driving the formation and maintaining the functions of the nuclear compartments.112 2736

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

Figure 4. Schematic representations of the DNA hybridization on a material surface (a) with a modest DNA probe density, providing maximum hybridization efficiency, or (b) with a high probe density, decreasing the amount and rate of the hybridization.

significance of the degree of molecular crowding on the hybridization rates and base pair stabilities, which needs more work to further improve gene analysis and molecular sensing applications using the solid materials, such as a DNA microarray chip. The hybridization kinetics obtained with DNA-conjugated gold nanoparticles increase by the addition of PEG (MW = 2 × 104), attributed to the surface blocking effect by PEG that competes with DNA for surface binding sites.138 More frequently, the surface is covalently modified with dextran, PEG, or a self-assembled monolayer (SAM) of a lipid or alkanethiol. In such cases, the DNA hybridization proceeds under the condition of being crowded with the coating molecules. It is also important to consider the fact that the mobility and arrangements of water near the surfaces are highly restricted, and thus the water activity and dielectric properties of the medium of a DNA probe are different from those of a dilute solution. These factors are the same as those considered for cellular reactions. The analysis of DNA hybridization on the solid surface would have advantages for the quantitative study of molecular crowding effects on the rates and equilibria of nucleic acid interactions.

Nuclear DNA is highly condensed and wrapped around histone proteins, forming the chromosomes.113,114 Histone proteins regulate the nucleosome assembly by causing a compaction of DNA, and the extent of the compaction changes during the cell cycle. The DNA compaction also plays roles in avoiding too much condensation and retaining flexibility and reversible structural modulations.115−117 Macromolecular crowding in the nucleoplasm is supposed to be a significant and major factor in the chromosome compaction.118 Moreover, it was found that the macromolecular crowding effects on gene interactions with transcription factors play a key role in transcriptional bursts that make the mRNA and protein levels much higher than that predicted by first-order kinetics.119 2.4. Nucleic Acids Immobilized on Solid Surfaces

The phenomenon of molecular crowding is related to the nature of nucleic acids immobilized on a material surface, and the environmental effects on the rate and equilibrium constant of DNA hybridization resemble those observed in biological media. DNA oligonucleotides can be immobilized on various types of material surfaces, such as a flat plate, nanoparticle, microfluid device, and polymer gel. The DNA molecules immobilized on a material surface are less flexible and sterically crowded, similar to the conditions of a living cell. There are several studies of oligonucleotide hybridization on a flat sensor surface120−125 and a nanoparticle surface.123,126−132 The hybridization on a flat surface can be quantified from the refractive index change monitored by the surface plasmon resonance (SPR) angle shift or from the mass change at the quartz crystal microbalance (QCM) sensor surface; these biosensors have the advantages of a high-throughput and of rapid and sensitive detections of a target sequence or an analyte molecule.133,134 The quantitative analysis methods can be used to investigate the effects of molecular crowding on DNA base pairing. In general, the greater loading of a probe oligonucleotide on the surface produces more responses, but too high of a probe density reduces the efficiency and rate of hybridization due to steric crowding (Figure 4).122,135,136 The control of the probe density is also important when analyzing the DNAbinding proteins that require a large space to accommodate the protein or their complexes.137 On the other hand, oligonucleotide hybridization on nanoparticles can be measured by monitoring the color change in the nanoparticle solution caused by the aggregation of DNA-conjugated gold nanoparticles of typically 10−100 nm diameters.126 As seen for the hybridization on flat surfaces, the DNA probe modestly loaded onto gold nanoparticles showed a greater hybridization stability compared to that in solution, but highly loaded DNA did not.130 The DNA hybridization events are indicative of the

2.5. Computer Simulations of Intracellular Environments

The computational modeling and dynamics simulations are important for the theoretical study of the molecular crowding effects. The studies consider the excluded volume of macromolecules and the long-range electrostatic and short-range interactions. There are a number of important insights from computational studies that assemble the intracellular environment in silico.139,140 The studies, often employing the Monte Carlo and Brownian dynamics simulations, can simulate the dynamic behavior of biomolecules under the conditions of macromolecular crowding. The simulation and atomistic modeling of protein conformational changes and a protein folding stability can provide results consistent with experimental studies.141−143 A kinetic simulation study suggests that macromolecular crowding influences the genetic network model of a bacterial virus, and the crowding effects are mainly caused by the shift in the association−dissociation equilibria rather than the slowing down of the diffusion of proteins.144 Several attempts to model the cytoplasmic crowding environment have been made.145 The simulation study is particularly useful for our understanding of molecular diffusion in the interior of cells. Stochastic dynamics simulations with the consideration of spherical particles having the masses, radii, and number densities similar to those of the experimentally obtained data on proteins have revealed the importance of molecular charge to the diffusion rate of macromolecules in a 2737

