The interaction of adriamycin and adriamycin analogues with nucleic acids in the B and A conformations.

The reinforced intercalative binding to DNA typical of adriamycin and daunomycin can still occur if there is epimerisation at C4' or if the O-methyl group is lost or if the 9-substituents are deleted or if the 4'-hydroxyl group is lost. In the latter two cases however, there is a reduction in affinity for the DNA, supporting the suggested role of the 9-hydroxyl and 4'-hydroxyl groups in secondary stabilization of the complex. Epimerisation at C-1' or at C-3' alters but does not abolish the intercalative mode of binding to DNA whereas epimerisation at C-7 precludes intercalation of the chromophore into the helix of DNA. In contrast to the interaction with the B-form found in DNA, the parent drugs do not intercalate into nucleic acids possessing the A-conformation and none of the above-mentioned structural changes will allow intercalation into A-form nucleic acids.     

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.     

Global mapping of nucleic acid conformational space: dinucleoside monophosphate conformations and transition pathways among conformational classes

A global conformational space of 6253 dinucleoside monophosphate (DMP) units consisting of RNA and DNA (free and protein/drug-bound) was 'mapped' using high resolution crystal structures cataloged in the Nucleic Acid Database (NDB). The torsion angles of each DMP were clustered in a reduced three-dimensional space using a classical multi-dimensional scaling method. The mapping of the conformational space reveals nine primary clusters which distinguish among the common A-, B- and Z-forms and their various substates, plus five secondary clusters for kinked or bent structures. Conformational relationships and possible transitional pathways among the substates are also examined using the conformational states of DNA and RNA bound with proteins or drugs as potential pathway intermediates.     

Protein and nucleic acid changes during growth and aging in the mosquito

1. Mosquito samples from the entire life span including senescence were analysed for weight, protein, RNA and DNA. 2. The results for each component indicate that maximal values are attained during larval development. No changes were observed during metamorphosis or the adult period. 3. The peak concentration of RNA and DNA was reached on the third day larval age. Protein content and weight were highest on the sixth day. 4. Except for the first 2 days of larval life the DNA/protein ratio was constant. Hence protein content is an index of cell number for this organism. 5. The distribution of the different biochemical components was determined in different body regions and in subcellular fractions. A high concentration of DNA was found in the soluble fraction of larval but not adult homogenates.     

A NMR strategy to unambiguously distinguish nucleic acid hairpin and duplex conformations applied to a Xist RNA A-repeat

All RNA sequences that fold into hairpins possess the intrinsic potential to form intermolecular duplexes because of their high self-complementarity. The thermodynamically more stable duplex conformation is favored under high salt conditions and at high RNA concentrations, posing a challenging problem for structural studies of small RNA hairpin conformations. We developed and applied a novel approach to unambiguously distinguish RNA hairpin and duplex conformations for the structural analysis of a Xist RNA A-repeat. Using a combination of a quantitative HNN-COSY experiment and an optimized double isotope-filtered NOESY experiment we could define the conformation of the 26-mer A-repeat RNA. In contrast to a previous secondary structure prediction of a double hairpin structure, the NMR data show that only the first predicted hairpin is formed, while the second predicted hairpin mediates dimerization of the A-repeat by duplex formation with a second A-repeat. The strategy employed here will be generally applicable to identify and quantify populations of hairpin and duplex conformations and to define RNA folding topology from inter- and intra-molecular base-pairing patterns.     

Study of the hydration characteristics of nucleic acid bases and their complementary pairs using the Monte-Carlo method

To elucidate the role of certain atomic groups in the formation of the nucleic acid hydrate shell, we simulated the systems involving a base or a complementary pair (the base molecules are methylated in N9 of purines and in N1 of pyrimidines) and 25 water molecules using the Monte-Carlo method. All hydrophilic centers, except for N1 purines and N3 pyrimidines in complementary pairs, form hydrogen bonds (H-bonds) with water molecules. The mean numbers of H-bonds formed by different centers, and distributions of the geometric characteristics of these bonds, which appeared similar to those in crystals, have been calculated. The formation of bridges of one, two of three water molecules between hydrophilic centers was shown. The probabilities of formation of these bridges have been calculated.     

