Polynucleotide Structures Research Articles

Subpicosecond dynamics in nucleotides measured by spontaneous Raman spectroscopy

The band widths in Raman spectra are sensitive to dynamics active on a time scale from 0.1 to 10 ps. The band widths of nucleotide vibrations and their dependence on temperature, concentration, and structure are reported. From the experimental band widths and second moments, it is derived that the adenine vibrations at 725, 1336, 1480, and 1575 cm-1, and the uracil vibration at 787 cm-1, are in the fast modulation limit. The correlation times of the perturbations are faster than 0.4 ps. Thermal melting of the helical structure in polynucleotides results in larger band widths, due to an increase in vibrational dephasing and energy relaxation as a consequence of the increased interaction of the base moieties with the solvent molecules. The band width of the 725 cm-1 adenine vibration is dependent on the type and structure of the backbone. It is found to be perturbed by movements of the sugar-phosphate moiety relative to the base. The band width of the 1575 cm-1 adenine vibration is found to be sensitive to the base-pairing interaction. From a comparison of the band widths in polynucleotides with a different base sequence (homopolymer vs alternating purine-pyrimidine sequence), it is concluded that resonant vibrational energy transfer between the base molecules is not important as a relaxation process for the vibrational band widths of nucleotides. Several theoretical models for the interpretation of band widths are discussed. The theory does not take into account the strong hydrogen-bonding nature water and hence fails to describe the observations in nucleotide-water systems. The bands of the carbonyl stretching vibrations are inhomogeneously broadened. The carbonyl groups have a strong dipolar interaction with the polar water molecules and are therefore strongly perturbed by coupling to the heatbath via hydrogen bonds.

Electrochemical analysis of formation of polynucleotide complexes in solution and at electrode surfaces

The formation of double- and triple-stranded complexes between the synthetic homopolyribonucleotides in solution and at the surfaces of the hanging mercury drop (HMDE) and carbon paste (CPE) electrodes was studied by means of voltammetric and chronopotentiometric methods. It was shown that the cyclic voltammetry reduction signal of poly(C) and the anodic signal of poly(G) obtained with HMDE, as well as the chronopotentiometric oxidation signal of poly(G) obtained with CPE can be used to detect the duplex and triplex formation in solution. In addition to these intrinsic polynucleotide signals, the indicator [Co(phen) 3] 3+ was used to differentiate between single-stranded and ordered structures at CPE. It was shown that the indicator binds preferentially to the duplex poly(G) · poly(C) (formed in solution followed by adsorption at CPE) displaying, however, no preferential binding to the triplex structures. If poly(C) or poly(G) were immobilized at the electrode surface (by adsorption forces) and hybridized with the complementary polynucleotide in solution (at neutral pH), only a small extent of complexation could be detected at HMDE under the given conditions. On the other hand, analogous experiments with CPE suggested formation of ordered polynucleotide structures at the carbon electrode surface. As a result of complexation of poly(C) with poly(G) at neutral pH, formation of more than one ordered structure, including the duplex, was indicated. Polynucleotide complexation performed at pH 3.3 (optimum for formation of the protonated triplex poly(C +) · poly(G) · poly(C) in solution) resulted in the formation of a mixture of structures including triplex and duplex structures. Binding of [Co(phen) 3] 3+ to the product of poly(A) complexation with poly(U) suggested that the yield of the duplex formed at the CPE surface may be influenced by the conformation of the single-stranded polynucleotide anchored at the surface prior to the hybridization. The implications of these findings to DNA biosensor development are discussed.

Macrocyclic metal complexes for selective recognition of nucleic acid bases and manipulation of gene expression