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

Figure 5. (a) Several molecules used as a cosolute in the molecular crowding studies of nucleic acids. (b) Comparison of the molecular sizes of PEGs of different average molecular weights, ethylene glycol, and a short DNA duplex. The gyration diameters of the PEG molecules are represented by a sphere.

model of the cytoplasm of E. coli.139 The simulation of E. coli cytoplasm proteins (50 different species of the most abundant proteins under experimentally measured concentrations) has successfully described the relative thermodynamic stabilities of the proteins measured in E. coli.146 It is also predicted that protein crowding affects the dielectric constant and the hydration structure surrounding a protein.93 Molecular simulation studies of nucleic acids under molecular crowding conditions are insufficient compared to those of proteins. It was reported that the macromolecular crowding by surrounding polymeric glucose chains stabilizes a hydrogen-bonded duplex between complementary DNA molecules in silico.147 It was also demonstrated that the structural confinement enhances the efficiency of the Na+- and Mg2+-induced collapse of a model RNA structure comprised of two RNA helices tethered by a flexible loop by affecting the conformation ensemble under confinement.148 However, the influences of the dielectric constant and the hydration structure are still unclear. It is remarkable that a simulation study of nucleic acid hybridization between probe and target molecules on a microarray surface provides guidelines for the probe length and surface density to maximize the microarray sensitivity and specificity.136 More computational perspectives are important in order to understand the behavior of nucleic acids under molecular crowding conditions.

3. THEORETICAL BACKGROUND OF THE MOLECULAR CROWDING EFFECTS ON NUCLEIC ACID INTERACTIONS 3.1. Water-Soluble Cosolutes for Creating the Molecular Crowding Conditions

Since systematic and quantitative studies of nucleic acid interactions within a living cell are difficult, in vitro experimental systems using buffered solutions containing a high concentration of background molecules are often used. Proteins, such as albumin, chymotrypsin, hemoglobin, and lysozyme, which are relatively easily available in high amounts, can be used as reagents to mimic the intracellular environment of being crowded with proteins. However, it is not easy to quantitatively analyze the reaction of interest, e.g., DNA hybridization, in the presence of a large amount of proteins. Moreover, the added proteins may bind to and degrade nucleic acids. Synthetic cosolutes are considered as convenient reagents for the molecular crowding studies, and used for preparing the mixed aqueous solutions with amounts of several to several tens of percent. Some compounds used for studying the molecular crowding effects on nucleic acids are presented in Figure 5a. These molecules create different molecular environments: polymer cosolutes create the condition of macromolecular crowding, and low molecular weight cosolutes create the condition of small-molecule crowding, although these effects of are not mutually exclusive. It is preferable that the cosolute molecules meet the following criteria: (1) they are highly soluble in water, (2) they do not cause precipitation of the nucleic acids, and (3) they do not strongly bind to nucleic acids and metal ions better than water, or there is little or no 2738

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

Figure 6. (a) Profiles of the activity coefficient of a spherical biomolecule B, γB, versus the fractional volume occupancy, ϕ, of a sphere molecule C. The values were calculated based on the effective hard spherical particle model by assuming the same density for both molecules having the molar mass, MB for B and Mc for C.40 (b) Relationships of γB versus the molecular size of B relative to C at different volume occupancy. (c) Relationships of the association equilibrium constant, K, of the reaction 2B ⇄ B2 versus ϕ, where K0 is the equilibrium constant without C.

thermodynamic activity of proteins. An approximate method based on theoretical calculations, assuming steric effects between hard spherical particles, provides good estimates of the activity coefficient of proteins.40,61,158−162 The calculation gives the activity coefficient of a protein in concentrated solutions of a cosolute molecule, and the value increases with the increasing fractional volume occupancy of cosolutes in a highly nonlinear fashion (Figure 6a). For example, the activity coefficient of a biomolecule of the same size as a cosolute exceeds 10 in the medium with the fractional volume occupancy of 0.2 and 100 in the medium of 0.3, which are close to the cellular level of the volume occupancy. Larger deviations in the activity coefficient from unity are predicted with larger biomolecules (Figure 6b). The increases in the activity coefficient have a significant advantage in molecular associations, and the association equilibrium constant increases at the high fractional volume occupancy of cosolutes (Figure 6c). An excluded volume theory of protein−polymer cosolute interactions, treating a rigid sphere particle and a flexible segmented polymer chain, has also been applied for the predictions of the excluded volume of proteins under the macromolecular crowding by poly(ethylene oxide) and other polymer cosolutes.61 Similar calculations might be made for nucleic acids, although nucleic acid strands cannot be assumed to have a spherical shape. The calculation will be useful to evaluate and discuss the excluded volume effects on nucleic acids.