Molecular dynamic simulations of environment and sequence dependent DNA conformations: the development of the BMS nucleic acid force field and comparison with experimental results

Molecular dynamic (MD) simulations using the BMS nucleic acid force field produce environment and sequence dependent DNA conformations that closely mimic experimentally derived structures. The parameters were initially developed to reproduce the potential energy surface, as defined by quantum mechanics, for a set of small molecules that can be used as the building blocks for nucleic acid macromolecules (dimethyl phosphate, cyclopentane, tetrahydrofuran, etc.). Then the dihedral parameters were fine tuned using a series of condensed phase MD simulations of DNA and RNA (in zero added salt, 4M NaCl, and 75% ethanol solutions). In the tuning process the free energy surface for each dihedral was derived from the MD ensemble and fitted to the conformational distributions and populations observed in 87 A- and B-DNA x-ray and 17 B-DNA NMR structures. Over 41 nanoseconds of MD simulations are presented which demonstrate that the force field is capable of producing stable trajectories, in the correct environments, of A-DNA, double stranded A-form RNA, B-DNA, Z-DNA, and a netropsin-DNA complex that closely reproduce the experimentally determined and/or canonical DNA conformations. Frequently the MD averaged structure is closer to the experimentally determined structure than to the canonical DNA conformation. MD simulations of A- to B- and B- to A-DNA transitions are also shown. A-DNA simulations in a low salt environment cleanly convert into the B-DNA conformation and converge into the RMS space sampled by a low salt simulation of the same sequence starting from B-DNA. In MD simulations using the BMS force field the B-form of d(GGGCCC)2 in a 75% ethanol solution converts into the A-form. Using the same methodology, parameters, and conditions the A-form of d(AAATTT)2 correctly converts into the B-DNA conformation. These studies demonstrate that the force field is capable of reproducing both environment and sequence dependent DNA structures. The 41 nanoseconds (nsec) of MD simulations presented in this paper paint a global picture which suggests that the DNA structures observed in low salt solutions are largely due to the favorable internal energy brought about by the nearly uniform screening of the DNA electrostatics. While the conformations sampled in high salt or mixed solvent environments occur from selective and asymmetric screening of the phosphate groups and DNA grooves, respectively, brought about by sequence induced ion and solvent packing.     

HIV-1 Gag extension: conformational changes require simultaneous interaction with membrane and nucleic acid

The retroviral Gag polyprotein mediates viral assembly. The Gag protein has been shown to interact with other Gag proteins, with the viral RNA, and with the cell membrane during the assembly process. Intrinsically disordered regions linking ordered domains make characterization of the protein structure difficult. Through small-angle scattering and molecular modeling, we have previously shown that monomeric human immunodeficiency virus type 1 (HIV-1) Gag protein in solution adopts compact conformations. However, cryo-electron microscopic analysis of immature virions shows that in these particles, HIV-1 Gag protein molecules are rod shaped. These differing results imply that large changes in Gag conformation are possible and may be required for viral formation. By recapitulating key interactions in the assembly process and characterizing the Gag protein using neutron scattering, we have identified interactions capable of reversibly extending the Gag protein. In addition, we demonstrate advanced applications of neutron reflectivity in resolving Gag conformations on a membrane. Several kinds of evidence show that basic residues found on the distal N- and C-terminal domains enable both ends of Gag to bind to either membranes or nucleic acid. These results, together with other published observations, suggest that simultaneous interactions of an HIV-1 Gag molecule with all three components (protein, nucleic acid, and membrane) are required for full extension of the protein.     

Interaction of nucleic acid bases with water molecules and formation of mismatched nucleotide pairs

The possibility of the inclusion of water molecules in the formation of mismatched nucleotide pairs was considered in relation to the mechanisms of point errors in template directed biosynthesis. Calculations of the intermolecular interaction energy for systems containing two bases and one water molecule were carried out by the method of atom-atom potential functions. There exist energy minima for each base pair, corresponding to a single N--H...O or N--H...N H-bond between the bases and H-bonding of the water molecule with both bases. The relative positions of glycosyl bonds in some of these minima are closer to those for Watson--Crick pairs, than the positions of minima for these pairs without water. For other minima, the H-bond formation between the water molecule and the two bases additionally stabilizes the relative base position in wobble-pairs with two H-bonds between the bases. The base and water positions in energy minima are compared with the positions in some pairs proposed on the basis of NMR and X-ray data for double helical oligonucleotides.     

Volume changes accompanying the complex formation of polyadenylic and polyuridylic acids

The complex formation of polyadenylic acid (poly A) and polyuridylic acid (poly U) in 0.1M NaCl solution containing 0.01M sodium cacodylate was followed by dilatometric measurements at various mixing ratios of poly A and poly U. The volume changes, ΔV, accompanying the formation of poly A. poly U and poly A.2poly U were + l.5 and + 2.5 ml per mole of the nucleotide residue, respectively. This increase in volume was probably due to the increased counterion binding when the single-stranded polynucleotides were converted into the double- and triple-stranded helices, since depletion of charged species from the solvent proper would lessen the effect of electrostriction, thus resulting in a positive ΔV. The conversion of a single-stranded poly A to a double-stranded helix in acidic solution led to a ΔV of + 3.8 ml per mole of the nucleotide residue. This increase in volume was attributed to the charge neutralization as a result of protonation of the adenine bases.     