Abstract

Toward the Accurate Modeling of DNA: The Importance of Long-Range Electrostatics

The relationship between DNA structure and function is fundamental to the understanding of biological processes. Currently, the most reliable source of biomolecular structural information comes from X-ray crystallographic data. Recent advances in theoretical modeling techniques have allowed molecular simulations to approach the accuracy obtainable from X-ray crystallography for proteins. We report the results of a 2.2 ns simulation of the B-DNA dodecamer d[CGCGAATTCGCG12 in a crystal unit cell, and demonstrate that, with rigorous accommodation of long-range forces, molecular simulation may be extended to provide atomic level accuracy of polynucleotide structures. Most of what is known about DNA structure has been derived from crystal structure analyses. A concern that has been expressed brings into question the degree to which the intrinsic structure of DNA is disrupted by the crystal lattice. It has been recently pointed out by Dickerson’ that lattice forces in DNA crystals are relatively weak, and that in fact these forces can be exploited to obtain useful information about DNA deformability. This notion strikes at the heart of a fundamental problem in structural biology: to understand the nature of DNA structure in the presence of environmental forces in vivo. A powerful theoretical technique for studying biomolecular structure is molecular simulation. The reliability of molecular simulation to the study of polynucleotide structure, however, has lagged behind that of proteins. This is largely because of the difficulty associated with accommodation of long-range electrostatic forces. In conventional macromolecular simulations, pairwise Coulomb interactions are evaluated using a finite cutoff to decrease computational effort. Ultimately, this truncation of the Coulomb potential leads to unrealistic behaviors2 The adverse effects of the cutoff methods are amplified in highly ionic systems such as DNA. In an attempt to circumvent these problems, artificial constructs are frequently introduced such as reduced phosphate charges on the nucleotide ba~kbone,~ modified Coulomb potentials in the form of distance dependent dielectric function^^,^ or switching function^,^,^ and distance restraints to enforce Watson-Crick hydrogen Perhaps the most studied DNA sequence both experimentally and theoretically is the dodecamer sequence d[CGCGAATTCGCG]2.8,9 Recent attempts to simulate the dodecamer in solution using full charges on the phosphates and explicit counterions to balance the charge led to structures that were

Interaction specificity of benzimidazol group compounds with AT-containing polynucleotides.

The interaction between polynucleotides: poly(dA)-poly(dT), poly(dA-dT), poly(am2dA-dT), and the AT-specific compounds of benzimidazol group has been studied. It is been shown that these compounds bind to poly(dA)-poly(dT) and poly(dA-dT) at low and high salt concentration in solution. Poly(am2dA-dT) interacts with AT-specific compounds only at low salt, where this polynucleotide is in a B-form, but not at high salt, when the polynucleotide converts to another conformation. Thus, the interaction specificity of the groove-binding ligands is influenced not only by the minor groove substituents, but the peculiarities of the secondary structure of polynucleotides.

Methylene blue intercalates with triplex poly(dT)*poly(dA)·poly(dT) but not duplex poly(dA)·poly(dT)

Methylene blue intercalates with triplex poly(dT*dA·dT) even though it binds to the precursor duplex poly(dA·dT) in the major groove; the switch may be related to conformational modifications of the polynucleotide structure upon binding of the third strand.

Repair of UV-damaged DNA by mammalian cells and Saccharomyces cerevisiae

Cells use many strategies to repair genomic damage caused by environmental agents and arising from the natural instability of the polynucleotide structure. Nucleotide excision repair is the most versatile DNA repair pathway and is the main defense of mammalian cells against UV-induced DNA damage. Defects in proteins involved in this pathway can lead to inherited disorders (such as xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy) that are associated with hypersensitivity to sunlight. Most of the proteins and genes involved in these syndromes have now been identified. Study of UV-sensitive yeast RAD mutants has greatly aided this process and has revealed strong conservation of the components of nucleotide excision repair in eukaryotes. It has recently become clear that some of the proteins involved in the DNA repair process have dual functions and also participate in basal transcription and DNA replication.

Berenil recognizes and changes the characteristics of adenine and thymine polynucleotide structures

Using circular dichroism, differential scanning calorimetry and susceptibility to DNAse I cleavage assays, we show that the interaction of berenil, a minor-groove binding drug, with poly(dA−dT) ṡ poly(dA−dT) and poly(dA) ṡ poly(dt) involves important changes in the polynucleotide conformation. The effect of berenil on poly(dA−dT) ṡ poly(dA−dT) comprises a clear alteration in CD spectra even at drug/DNA ratios smaller than the stoichiometric value. Berenil recognizes and binds to the alternating-B conformation of DNA changing it to a new conformation which appears to show some of the peculiarities of poly(dA) ṡ poly(dT), possibly through a modification in the helical parameters at the TpA and ApT steps. Such alteration is accompanied by a small calorimetric enthalpy change. Moreover, the calorimetric enthalpy does not change significantly whatever the input ratio of drug to poly(dA−dT) ṡ poly(dA−dT), indicating that berenil binding does not substantially alters the enthalpy of transition. In addition to increasing the melting temperature of the polynucleotide, berenil reduces the cooperativity of the poly(dA−dT) ṡ poly(dA−dT) transition slightly more than either distamycin or netropsin.