significant difference in the binding before and after a given nucleic acid reaction. Neutral or net neutral molecules are usually used, and PEG and polysaccharides are often employed as the macromolecular crowding reagents although they may cause precipitation and preferentially interact with proteins or nucleic acids. The gyration diameters of PEG molecules have been evaluated,149 and the sizes of PEG, ethylene glycol, and a short oligonucleotide are compared in Figure 5b. The large PEG generates an area inaccessible to other molecules and increases the solution viscosity. The excluded volume effect that increases the thermodynamic activity of biomolecules may increase their association rates; however, the increased viscosity that reduces the diffusion rates overrides the excluded volume effect when the reaction probability is close to unity.150 In contrast, small cosolute molecules do not act as obstacles, but effectively change the solution property. In particular, ethylene glycol and small PEG molecules decrease the water activity and generate an osmotic pressure (osmotic stress). Since the inclusion of organic compounds with a low dielectric value lowers the dielectric constant of a solution, the addition of cosolutes in many cases decreases the dielectric constant.93,151,152 Accordingly, the molecular environment is markedly different and can be modulated by the type and size of the cosolutes used. 3.2. Excluded Volume Effect by Polymer Cosolutes

Polymer cosolutes generate a space inaccessible to other molecules, and the excluded volume per cosolute increases as the size of a cosolute increases. The excluded volume effect by cosolutes also increases as the size of biomolecules increases. From a thermodynamic viewpoint, steric crowding favors the reactions that reduce the net volume, such as the formation of more compact and ordered structures. The steric effects explain many protein reactions studied using polymer cosolutes. For example, PEG and polysaccharides used as the macromolecular crowding reagents increase the folding stability of DNA kinase, lysozyme, and RNase A153−156 and favor folding of the histone H1 peptide into a molten globule that increases the transition rate toward the DNA-bound state.157 A molecular dynamics simulation study also showed reductions in the population of an open state in equilibrium with a closed state of several tested proteins (yeast protein disulfide isomerase, adenylate kinase, orotidine phosphate decarboxylase, tryptophan repressor, hemoglobin, DNA β-glucosyltransferase, and Ap(4)A hydrolase), which were attributed to the excluded volume effect.142 Protein associations under macromolecular crowding are also often explained by the excluded volume effect that increases the

3.3. Influences of Polymer Cosolutes on DNA−Protein Interactions

There are several reviews describing the macromolecular crowding effects on proteins, including the thermal stability, multimerization, fibril formation, folding pathways, and enzyme activities.42,43,163−167 We now introduce the proteins that interact with nucleic acids. The addition of PEG or polyvinylpyrrolidone in a solution enhances the binding between DNA and proteins extracted from E. coli.168 PEG (MW = 8 × 103), dextran (MW = 7 × 104), Ficoll (MW = 7 × 104), and bovine plasma albumin (MW = 6.7 × 104) accelerate the rate of the DNA ligation catalyzed by T4 DNA ligase.169 These polymer cosolutes were also found to enhance the polymerization activity of T4 DNA polymerase and the polymerization and exonuclease activities of the large fragment of E. coli DNA polymerase I (Klenow fragment) due to the increased binding to DNA.170 Accelerations of DNA polymerization by T7 and Taq DNA polymerases and DNA hydrolysis by DNase I and S1 nuclease in the solution crowded with PEG 2739

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

Figure 7. (a) Dependence of the association equilibrium constant, K, of a reaction involving water molecules on the water activity, aw. The figure was constructed assuming that the linear slope of the plot (∂ log K/∂ log aW) represents the hydration change during a reaction, −Δnw, in which waterrelease reactions have positive Δnw values and water-uptake reactions have negative values. The parameters of aw,0 and K0 indicate the water activity and the association equilibrium constant, respectively, in a dilute solution. (b) Relationships of log(K/K0) versus Δnw for different water activities. When the water activity is 0.955 or 0.912, the value of log(aw/aw,0) becomes −0.02 or −0.04, respectively. (c) The energy diagrams for the binding of biomolecules in the absence and presence of a cosolute that preferentially binds to the bound state by replacing the hydrated water molecules. The same diagram can be constructed for intramolecular biomolecular reactions.

