Hydration Of Nucleic Acids Research Articles

Synthesis and Evaluation of Thiocyanate and Selenocyanate Derivatives of Adenosine and Phenylalanine as Vibrational Reporters

Two novel modified 2’-deoxyadenosine (dA) molecules, Si2-dA-SCN and Si2-dA-SeCN, and two novel modified phenylalanine (Phe) molecules, Boc-Me-PheCH2SCN and Boc-Me-PheCH2SeCN have been synthesized and the -SCN and -SeCN functional groups evaluated as vibrational reporters. The syntheses of Si2-dA-SCN and Si2-dA-SeCN were accomplished in three steps in 16% and 32% overall yields, respectively, from commercially available adenine 9-β-D-arabinofuranoside. Similarly, the syntheses of Boc-Me-PheCH2SCN and Boc-Me-PheCH2SeCN were completed in four steps in 8.9% and 2.3% overall yields, respectively, using commercially available N-(tert-Butoxycarbonyl)-4-iodo-L-phenylalanine as the starting material. The absorbance bands resulting from the -SCN and -SeCN stretch vibrational modes are in a clear region of the IR spectrum, and are narrow and symmetrical suggesting that they are not complicated by anharmonic coupling. These stretch vibrational modes are also sensitive to local environment as shown by change in position and full width half maximum (fwhm) of the absorbance bands in different solvents, including tetrahydrofuran (THF), H2O/THF mixtures, and ethanol (EtOH). For example, the -SeCN absorbance band of Boc-Me-PheCH2SeCN shifted from 2151.2 to 2150.7 cm−1, and broadened from 7.1 to 11.0 cm−1, going from 0-40% (v/v) H2O/THF. In addition, the same band in ethanol shifted to 2154.1 cm−1 and the fwhm increased to 12.7 cm−1. These reporters also have longer vibrational lifetimes than other commonly used probes as determined by two-dimensional infrared spectroscopy (2D IR) experiments using Si2-dA-SCN and Si2-dA-SeCN. The increased lifetimes and the solvent sensitivity make -SCN and -SeCN moieties highly promising for their utility in studying several processes, including protein and nucleic acid hydration dynamics.

Determination of nucleic acid hydration using osmotic stress.

Water plays an important role in structure and molecular recognition of biopolymers. Understanding hydration of biopolymers is a significant problem in structural chemistry and biology. However, hydration is a dynamic process that is difficult to study. While X-ray crystallography, NMR, and molecular modeling have provided structural detail on nucleic acid hydration and valuable insights into water dynamics, the thermodynamic contribution of water molecules to conformational equilibria and recognition of nucleic acids remains poorly understood. This unit describes a thermodynamic analysis of nucleic acid hydration using osmotic stress. Osmotic stress monitors the depression of melting temperature upon decreasing water activity, and calculates the number of thermodynamically unique water molecules associated with the double helix and released from single strands upon melting. Comparison of the number of water molecules released upon melting of nucleic acids with different sequences and chemical modifications provides insights that complement and enhance information obtained by other methods.

25 Years Full of Chemical Discovery

25 Years Full of Chemical Discovery

DNA polymerase activity in water-structured and confined environment of reverse micelles

DNA polymerases are the key enzymes of DNA replication and repair. These proteins in living cells are functioning not as isolated entities but in multiprotein complexes, and their work are carefully regulated. The main factors determining the enzyme activity are the structure and dynamics of ‘biological’ water. Reverse micelles (nano-sized water droplets dispersed in a continuous oil phase) are the simple model systems where the structure and dynamics of water are controlled. In this work, the activities and processivity of Klenow fragment of E. coli DNA polymerase I, thermostable Tte DNA polymerase, and HIV-1 reverse transcriptase are investigated as a function of the water pool size. Klenow fragment was more active on poly(rA)-oligo(dT) in reverse micelles compared with the water buffer with activity being increased upon increasing water content. Tte polymerase was more active at low water content. HIV-1 reverse transcriptase revealed comparable activity and processivity on poly(rA)-oligo(dT) in the water buffer and reverse micelles at water content of 15–40%. Thus, the polymerase activity appears in certain range of water concentration and depends on the local polarity determining the protein ‘expansion’, microviscosity inside nano-droplets determining the enzyme dynamics, and nucleic acid hydration degree.