Differential effects of spermine and its analogues on the structures of polynucleotides complexed with ethidium bromide

The interactions of spermine and polyamine analogues with synthetic polynucleotides of various base sequences complexed with ethidium bromide (EB) were investigated using measurements of fluorescence intensity and steady-state fluorescence polarization. Spermine and polyamine analogues displaced some but not all of the EB bound to poly(dA-dT).poly(dA-dT) or poly(dG-dC).poly(dG-dC), suggesting that polyamines may stabilize these polynucleotides in a conformation with reduced affinity for EB. Modifications of the aliphatic backbone of spermine have pronounced effects on its ability to displace EB from poly(dA-dT).poly(dA-dT) but not from poly-(dG-dC).poly(dG-dC). Spermine and some but not all of the polyamine analogues caused fluorescence depolarization when they interacted with the complex of EB and poly(dA-dT).poly-(dA-dT). Neither spermine nor any of the analogues, however, induced fluorescence depolarization in the complex of EB with poly(dG-dC).poly(dG-dC) or poly(dA).poly(dT). This suggests that spermine and some spermine analogues induce structural changes specific to alternating A-T sequences.

Demonstration of Z-d(5BrCGAT5BrCG) and B-d(CGCGATCGCG) form crystal structures in DNA-cobalt hexammine complexes by Kr 647.1 nm excitation of Raman spectra.

Cobalt hexammine [Co(NH3)6(3+)] is an efficient DNA complexing agent which significantly perturbs nucleic acid secondary structure. We have employed red excitation (647.1 nm) from a krypton laser to obtain Raman spectra of the highly colored complexes formed between cobalt hexammine and crystals of the DNA oligomers, d(5BrCGAT5BrCG) and d(CGCGATCGCG), both of which incorporate out-of-alternation pyrimidine/purine sequences. The Co(NH3)6(3+) complex of d(5BrCGAT5BrCG) exhibits a typical Z-form Raman signature, similar to that reported previously for the alternating d(CGCGCG) sequence. Comparison of the Raman bands of d(5BrCGAT5BrCG) with those of other oligonucleotide and polynucleotide structures suggests that C3'-endo/syn and C3'-endo/anti thymidines may exhibit distinctive nucleoside conformation markers, and tentative assignments are proposed. The Raman markers for C2'-endo/anti adenosine in this Z-DNA are consistent with those reported previously for B-DNA crystals containing C2'-endo/anti dA. Raman bands of the cobalt hexammine complex of d(CGCGATCGCG) are those of B-DNA, but with significant differences from the previously characterized B-DNA dodecamer, d(CGCAAATTTGCG). The observed differences suggest an unusual deoxyguanosine conformer, possibly related to a previously characterized structural intermediate in the B-->Z transition. The present results show that crystallization of d(CGCGATCGCG) in the presence of cobalt hexammine is not alone sufficient to induce the left-handed Z-DNA conformation. This investigation represents the first application of off-resonance Raman spectroscopy for characterization of highly chromophoric DNA and illustrates the feasibility of the Raman method for investigating other structurally perturbed states of DNA-cobalt hexammine complexes.

Study of polynucleotide conformation by resolution-enhanced ultraviolet spectroscopy poly(rC) and poly(dC).

Self-deconvolution and the fourth derivative of ultraviolet absorption spectra have been used to study stacked single-stranded and double-helix structures of different cytosine-containing polynucleotides for the first time. These compounds were studied under different solution conditions (pH and organic solvents) and at low temperatures. The red shift of the lower band (B2u band plus possibly some n-->pi* transition) of the absorption spectra in the cytosine-containing polynucleotides and the appearance of new peaks in the deconvoluted and derivative spectra in the 280-310 nm region are attributed mainly to cytosine-cytosine stacking interactions. In particular, the fourth-derivative peaks at wavelengths higher than 290 nm can be associated to coupling of electronic transitions of cytosine bases. The nature of the electronic transitions producing the absorption bands which are resolved in the aforementioned fourth-derivative peaks is discussed. It is concluded that the resolution-enhancement techniques used in this work, i.e. self-deconvolution and fourth derivative, complement each other and are useful methods to study structural changes of single-stranded and double-stranded polynucleotides allowing, at the same time, more information to be obtained about specific stacking interactions than classical absorption spectrophotometry.

The biological chemistry of the elements: the inorganic chemistry of life

Part 1 The chemical and physical factors controlling the elements of life: the chemical elements in biology the principles of the chemical uptake and speciation of the elements in biology physical separation of the elements - compartments and zones in biology kinetic considerations of element reactions energy in biological systems and hydrogen biochemistry the functional value of the chemical elements in biological systems. Part 2 The roles of individual elements in biology: the role of biological macromolecules and polymers sodium and potassium - osmotic control, electrolyte equilibria and currents magnesium - primary metabolism and polynucleotide structures calcium - controls and triggers zinc - Lewis acid catalysis and regulation non-haem iron redox reactions and controls haem iron - coupled redox reactions manganese copper - extracellular redox reactions and matrix formation nickel and cobalt - primitive hydrogen and organmetallic biochemistry molybdenum and vanadium - nitrogen fixation and extracellular oxygen transfer phosphorus and silicon - condensation reactions of non-metals redox non-metals - sulphur, selenium and halogens. Part 3 The co-operative interaction of elements in living systems: biomineralization and shape homeostasis and morphogenesis man's interference in element distribution - medicines and pollutants.