were also reported.171,172 The cosolute effects enhancing the polymerase activities were utilized to improve the performance of polymerase chain reaction (PCR) and reverse transcription PCR.173,174 Moreover, the DNA supercoiling of closed circular plasmid DNAs catalyzed by DNA gyrase are stimulated by RNA transcripts as well as by the polymer cosolutes of PEG (MW = 8 × 103) and polyvinyl alcohol (MW = 3 × 104−7 × 104).175 It is possible that the intracellular macromolecular crowding has the similar effects.

equilibrium constant of a reaction accompanied by the release (or uptake) of 50 or 100 water molecules in a solution with the water activity of 0.955 becomes approximately 10- or 100-times higher (or lower) than that obtained in a pure water solution (Figure 7b). Nucleic acids have many hydration sites, and the sugar− phosphate backbone and the bases are well hydrated. Crystal structure data and molecular dynamics simulations have revealed that water molecules occupy about 20 defined sites per base pair in a DNA duplex; that is, the phosphate anionic oxygen atoms, phosphodiester oxygen atoms, and the furanose O4′ atoms participate in the binding of water molecules that directly or indirectly interact with DNA bases, and in particular, there are well-ordered water molecules forming a spine of hydration in the DNA minor groove.20,27,178−180 More water molecules fluctuating between the transient contact with nucleotides and being a part of the bulk water also exist. Since nucleic acids are highly hydrated in aqueous solutions, most reactions involving nucleic acids are accompanied by the release or uptake of water, and thus, water interactions play an important role in the structural stability and dynamics of nucleic acids. Hydrophilic molecules are effective for changing the water activity, and small cosolutes, such as glycerol, sucrose, ethylene glycol, and small PEG molecules, are frequently used. The osmotic pressure experiments provide insights regarding the stability of nucleic acid structures under osmotic pressure and the hydration changes before and after a given reaction. When studying the osmotic pressure effects, it is important to recognize that the simple linear regression analysis shown in

3.4. Effect of Osmotic Pressure Created by Cosolutes

There are a limited number of free water molecules in cells, creating an osmotic pressure. The molecular environment can be reproduced using a mixed solution with cosolutes since they act as osmolytes that lower the activity of water, aw, to less than unity.54,176 The chemical potential of water decreases by RT ln aw, where R is the gas constant and T is the absolute temperature, whereas the water activity of pure water is 1.0. For example, a solution with the water activity of 0.95 can be prepared using cosolute molecules in the amount of 5−30 wt %, depending on the cosolute used. The osmotic pressure effect is determined by how the water molecules participate in a reaction (Figure 7a). A simple thermodynamic model considers that the dependence of the equilibrium binding constant, K, of a biomolecular reaction on the water activity is related to the number of water molecules released during the reaction, represented by the equation, −Δnw = (∂ log K/∂ log aW), if the effects of other cosolute interactions (e.g., cosolute−biomolecule interactions) are less significant.177 Water-release reactions have positive Δnw values, and water-uptake reactions have negative values. Based on the correlation equation, the 2740

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

Table 3. Hydration Changes for DNA−Protein Interactions Determined by Osmotic Pressure Experiments protein

DNA sequence

cAMP receptor

lac promoter (embedded in a 216-bp DNA)

ultrabithorax

TTAATGGCC (in a 40-bp)

deformed

TTAATGAAC (in a 40-bp)

tryptophan repressor HhaI with S-adenosylmethionine

operator sequence (in a 25-bp) GCGC (target in a 37-bp)

HhaI with S-adenosylhomocysteine

GCGC (target in a 37-bp)

glutaminyl-tRNA synthetaseb glutamyl-tRNA synthetaseb EcoRI

tRNAGln tRNAGlu GAATTC (target site in a 322-bp)

EcoRI

BamHI

GAATTC (target site in a 14-bp) TAGACG (nontarget in a 14-bp) TAATTC (star site in a 14-bp) GGATCC (target site in a 30-bp)

BamHI

CCTAGG (nontarget in a 30-bp)

gal repressor

operator sequence (target sequence in a 284-bp)

TATA-binding protein

Poly(dI-dC) (nontarget)c TATAAAA (target in a 14-bp) TITIIII (nontarget in a 14-bp)c

cosolute

Δnw

ref

acetamide ethylene glycol glycerol triethylene glycol sucrose PEG (MW = 8000) PEG (MW = 1450) PEG (MW = 400) triethylene glycol sucrose glycine betaine glycerol triethylene glycol sucrose glycine betaine glycerol glycine betaine glycerol ethylene glycol glycerol ethylene glycol triethylene glycol triethylene glycol glycerol triethylene glycol ethylene glycol ethylene glycol ethylene glycol methyl glucoside sucrose stachyose glycine betaine DMSO triethylene glycol methyl glucoside sucrose stachyose glycine betaine DMSO triethylene glycol sucrose glycine betaine triethylene glycol sucrose triethylene glycol triethylene glycol