Hydration of nucleic acid components in dependence on nucleotide composition and relative humidity: a Monte Carlo simulation

What role does the nucleotide composition play in the process of formation of hydrated environment of nucleic acids? Can one estimate the hydration of nucleic acids on the level of their components? In order to resolve these questions we have completed an extensive computer simulation of the hydration of nucleic acids components - deoxynucleoside monophosphates distinguished by nucleotide composition. The energetic characteristics of systems containing deoxynucleoside monophosphates and water clusters of various dimensions are received. Our results demonstrate that deoxynucleoside monophosphates containing guanine and/or cytosine residues hydrated more strongly because of formation of more hydrogen bonds with water molecules in small water clusters, i.e. at low values of relative humidity. With increasing of number of water molecules in a water cluster the energetic preference of deoxynucleoside monophosphates containing guanine and/or cytosine residues decreases, and for water clusters corresponding to a state of a dilute aqueous solution the hydration of all types of deoxynucleoside monophosphates does not differ in a great degree. Deoxynucleoside monophosphates containing guanine and/or cytosine residues cause the greater destruction of the water structure compensated by the greater interaction with the nearest water molecules for all levels of relative humidity.

Volumetric characterization of the hydration properties of heterocyclic bases and nucleosides

We have determined the partial molar volumes, expansibilities, and adiabatic compressibilities of six heterocyclic nucleic acid bases, five ribonucleosides, and six 2′-deoxyribonucleosides within the temperature range 18–55°C. We interpret the resulting data in terms of the hydration of the component hydrophobic and polar atomic groups. From our temperature-dependent volumetric studies, we found that the total contraction of water caused by polar groups of each individual heterocyclic base and nucleoside depends on the proximity and chemical nature of other functional groups of the solute. In addition, the compressibility contributions of polar groups vary greatly in sign and magnitude depending on the surrounding functional groups. In agreement with previous studies, our results are suggestive of little or no interaction between the sugar and base moieties of a nucleoside. In general, our data shed light into the hydration properties of individual heterocyclic bases and nucleosides, which may have significant implications for the sequence-dependent hydration of nucleic acids. We discuss the potential importance of our results for developing an understanding of the role that solvent plays in the stabilization/destabilization of nucleic acid structures.

Molecular dynamics simulation of nucleic acids: Successes, limitations, and promise

In the last five years we have witnessed a significant increase in the number publications describing accurate and reliable all-atom molecular dynamics simulations of nucleic acids. This increase has been facilitated by the development of fast and efficient methods for treating the long-range electrostatic interactions, the availability of faster parallel computers, and the development of well-validated empirical molecular mechanical force fields. With these technologies, it has been demonstrated that simulation is not only capable of consistently reproducing experimental observations of sequence specific fine structure of DNA, but also can give detailed insight into prevalent problems in nucleic acid structure, ion association and specific hydration of nucleic acids, polyadenine tract bending, and the subtle environmental dependence of the A-DNA-B-DNA duplex equilibrium. Despite the advances, there are still issues with the methods that need to be resolved through rigorous controlled testing. In general, these relate to deficiencies of the underlying molecular mechanical potentials or applied methods (such as the imposition of true periodicity in Ewald simulations and the need for energy conservation), and significant limits in effective conformational sampling. In this perspective, we provide an overview of our experiences, provide some cautionary notes, and provide recommendations for further study in molecular dynamics simulation of nucleic acids.

Clathrate hydrates are poor models of biomolecule hydration.

Clathrate hydrates form the basis of a general model of biomolecule hydration. In clathrate hydrate crystal structures, the size of hydrogen-bonded water rings is highly constrained to five members. The clathrate hydrate model predicts that the size of water rings near biomolecule surfaces is similarly constrained to five members. This report describes a test of this model of biomolecule hydration. We have demonstrated that five-membered water rings are not a general feature of protein or nucleic acid hydration. The clathrate hydrate model appears to be inappropriate for soluble biomolecules.

NMR studies of the hydration of biological macromolecules

The information on the hydration of proteins and nucleic acids in aqueous solution that can be obtained using high resolution nuclear magnetic resonance (NMR) spectroscopy is largely complementary to the information obtained by diffraction experiments with single crystals, which is the only other approach enabling studies of hydration at atomic resolution. The presently discussed NMR studies are primarily focused on the rate processes governing the exchange of water molecules between the bulk solvent and the hydration sites on the biological macromolecules. A novel NMR experiment for observation of protein and nucleic acid hydration is described, and long-time molecular dynamics (MD) simulations used to support the analysis of NMR data on hydration water in the intermolecular interface of protein–DNA complexes are presented.

Comparative study of three systems of potential functions for simulation of nucleic acid hydration

Three systems of potential functions (PF) which are used in simulations of hydration of bioorganic molecules have been compared: Poltev and Malenkov (PM), Weiner and Kollman (WK) (used in the widespread AMBER program), and OPLS (optimized potentials for liquid simulations) of Jorgensen (J). The values of interaction energies of individual water molecules with single molecules of nucleic bases calculatedvia PM potentials are in somewhat better accord with mass spectrometric data than those calculatedvia WK PF. OPLS give much smaller energy values for all compounds considered; therefore they were not used in further computations. Monte Carlo simulation of hydration of 9-methyladenine, 1-methyluracil, and 1-methylthymine in systems with 300 water molecules and periodic boundary conditions have been performed. Simulations with PM potentials give better agreement with the experimental data on hydration energies than those with WK PF that allows to prefer PM PF for simulation of hydration of nucleic acids.