Complex between single-stranded DNA and gene 5 protein of bacteriophage M13 studied with linear dichroism and ultraviolet absorption

We have studied complexes between the gene 5 protein (gp5) of bacteriophage M13 and various polynucleotides, including single-stranded DNA, using ultraviolet absorption and linear dichroism. Upon complex formation the absorption spectra of both the protein and the polynucleotides change. The protein absorption changes indicate that for at least two of the five tyrosine residues per protein monomer the environment becomes less polar upon binding to the polynucleotides but also to the oligonucleotide p(dT) 8. All gp5-polynucleotide complexes give rise to intense linear dichroism spectra. These spectra are dominated by negative contributions from the bases, but also a small positive dichroism of the protein can be discerned. The spectra can be explained by polynucleotide structures, which are the same in all complexes. The base orientations are characterized by a substantial inclination and propellor twist. The number of possible combinations of inclination and propeller twist values, which are in agreement with the linear dichroism results, is rather limited. The base orientations with respect to the complex axis are essentially different from those in the complex with the single-stranded DNA-binding protein gp32 of bacteriophage T4.

Block‐units method for conformational calculations of large nucleic acid chains. I. Block‐units approximation of atomic structure and conformational energy of polynucleotides

A new block-units method for rapid conformational calculation of large nucleic acid fragments has been developed. Atomic structure of polynucleotides has been approximated by the block-units structure. Each monomer of the polynucleotide consists of three one-center block units: phosphate, ribose, and nucleic base. Full conformational energy of the polynucleotide is separated into two parts. The first part is the energy of the short-range interactions between adjacent block units and is calculated on the basis of the potential energy surface model of the dinucleotide fragment pXp (where X = A, G, T, U, C). The second part is the energy of the middle- and long-range interactions between separated block units, and is calculated as a sum of the effective interaction energies between centers of the block units. The present block-units method is in agreement within the range of +/- 0.5 kcal/mol with the method of the atom-atom potentials, but the former is 30-100-fold faster. The block-units method is recommended for screening of the probable conformations of the large polynucleotide systems.

THE INTERACTIONS OF PYRENYLMETHYL TRIBUTYLPHOSPHONIUM BROMIDE WITH SINGLE STRAND POLYNUCLEOTIDES

The binding of the water soluble derivative of pyrene, pyrenylmethyl tri-n-butylphosphonium bromide (PMTP), to single strand polynucleotides has been characterised by changes in the absorption spectra, fluorescence spectra, the fluorescence lifetime and the time-resolved fluorescence anisotropy of PMTP. For polyguanylic acid at low ionic strength, there are two modes of binding; external ionic binding and intercalation between the bases. The former results in PMTP excimer formation and the latter involves a charge-transfer complex between PMTP and guanine bases. Polyadenylic acid and polyinosinic acid show only external binding and poly[C] exhibits an interaction which is a combination of external binding and partial intercalation. Single strand DNA binds PMTP by intercalation and external binding at low ionic strength. In the presence of 0.2 M sodium chloride, only binding by intercalation between the bases occurs for poly[G]. Single strand DNA bound PMPT by partial intercalation, as well as full intercalation between the bases, but polyadenylic acid, polycytidylic acid and polyinosinic acid showed no significant binding in the presence of 0.2 M sodium chloride. The differences in the binding of PMTP are attributed to variations in the rigidity and form the structures of single strand polynucleotides adopt in solution. A full analysis of the binding isotherms has been made using methods based on Scatchard plots and the McGhee and von Hippel approach, which are critically compared.

Singlet oxygen takes part in 8-hydroxydeoxyguanosine formation in deoxyribonucleic acid treated with the horseradish peroxidase-H2O2 system.

Treatment of calf thymus deoxyribonucleic acid (DNA) with the horseradish peroxidase-H2O2 system resulted in efficient formation of 8-hydroxydeoxyguanosine (8-OH-dG) residues. It was concluded that singlet oxygen was the reactive species involved, based on experiments using active oxygen scavengers and D2O. For 8-OH-dG formation, a higher-ordered polynucleotide structure seems to be required: double stranded DNA was a better substrate for the reactive species than single stranded DNA, and monomeric deoxyguanosine underwent C8-hydroxylation to a lesser extent.