80 76 78 81 84 1677a 1034a 518a 27 23 22 25 5 5 5 5 250 8.1 8.4 10.1 10.4 0−4 0−4 90 120 146 76 216 109 46 467 135 158 183 244 172 612 267 278 328 130 100 180 ∼0 18.3 −2.9

186

187

187

188 189 189 190 190 191 193

195

195

196

197

a

The high values are supposed to be due to the excluded volume effect that significantly contributes to values of the observed binding constant. In this case, the osmotic pressure experiments are not a measure of water binding during protein binding. bThe proteins are the mitochondrial aminoacyl-tRNA synthetases bound to tRNA. cI represents inosine.

Figure 7a may not be applied when cosolutes preferentially interact with nucleic acids. The interaction of cosolute molecules with a biomolecule can be favorable or unfavorable.181 Favorable binding (preferential interaction) is the interaction that the binding between a cosolute and a biomolecule is preferred, and hence, the cosolute accumulates in the vicinity of the biomolecule. Unfavorable binding (preferential exclusion) is the interaction that a cosolute is enriched in the bulk solvent with respect to the vicinity of a biomolecule, resulting in the phenomenon that the biomolecule

is preferentially hydrated. It has been discussed that the preferential binding of cosolute molecules to the surface of native proteins or exclusion in an unfolded state increases the protein folding stability, resulting from the difference in the interaction energies with cosolutes between the unfolded and folded states (Figure 7c).56,60,182 The biomolecule−solvent interactions can be described in terms of a three-component thermodynamic model, i.e., water, a biomolecule, and a cosolute. The treatment allows us to discuss nucleic acid hydration and the interaction with cosolute molecules.19,51,54,56 2741

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

Figure 8. Different contributions of water to the EcoRI binding to the target and nontarget DNA sequences.

Figure 9. (a) Chemical structures of the ligands that bind to a DNA duplex. (b) Contributions of water to the DNA binding of netropsin, daunomycin, and ethidium bromide.

3.5. Cosolute Studies for DNA−Protein Interactions

negative slope values, were not appropriate for the osmotic pressure experiment probably due to the significant contribution of the excluded volume effect to the observed binding constant. The osmotic pressure studies for other DNA-binding proteins (ultrabithorax and deformed homeoproteins, tryptophan repressor protein, HhaI, and glutaminyl- and glutamyltRNA synthetases) also showed increased stabilities in the presence of cosolutes and decreased hydration after the association with their target sequences.187−190 It is remarkable that different water contributions to the target and nontarget DNA binding were reported for several proteins. The binding of cAMP receptor protein to a nontarget DNA sequence is accompanied by the net release of 2−44 water molecules that are much fewer than the binding to the target lac promoter.186 In the case of EcoRI, the difference in the free energy changes between the bindings to the target and nontarget sequences linearly increase with the increasing amount of triethylene glycol.191 This observation was attributed to the benefits of the reduced water activity on the targetselective binding, resulting from a release of more water for target site binding and fewer for nontarget site binding (Figure 8).192,193 On the other hand, the formation of the BamHI− target DNA complex releases a smaller number of water molecules compared to the formation of a nontarget complex, consistent with the different cavity volumes found in the crystal structures of the target and nontarget complexes.194,195 Target DNA binding of the transcription factors, such as the gal repressor and TATA-binding protein, releases more water than the binding to nontarget sequences.196,197 These observations suggest that the osmotic pressure at the cellular level could influence not only the binding affinity, but also the target specificity of many DNA−protein complexes.

Since the water contribution is important for the binding of proteins to DNA,183−185 the molecular crowding influences the DNA−protein binding. The differences in the numbers of bound water molecules between the DNA−protein complex and the free DNA and protein can be estimated from the linear dependence of the log K for the protein binding on log aW, (∂ log K/∂ log aW), as shown in Figure 7 or, equivalently, from the linear dependence of the logarithm of K on the cosolute osmolal concentration ([osm]) because water activity and cosolute concentration are linked by an approximate equation of d ln aW ≈ −d[osm]/55.5. Table 3 lists the hydration changes, Δnw, for DNA−protein interactions determined by the osmotic pressure experiments using cosolutes. For example, the binding between the E. coli. cyclic-AMP (cAMP) receptor protein and a lactose promoter (lac promoter) sequence fragment was measured in the mixed solutions containing acetamide, ethylene glycol, glycerol, triethylene glycol, sucrose, or PEGs.186 The inclusion of the cosolutes in the reaction mixture increased the observed binding constant K (e.g., 2.3 × 109 M−1 with no added cosolute and 2.9 × 1010 M−1 with 0.8 M sucrose), and the logarithm of K was linearly correlated with that of the water activity. The linear regression analysis gave very similar slope values, (∂ log K/∂ log aW), ranging from −76 to −81 for these small cosolutes, suggesting that specific cosolute−protein and cosolute−DNA interactions are less likely to contribute to the analysis. The data provide an estimate of the Δnw around 79 water molecules, on average, released upon the protein binding. The water release-reaction model is consistent with the structural feature that the wateraccessible surface area is significantly reduced after the binding.186 In the study, PEG molecules, providing large 2742