Protein and nucleic acid hydration and cosolvent interactions: establishment of reliable baseline values at high cosolvent concentrations

Hydration and cosolvent interactions of biological macromolecules can be derived, subject to excluded volume corrections, from studies of density increments at constant chemical potentials of diffusible solutes through a semipermeable membrane. In addition to precision density determinations of solutions dialyzed to equilibrium, the analytical ultracentrifuge, static and dynamic light and small angle X-ray and neutron scattering, and combined pairwise use of, for instance, ultracentrifugation and neutron scattering, considerably strengthen the experimental analysis and its interpretation. We have examined hydration of bovine serum albumin (BSA) in the native and denatured states, and binding of the denaturant guanidinium chloride (GdmCl) to the latter form; hydration of DNA and interaction with NaCl and CsCl; revised values of the halophilic malate dehydrogenase (hMDH) tetramer hydration and ‘binding’ of salts; probing of nucleosome core particle hydration as distinct from and additionally to the evaluation of volume exclusion (holes), by use of variously sized sugar related probes. Conclusions presented are compared to results from precision calorimetry and from X-ray crystallography structures, whenever applicable, and comparisons made with alternative interpretations and experimental approaches.

Evidential study of correlated events in biochemistry: Physicochemical mechanisms of nucleic acid hydration as revealed by factor analysis

AbstractUsing a factor analysis technique, the experimental physicochemical data on the hydration of mononucleotides, several polynucleotides, their double‐helical complexes and natural DNAs were studied. The information about the factors determining the changes in physicochemical parameters vs the hydration was obtained. This work discusses a possible physical sense of the factors obtained and the expedience of using factor analysis to interpret the molecular‐biophysical experiments.

Modeling nucleic acid hydration

Modeling nucleic acid hydration

Aqueous hydration of nucleic acid constituents: Monte Carlo computer simulation studies.

Monte Carlo computer simulations were performed on dilute aqueous solutions of thymine, cytosine, uracil, adenine, guanine, the dimethyl phosphate anion in the gauche-gauche conformation and a ribose and deoxyribose derivative. The aqueous hydration of each molecule was analysed in terms of quasi-component distribution functions based on the Proximity Criterion, and partitioned into hydrophobic, hydrophilic and ionic contributions. Color stereo views of selected hydration complexes are also presented. A preliminary discussion of the transferability of functional group coordination numbers is given. The results enable to comment on two current problems related to the hydration of nucleic acids: a) the theory of Dickerson and coworkers on the role of water in the relative stability of the A and B form of DNA and b) the idea of water bridges and filaments emerging from the computer simulation results on the hydration of DNA fragments by Clementi.

Isopycnic sedimentation of DNA. in metrizamide: the effect of low concentrations of ions on buoyant density and hydration

Metrizamide, an inert, non-ionic organic compound, dissolves in water to give a dense solution in which DNA bands isopycnically at a density corresponding to that of fully hydrated DNA. Density-gradient centrifugation in solutions of metrizamide has been used to determine the effects of very dilute solutions of salts on the buoyant density of native and denatured DNA. It has been shown that the buoyant density of DNA is dependent on both the counter-cation and the anion present. Interpretation of the data in terms of the degree of hydration of the macromolecule indicates that (i), NaDNA is more highly hydrated than CsDNA; and (ii), the hydration of NaDNA varies with anion in the order sulphate< fluoride< chloride< bromide< iodide. It is suggested that isopycnic centrifugation in metrizamide is a simple method for determining the effects of salts (and other small molecules) on the hydration of nucleic acids under conditions of high ratios of salt to DNA (> 5 x 10(3) moles/mole) while high (0.999) water activity is maintained.

Buoyant densities and hydration of nucleic acids, proteins and nucleoprotein complexes in metrizamide

Metrizamide is an inert, non-ionic organic compound which dissolves in water to give dense solutions of relatively low viscosity. DNA, RNA, protein and unfixed nucleoprotein complexes all band isopycnically in density gradients of metrizamide. The buoyant densities of nucleic acids are extremely low in metrizamide and correspond to those of fully hydrated DNA and RNA. The buoyant densities of proteins are higher than those of the nucleic acids, but also correspond to those of fully hydrated molecules. Nucleoproteins band at densities which are dependenton the proportions of nucleic acid and protein in the complex. While nucleic acids formed single bands in metrizamide gradients, all proteins tested showed a bimodal distribution. The reason for this is unknown. Metrizamide does not inactivate proteins since fully active catalase can be recovered after centrifugation to equilibrium in metrizamide. Chromatin also is unaffected by exposure to metrizamide.