Existence of the C Structure in Poly(dA-dC) · Poly(dG-dT)

We show that the lithium salt of calf-thymus DNA can assume the C structure in nonoriented, hydrated gels. The transitions between the B and C structures showed little hysteresis and none of the metastable structural states which occur in oriented gels. Therefore crystal-lattice forces are not needed to stabilize the C structure. The occurrence of the alternative structures of the Li, Na and K salts of poly(dA-dC).poly(dG-dT) was measured as a function of hydration for nonoriented gels. Poly(dA-dC).poly(dG-dT).Li exists in the B structure at high hydrations and in the C structure at moderate hydrations with no A or Z structure at any hydration tested. The Na salt of poly(dA-dC).poly(dG-dT) exists in the B structure at high hydration, as mixtures of B and C at moderate hydrations and in the A structure at lower hydrations. The potassium salt behaves similarly except that mixtures of the C and A structures exist at lower hydrations. ZnCl2 and NaNO3, which promote the Z structure in duplex poly(dG-dC), promote the C structure in poly(dA-dC).poly(dG-dT). Information contained in the sequence of base pairs and not specific ionic interactions appear to determine the stability of the alternative structures of polynucleotides as hydration is changed.

Changes in DNA and surface properties of peripheral blood leukocytes in monkeys after whole-body ?-irradiation

Changes of Macaca nemestrina and Rhesus blood DNA have been studied up to 5 days after the total uniform gamma-irradiation in doses 6.2 and 6.5 Gy. The content of nucleotide ATrich DNA has been evaluated in the fractions of leucocytes with the various surface adherence properties. The dynamics of the content nucleotide AT-DNA and blood leucocytes were similar at the both monkey species. The evaluation of structural state DNA in the blood nucleotide with the fluorescent dyes (ethidium bromide and 4; 6-diamidine-2-phenylindole) demonstrated that the important changes in the polynucleotide structure occurred from 6 to 24 h after irradiation and maintained up to 5 days. Adhesive capacity changes were reversible but they preceded the DNA structural changes. At 24 h postirradiation non-adhesive cells with relative higher AT-DNA content were found.

Modification of Bacillus subtilis elongation factor Tu by N-tosyl-L phenylalanyl chloromethane abolishes its ability to interact with the 3′-terminal polynucleotide structure but not with the acyl bond in aminoacyl-tRNA

Modification of B. subtilis EF-TU by N-tosyl-L-phenylalanyl chloromethane destroyed its ability to promote protein synthesis and resulted in selective dissociation of the two binding activities of the protein for aminoacyl-tRNA. The modified EF-Tu was completely ineffective in the protection of the 3′-terminal CCA structure of tRNA against pancreatic ribonuclease, while remaining almost fully active in the protection of the ester bond between the 3′-terminal adenosine and the amino acid residue in aminoacyl-tRNA.

Anomalous structure and properties of poly (dA).poly(dT). Computer simulation of the polynucleotide structure with the spine of hydration in the minor groove.

The results of the search for low-energy conformations of poly(dA).poly(dT) and of the poly(dA).poly(dT) "complex" with the spine of hydration similar to that found by Dickerson and co-workers (Kopka, M.L., Fratini, A.V., Drew, H.R. and Dickerson, R.E. (1983) J. Mol. Biol. 163, 129-146) in the minor groove of the CGCGAATTCGCG crystals are described. It is shown that the existence of such a spine in the minor groove of poly(dA).poly(dT) is energetically favourable. Moreover, the spine of hydration makes the polynucleotide conformation similar to the poly(dA).poly(dT) structure in fibers and to the conformation of the central part of CGCGAATTCGCG in crystals; it also acquires features characteristic of the structure of poly(dA).poly(dT) and DNA oligo(dA)-tracts in solution. It is shown that the existence of the TpA step in conformations characteristic of the poly(dA).poly(dT) complex with the spine of hydration is energetically unfavourable (in contrast to the ApT step) and therefore this step should result in destabilization of the spine of hydration in the DNA minor groove. Thus, it appears that the spine of hydration as described by Dickerson and co-workers is unlikely to exist in the poly d(A-T).poly d(A-T) structure. The data obtained permit us to interpret a large body of experimental facts concerning the unusual structure and properties of poly(dA).poly(dT) and oligo(dA)-tracts in DNA both in fibers and in solution. The results provide evidence of the existence of the minor groove spine of hydration both in fibers and in solution on A/T tracts of DNA which do not contain the TpA step. The spine plays an active role in the formation of the anomalous conformation of these tracts.