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

Table 4. Hydration Change in DNA−Ligand Interactions Determined by Osmotic Pressure Experiments ligand

a

DNA sequence

Minor Groove Binder netropsin analog

chicken erythrocyte DNA

Hoechst 33258

d(CGCGCAATTGCGCG)

DAPI

d(CGCGCAATTGCGCG)

pentamidine

d(CGCGCAATTGCGCG)

netropsin

d(CGCGCAATTGCGCG)

Intercalator daunomycin propidium proflavine 7-aminoactionomycin D ethidium bromide daumocycin adriamycin actinomycin D TMPyP

calf thymus DNA calf thymus DNA calf thymus DNA calf thymus DNA calf thymus DNA calf thymus DNA calf thymus DNA calf thymus DNA d(G3T2AG3T2AG3T2AG3)

Δnw

cosolute glycine betaine sorbitol triethylene glycol triethylene glycol tetraethylene glycol acetamide glycine betaine triethylene glycol, actamide, glycine betaine, TMAO triethylene glycol, actamide, glycine betaine, TMAO triethylene glycol, actamide, glycine betaine, TMAO sucrose, sucrose, sucrose, sucrose, sucrose, sucrose, sucrose, sucrose glycerol

triethylene triethylene triethylene triethylene triethylene triethylene triethylene

glycol, glycol, glycol, glycol, glycol, glycol, glycol,

glycine glycine glycine glycine glycine glycine glycine

betaine betaine betaine betaine betaine betaine betaine

ref

−53 −57 −54 −78 −67 −51 −51 −35a

202

−34a

202

−26a

202

−18a −6.4a −30a −32a −0.25a −17.8a −35.8a −39 −30

203, 204 204 204 204 203, 204 205 205 206 209

199

200

The number was determined using several different osmolytes.

3.6. Cosolute Studies for DNA−Ligand Interactions

33258 complex but participate in the hydrogen bond network rearranged after the complex formation. The experiments using triethylene glycol, acetamide, glycine betaine, and TMAO (trimethylamine N-oxide) also showed a decreased DNA binding affinity of the minor groove binding DAPI (4′,6diamidino-2-phenylindole), pentamidine, and netropsin, attributed to the water association reactions, in which the waters located at the amino groups of the ligands mediating the interactions with DNA were supposed to be partly responsible for the 26−35 water molecules increased after the complex formation.202 For the intercalating ligands, the binding affinity between calf thymus DNA and daunomycin, propidium, proflavine, or 7aminoactinomycin D decreases in solutions containing sucrose, triethylene glycol, or glycine betaine, and the binding is to be accompanied by an uptake of 6−32 water molecules depending on the studied intercalators.203,204 On the other hand, the affinity for ethidium binding is only slightly changed by these cosolutes, suggesting that a very small net number of water molecules is involved in the ethidium intercalation (Figure 9b).203 The osmotic pressure created by small cosolutes is also unfavorable for the binding between calf thymus DNA and the intercalator daunomycin, adriamycin, or actinomycin D, used in the clinic for the treatment of cancers and tumors, and the increased hydration of approximately 18−39 after the binding were reported.205,206 These studies suggest that a slight difference in the ligand structures may have a significant influence on the hydrogen bonding network of water. It appears that drug binding to a DNA duplex are, in many cases, accompanied by water binding and negative Δnw values, and hence the drug molecule binding becomes less favorable under the environment of more osmotic pressure. The compounds that selectively bind to the telomeric Gquadruplexes and inhibit the activity of telomerase are

DNA is the target of some drug molecules, including antitumor antibiotics.198 It is important to develop the DNA binding ligands that selectively and tightly bind to target sites inside cells. Since small ligand molecules are not significantly affected by the steric effects, consideration of the osmotic pressure effect is often more important.183 The DNA binding ligands are classified into the groove binders and intercalators (Figure 9a). The binding mode, molecular size, and chemical structure decide the participation of water molecules, affecting the ligand binding under osmotic pressure. The small-molecule crowding influences the ligand binding affinity, and Table 4 lists the hydration changes determined by the osmotic pressure experiments. Since the DNA helical grooves are highly hydrated, the binding of the groove binders is expected to be accompanied by a release of water from the DNA surface. Specifically, there are distinct water molecules creating a spine of hydration in the minor groove.180 However, the binding affinity of a minor groove binding netropsin analogue was found to be reduced by the addition of glycine betaine, sorbitol, or triethylene glycol, and the destabilizations were attributed to the increased hydration after the complex formation with chicken erythrocyte DNA, producing negative Δnw values ranging from −53 to −57.199 The affinity of the minor groove binding Hoechst 33258, known as a potent antitumor antibiotic and also commonly used as a DNA stain, to a short DNA duplex decreases with the increasing concentration of the osmolytes, such as triethylene glycol, tetraethylene glycol, acetamide, and glycine betaine.200 In the binding, negative Δnw values ranging from −51 to −78 were obtained, which are comparable to those determined by measurements of the ultrasonic velocity of the solutions.201 The acquired water molecules would include the water molecules that are not in the vicinity of the DNA−Hoechst 2743

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

promising as drugs against a variety of cancer types.207,208 Under the molecular crowding conditions with PEG (MW = 200) and glycerol, the G-quadruplex-binding ligands having aromatic rings and positive charges, such as TMpyP (5,10,15,20-tetra(N-methyl-4-pyridyl)porphine), BMVC (3,6bis(1-methyl-4-vinylpyridinium)carbazole diiodide), and Hoechst 33258, are less effective for stabilizing the Gquadruplex structure and inhibiting the telomerase activity.209 On the other hand, the derivatives of isoflavone and triazatruxene with a neutral charge showed an increased affinity and specificity to the G-quadruplexes in the solutions crowded with PEGs (MW = 200 and 4 × 103).210,211 Since the molecular crowding conditions change the folding topology of some Gquadruplex structures,212−216 it is not straightforward to develop the G-quadruplex-binding ligands. This section has described the interactions of DNA with proteins and small ligands under molecular crowding conditions with cosolutes. These studies implicate an important role of the modulation of the degree of intracellular molecular crowding and osmolality on cellular functions.78,79 The studies using polymer cosolutes suggest that macromolecular crowding inside a cell affects many DNA−protein interactions. Furthermore, an enhanced stability of DNA−protein complexes with an increased osmolality could contribute to the ability of living cells to tolerate dehydration and a change in the external salt concentration. The studies using small cosolutes also have demonstrated that the intracellular osmotic pressure significantly affects the DNA interactions with proteins and small ligands. In particular, many groove binding and intercalating drug molecules bind less strongly to their target DNA sites under osmotic pressure, suggesting that the intracellular environment takes disadvantage of most drug binding. Accordingly, considerations of the involvement of waters in binding with DNA would be important for the developments of drugs and functional probe molecules that play a beneficial function in cells.

by environmental factors could play an essential role in regulating stable on−off switching of genes in cells.224 Increasing the amount of a single-stranded RNA is also capable of inducing the compaction of large DNA molecules, suggesting the interplay between the dynamics of chromosomal DNA and the production of RNA in the cytoplasmic environment.225 On the other hand, the additions of glycerol, ethylene glycol, 1,2dimethoxyethane, primary alcohols, and acetone that do not cause significant steric effects also induce the compaction of the T4 bacteriophage DNA.226 In such cases, the reduction of the solvent dielectric constant was supposed to be dominant. Significances of the bulk dielectric constant were also reported about the twist energy of a circular plasmid DNA in solutions containing ethylene glycol, glycerol, sorbitol, sucrose, or glycine.227 During the condensation of DNA molecules, the rate of a DNA strand exchange increases. It was reported that dextran sulfate (MW = 5 × 105) and PEO (MW = 8 × 103) around 10−30 wt % significantly accelerate the kinetics of the renaturation of denatured DNA fragments and increase the strand exchange between single-stranded and double-stranded DNAs.228 This result was attributed to the limited space to search for a complementary sequence in the condensed phases. A DNA strand exchange of 20-mer length is also accelerated by several orders of magnitude in a solution crowded with PEG (MW = 6 × 103) at 50%, but the acceleration was caused by the more duplex breathing by hydrophobic interactions between the DNA bases and the PEG.229 It is possible that the cosolute effects and their determinants on DNA condensation are different depending on the DNA length and the studied cosolutes. 4.2. Stability of the Polynucleotide Duplexes

The effects of cosolutes on the thermodynamic stability of DNA and RNA duplexes are well studied. The thermal melting temperature, Tm, at which one-half of the folded structure is dissociated, is useful to evaluate the strength of the base pairs.230 PEG and polysaccharides are highly soluble in water and have a relatively low vapor pressure. These properties are important for performing the experiments in which the nucleic acid structures are thermally melted to measure the thermodynamic stability. The Tm of a homopolymer RNA duplex, poly(I)•poly(C) (polyinosine associating with polycytidine), increases by 1.8− 2.0 °C by PEGs (MW = 4 × 103 and 2 × 104) and 0.7 °C by dextrans (MW = 1 × 104 and 7 × 104) at 10 wt %.231 The Tm of a DNA duplex, poly(dA)•poly(dT), also increases as the amount and molecular weight of the PEG increase (for example, by around 5 °C at 19 wt % PEG with a molecular weight of 8 × 103).232 These polynucleotide duplexes exhibited a highly cooperative melting transition, indicative of a long length of base pairs and occupation of a large volume. The increases in the Tm are consistent with the steric effects such that the presence of large cosolutes favors the reactions that decrease the net volume. Indeed, the hydrostatic pressure study has shown a positive volume change accompanying the denaturation of poly(dA)•poly(dT) consisting of around 1 × 103 base pairs.233 On the other hand, ethylene glycol, glycerol, acetamide, and sucrose, having a lower steric hindrance, decrease the Tm of poly(dA)•poly(dT) and E. coli DNA by 1−5 °C, and the Tm decreases have been discussed in the light of a reduced water activity.232,234 Decreases in the Tm of calf thymus DNA by 1.1−8.3 °C were also observed with polyols,

4. COSOLUTE EFFECTS ON THE STRUCTURES AND INTERACTIONS OF NUCLEIC ACID DUPLEXES 4.1. DNA Condensation

There are increasing reports about the thermodynamics and kinetics for the structure formation of nucleic acids under molecular crowding conditions with cosolutes. These studies have demonstrated that the crowding effects are different depending on the folded structure and the length of nucleic acids, as well as the cosolutes used. In this section, the cosolute effects on the Watson−Crick base-paired structures are described. DNA exists as a compact structure inside bacteriophage heads, bacteria nucleoids, and eukaryotic nuclei.217,218 The DNA compaction is induced by the condensation of DNA molecules mediated by the binding of counterions. The compaction occurs in vitro with high concentrations of multivalent ions that effectively neutralize the charge of condensed DNA.219,220 Large DNA molecules, such as calf thymus DNA, bacterial DNAs, bacteriophage DNAs, and supercoiled and linearized plasmid DNAs, are also compacted by the addition of PEG (MW = 2 × 104) or PEO (polyethylene oxide; MW = 7.5 × 103 or 2 × 104).221,222 The DNA compaction affects the transcriptional activity, and the transcription is completely inhibited after adding PEG.223 It was proposed that the transition to the compact structure induced 2744

dx.doi.org/10.1021/cr400113m | Chem. Rev. 2014, 114, 2733−2758

Chemical Reviews

Review

such as glycerol and the monosaccharides of i-erythritol, Dsorbitol, D-mannitol, myo-inositol, D-arabitol, and L-arabitol, but the results were attributed to an increased electrostatic repulsion among the DNA phosphates and the modification of the electrostatic interactions with counterions.235 The identification of the determinant factors that have caused the cosolute effects is not easy because of the simultaneous effects of cosolutes to reduce the water activity and change the dielectric constant.

the thermodynamic properties of nucleic acid structures under the molecular crowding conditions. Cosolutes also have an influence on the kinetics of base pairing. Small PEGs (MW = 200 and 600) changed the association and dissociation rate constants of DNA duplexes of 10-mer length, while the large PEG (MW = 8 × 103) was less effective.241 The large dextrans (MW = 1.2 × 104 − 8.7 × 105) and Ficoll (MW = 7 × 104) also did not change the kinetics of DNA duplexes of 16-mer and 12-mer lengths.98 These observations suggest less significant contributions of the excluded volume effect and the increased viscosity on the base-pair kinetics. Since a hydrostatic pressure study has suggested the significance of the hydration in the rates of a 22mer DNA duplex formation,242 the osmotic pressure effect may be responsible for the altered kinetics of base pairing in the presence of small cosolutes. Unfortunately, our knowledge of the crowding effects on the kinetics of base pairing is quite limited.

4.3. Structure, Thermodynamics, and Kinetics of the Short Duplexes

Further insights have been obtained by the studies using short synthetic sequences. The investigations using oligonucleotides have the following benefits: a double helix of a short nucleotide length (e.g.,