Two carbocyclic locked nucleic acid analogues give structural information about the role of hydration in A-type duplexes

Two locked nucleic acid (LNA) analogues with three-carbon 2'-4' linkages, saturated or unsaturated, are synthesized using a ring-closing metathesis based strategy. Strongly stabilized duplexes with complementary RNA and slightly destabilized duplexes with complementary DNA are observed. CD-spectroscopy indicates a less pronounced shift toward A-type duplexes compared to LNA. These results combining a strong N-type conformation with the absence of a 2'-oxygen demonstrate a stronger importance of minor groove hydration in an intermediate duplex type than in an A-type duplex.     

Cyclic AMP-induced differentiated neuroblastoma cells: changes in total nucleic acid and protein contents

The total DNA contents of neuroblastoma cells “differentiated” by dibutyryl cyclic AMP, prostaglandin E₁ and 4-(-3-butoxy-4-methoxybenzyl)-2-imidazolidinone treatment was about 50 percent that of control cells, indicating that cells were accumulated in the G₁-phase of the cell cycle. Sodium butyrate-treated cells were also accumulated in the G₁-phase; however, the expression of “differentiated” phenotype did not occur indicating that inhibition of cell division is not sufficient for morphological differentiation. A marked increase in RNA and protein contents of cyclic AMP-induced “differentiated” cells is consistent with an increase in the size of soma and nucleus.     

Biphasic gene expression changes elicited by Phakopsora pachyrhizi in soybean correlate with fungal penetration and haustoria formation

Inoculation of soybean (Glycine max) plants with Phakopsora pachyrhizi, the causal organism of Asian soybean rust, elicits a biphasic response characterized by a burst of differential gene expression in the first 12 h. A quiescent period occurs from 24 to 48 h after inoculation, in which P. pachyrhizi continues to develop but does not elicit strong host responses, followed by a second phase of intense gene expression. To correlate soybean responses with P. pachyrhizi growth and development, we inoculated the soybean cultivar Ankur (accession PI462312), which carries the Rpp3 resistance gene, with avirulent and virulent isolates of P. pachyrhizi. The avirulent isolate Hawaii 94-1 elicits hypersensitive cell death that limits fungal growth on Ankur and results in an incompatible response, while the virulent isolate Taiwan 80-2 grows extensively, sporulates profusely, and produces a compatible reaction. Inoculated leaves were collected over a 288-h time course for microarray analysis of soybean gene expression and microscopic analysis of P. pachyrhizi growth and development. The first burst in gene expression correlated with appressorium formation and penetration of epidermal cells, while the second burst of gene expression changes followed the onset of haustoria formation in both compatible and incompatible interactions. The proliferation of haustoria coincided with the inhibition of P. pachyrhizi growth in the incompatible interaction or the beginning of accelerated growth in the compatible interaction. The temporal relationships between P. pachyrhizi growth and host responses provide an important context in which to view interacting gene networks that mediate the outcomes of their interactions.     

A systematic study of patterns of hydration in nucleic acids:(I) guanine and cytosine

The hydration sites of guanine and cytosine are defined by examination of the crystal structures of bases, nucleosides, nucleotides, and three dinucleoside phosphate salts. The patterns of hydration for two guanine and cytosine containing oligonucleotides are then predicted. The relationship between these structural motifs and thermodynamic parameters is discussed.     

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

Using 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.     

Thermodynamic, counterion, and hydration effects for the incorporation of locked nucleic acid nucleotides into DNA duplexes

A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O, 4'-C-methylene bridge added to the ribose ring. LNA-modified oligonucleotides are known to exhibit enhanced hybridization affinity toward complementary DNA and RNA. In this work, we have evaluated the hybridization thermodynamics of a series of LNA-substituted DNA octamers, modified to various extents by one to three LNA substitutions, introduced at either adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) nucleotides. To understand the energetics, counterion effects, and the hydration contribution of the incorporation of LNA modification, a combination of spectroscopic and calorimetric techniques was used. The CD spectra of the corresponding duplexes showed that the modified duplexes adopt an A-type conformation. UV and DSC melting studies revealed that each type of duplex unfolds in a two-state transition. A complete thermodynamic profile at 5 degrees C indicated that the net effect of modification on thermodynamic parameters might be positional and that the neighboring bases flanking the modification might influence the favorable formation of the modified duplexes. Furthermore, relative to the formation of the unmodified reference duplexes, the formation of modified duplexes is accompanied by a higher uptake of counterions and a lower uptake of water molecules.     

Evaluation of the proposed interaction of nucleic acid with Clostridium difficile toxins A and B and the effects of nucleases on cytotoxicity

Both DNA and RNA were found to co-purify with Clostridium difficile toxin B but not toxin A. DNAase treatment greatly reduced the cytotoxicity of toxin B but not of toxin A. RNAase had no effect on either toxin. The effects on toxin B were shown to be due to a contaminating protease and could be inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride.