splatche: a program to simulate genetic diversity taking into account environmental heterogeneity

We present a program called splatche (SPatiaL And Temporal Coalescences in Heterogeneous Environments) to simulate the molecular diversity of samples of genes in an environmentally heterogeneous world. Simulations are performed by, first, simulating the colonization of the world using environmental information to constrain migrations and local densities. These simulated densities and migration rates recorded over time and space are then used to simulate genetic diversity under a coalescent framework. The program thus virtually allows the translation of ecological information into molecular diversity, a novel approach that can be used to study the effect of climatic change on genetic diversity.     

Sequence-dependent anisotropic flexibility of B-DNA. A conformational study

Bending flexibility of the six tetrameric duplexes was investigated d(AAAA):d(TTTT), d(AATT)2, d(TTAA)2, d(GGGG):d(CCCC), d(GGCC)2 and d(CCGG)2,. The tetramers were extended in the both directions by regular double helices. The stiffness of the B-DNA double helix when bent into the both grooves proved to be less than that in the perpendicular direction by an order of magnitude. Such an anisotropy is a property of the sugar-phosphate backbone structure. The calculated fluctuations of the DNA bending along the dyad axis, 5-7 degree, are in agreement with experimental value of the DNA persistence length. Anisotropy of the double helix is sequence-dependent: most easily bent into the minor groove are the tetramers with purine-pyrimidine dimer (RY) in the middle. In contrast, YR dinucleotides prefer bending into the major groove. Moreover, they have an equilibrium bend of 6-12 degree into this groove. The above inequality is caused by stacking interaction of the bases. The bend in the central dimer is distributed to some extent between the adjacent links, though the main fraction of the bend remains within the central link. Variation of the sugar-phosphate geometry in the bent helix is inessential, so that DNA remains within the B-family of forms: namely, when the helical axis is bent by 20 degree. the backbone dihedral angles vary by no more than 15 degree. The obtained results are in accord with x-ray structure of the B-DNA dodecamer; they further substantiate our early model of DNA wrapping in the nucleosome by means of "mini-kinks" separated by a half-pitch of the double helix, i.e. by 5-6 b.p. Sequence-dependent anisotropy of DNA presumably dictates the three-dimensional structure of DNA in solution as well. We have found that nonrandom allocation of YR dimers leads to the systematic bends in equilibrium structure of certain DNA fragments.     

Recent advances in the study of nucleic acid flexibility by molecular dynamics

The recent use of molecular dynamics (MD) simulations to study flexibility of nucleic acids has been reviewed from an analysis of the publications appearing in the past two years (from 2005 till date). Despite the existence of some unsolved problems in the methodologies, these years have been witness to major advances in the field. Based on a critical review of the most recent contributions, excitement exists on the expected evolution of the field in the next years.     

Towards a molecular dynamics consensus view of B-DNA flexibility

We present a systematic study of B-DNA flexibility in aqueous solution using long-scale molecular dynamics simulations with the two more recent versions of nucleic acids force fields (CHARMM27 and parmbsc0) using four long duplexes designed to contain several copies of each individual base pair step. Our study highlights some differences between pambsc0 and CHARMM27 families of simulations, but also extensive agreement in the representation of DNA flexibility. We also performed additional simulations with the older AMBER force fields parm94 and parm99, corrected for non-canonical backbone flips. Taken together, the results allow us to draw for the first time a consensus molecular dynamics picture of B-DNA flexibility.     

A theoretical study of the sequence specificity in binding of lexitropsins to B-DNA

A theoretical study is presented on the binding to B-DNA of a series of lexitropsins, these ligands being netropsin derivatives in which one or both of the pyrrole rings have been replaced by imidazoles. The best complexes have been located by energy minimisation taking into account nucleic acid flexibility, ligand flexibility, explicit, mobile counterions and solvent dielectric effects. Calculations have been performed for two homopolymeric DNA receptor sequences, AT base sequence, which only decreases in the imidazole derivatives. These results emphasize the decisive role of the molecular electrostatic potential of the nucleic acid in determining the sequence selectivity of these ligands, as opposed to the postulated role of adenine C2 - pyrrole beta hydrogen contacts.     

Observation of an A-DNA to B-DNA transition in a nonhelical nucleic acid hairpin molecule using molecular dynamics.

One of the truly challenging problems for molecular dynamics (MD) simulations is demonstrating that the trajectories can sample not only in the vicinity of an experimentally determined structure, but also that the trajectories can find the correct experimental structure starting from some other structure. Frequently these transitions to the correct structure require that the simulations overcome energetic barriers to conformational change. Here we present unrestrained molecular dynamics simulations of the DNA analogs of the RNA 5'-GGACUUCGGUCC-3' hairpin tetraloop. In one simulation we have used deoxyuracil residues, and in the other we have used the native DNA deoxythymine residues. We demonstrate that, on a nanosecond time scale, MD is able to simulate the transitions of both of the A-DNA stems to B-DNA stems within the constraints imposed by the four-base loop that caps the helix. These results suggest that we are now in a position to use MD to address the nature of sequence-dependent structural effects in nonduplex DNA structures.     

Quinoxaline antibiotics enhance peptide nucleic acid binding to double-stranded DNA

The effects of a wide range of DNA binding drugs on peptide nucleic acid (PNA) binding to double-stranded DNA by strand displacement have been investigated using a gel retardation assay. The bis-PNA [H-(Lys)-TTJTTJTTTT-(eg)(3)-TTTTCTTCTT-Lys-NH(2)] was used together with a 248 bp DNA fragment containing an appropriate target for the PNA. Most of the ligands that were studied, including DNA minor groove binders as well as intercalators and bis-intercalators, either have no effect or strongly inhibit PNA binding to DNA. By contrast, quinoxaline antibiotics facilitate PNA-DNA complex formation. The "PNA-helper" effect of echinomycin was studied in more detail using time and temperature dependence experiments to elucidate the mechanism. PNA binding to DNA follows pseudo-first-order kinetics, but the initial rate of binding is accelerated more than 10-fold in the presence of 10 microM echinomycin. The activation energy for PNA binding to dsDNA is lowered 2-fold by the antibiotic (45 vs 90 kJ/mol in the control). The reasons why quinoxalines promote the binding of PNA to DNA are not entirely clear but may well include distortions (opening) of the double helix that facilitate PNA invasion. This study establishes that the efficacy of DNA-targeted PNA antigene molecules could potentially be enhanced by judiciously adding certain DNA-interactive ligands.     

Molecular-modeling calculations of enzymatic enantioselectivity taking hydration into account

A new molecular-modeling methodology has been applied to explain enzymatic enantioselectivity in water. This methodology, which combines vacuum molecular mechanics and the continuum solvation method, should provide a more realistic view of the solvent-enzyme and solvent-substrate interactions than the heretofore used approaches involving the vacuum molecular mechanics only. The methodology described herein has been validated using the experimental data on alpha-chymotrypsin's enantioselectivity in the hydrolysis of four chiral substrates. The reasons why the vacuum molecular mechanics, although not taking hydration into account, still in most cases provide a satisfactory approximation of reality are discussed.     

Spermine-nucleic acid interactions: a theoretical study

The interaction of spermine with nucleic acids is simulated theoretically using refined semi-empirical energy formulae and an advanced minimization procedure. Various nucleic acids are considered: model homopolymeric DNA's, a dodecamer (CGCGAATTCGCG) of type B-DNA, as well as a transfer RNA, tRNA^Phe. The dominant role of electrostatic potential in determining the preferential binding sites of spermine is demonstrated in each of these cases and the role of counterions, nucleic acid structure, and base-pair sequence is analyzed.     

The solvation contribution to the binding energy of DNA with non-intercalating antibiotics.

The influence of the solvent on the binding energies to DNA of six non-intercalating antibiotics - netropsin, distamycin-3, distamycin-2, SN 18071, berenil and stilbamidine - is evaluated by combining the effect of the first hydration shell with that of bulk water. The first effect is computed by a methodology based on a spherical/point dipole model of water and limited to electrostatic interaction energies. Hydration shells are obtained which are energy optimized with respect to both water-solute and water-water interactions for the complexes and for the isolated DNA oligomers and ligands. The method allows even very large complexes to be studied in reasonable computation times. The second effect is introduced via a cavity treatment. It is shown that if the vacuum interaction energies already predict correctly the preference of the ligands for the minor groove of AT sequences of B-DNA, the introduction of the solvation effect is indispensable for reproducing the order of affinity of the ligands and for bringing the values of the complexation energies into close agreement with experimental data.     

Towards a methodology for taking physical degradation of ecosystems into account in LCA

An analytic procedure has been followed to select adequate methods to express ecosystem degradation in LCA. This procedure consisted of problem definition, identification of relevant issues, of a quantitative expression for ecosystem degradation and of possible nature value indicators and building a framework of criteria for selecting adequate methods. With the selection framework a first screening of methods was performed. For full quantification the following formula is proposed: ED = L (N_r- N_a), with land use I. = A t and nature value change (N_r- N_a) Degradation due to an activity appears difficult to operationalise, but ecosystem suppression by activities can well be assessed. N_r is then the natural background or would-be natural situation. N_a can best be described by the actual state during the activity, unless hard data on restoration is available. N_a and N_r can be expressed in the biomass production indicator NPP - NCR Biodiversity and/or erosion may be added to include irreversible effects.     

A precise method for the quantitation of proteins taking into account their amino acid composition.

A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-μl solutions containing 1 to 100 μg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per aliquot of hydrolysate analyzed can be determined.     

The relative flexibility of B-DNA and A-RNA duplexes: database analysis

An extensive analysis of structural databases is carried out to investigate the relative flexibility of B-DNA and A-RNA duplexes in crystal form. Our results show that the general anisotropic concept of flexibility is not very useful to compare the deformability of B-DNA and A-RNA duplexes, since the flexibility patterns of B-DNA and A-RNA are quite different. In other words, ‘flexibility’ is a dangerous word for describing macromolecules, unless it is clearly defined. A few soft essential movements explain most of the natural flexibility of A-RNA, whereas many are necessary for B-DNA. Essential movements occurring in naked B-DNAs are identical to those necessary to deform DNA in DNA–protein complexes, which suggest that evolution has designed DNA–protein complexes so that B-DNA is deformed according to its natural tendency. DNA is generally more flexible, but for some distortions A-RNA is easier to deform. Local stiffness constants obtained for naked B-DNAs and DNA complexes are very close, demonstrating that global distortions in DNA necessary for binding to proteins are the result of the addition of small concerted deformations at the base-pair level. Finally, it is worth noting that in general the picture of the relative deformability of A-RNA and DNA derived from database analysis agrees very well with that derived from state of the art molecular dynamics (MD) simulations.     

Binding of Hoechst 33258 to the minor groove of B-DNA

An X-ray crystallographic structure analysis has been carried out on the complex between the antibiotic and DNA fluorochrome Hoechst 33258 and a synthetic B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-C-G-C-G. The drug molecule, which can be schematized as: phenol-benzimidazole-benzimidazole-piperazine, sits within the minor groove in the A-T-T-C region of the DNA double helix, displacing the spine of hydration that is found in drug-free DNA. The NH groups of the benzimidazoles make bridging three-center hydrogen bonds between adenine N-3 and thymine O-2 atoms on the edges of base-pairs, in a manner both mimicking the spine of hydration and calling to mind the binding of the auti-tumor drug netropsin. Two conformers of Hoechst are seen in roughly equal populations, related by 180 degrees rotation about the central benzimidazole-benzimidazole bond: one form in which the piperazine ring extends out from the surface of the double helix, and another in which it is buried deep within the minor groove. Steric clash between the drug and DNA dictates that the phenol-benzimidazole-benzimidazole portion of Hoechst 33258 binds only to A.T regions of DNA, whereas the piperazine ring demands the wider groove characteristic of G.C regions. Hence, the piperazine ring suggests a possible G.C-reading element for synthetic DNA sequence-reading drug analogs.     

The flexibility of the nucleic acids: (III). The interaction of an aliphatic diamine, putrescine, with flexible B-DNA

A theoretical modelling of the interaction of putrescine (H3+N-(CH2)4-(+NH3) with DNA is carried out, introducing two new features which make the simulation of this interaction considerably more realistic. Firstly, the DNA to which putrescine is bound is fully flexible and thus able to respond to the distorting influence of the ligand. Secondly, the effect of changing the ratio of DNA base pairs per bound ligand is explicitly modelled. In this way, we have been able to confirm the experimentally known preference of putrescine binding with AT base pairs in B-DNA, but we also show, through the new features introduced, that the nature of the binding site of the ligand and the resulting impact on DNA conformation is strongly modified by the ligand binding density.     

Interactions of molecules with nucleic acids. I. An algorithm to generate nucleic acid structures with an application to the B-DNA structure and a counterclockwise helix.

An algorithm is developed that enables the routine determination of backbone conformations of nucleic acids. All atomic positions including hydrogen are specified in accord with experimental bond lengths and angles but with theoretically determined conformational angles. For two Watson-Crick base pairs at a separation of 3.38 Å, and perpendicular to a common helical axis, minimum energy configurations are found for all 10 combinations at helical angles of α ～ 36°–38°, corresponding to the B-DNA structure with C(2′)-endo sugar puckers. Backbone configurations exist only within the range 35.5° ? α ? 42°, which suggests the origin of the 10-fold helix. Calculated stacking energies for the B-DNA structure increases for each of the clustered groups of base pairs: G·C with G·C, G·C with A·T, and A·T with A·T, and they are in approximate agreement with experimental observations. The counter-clockwise helix is examined, and physically meaningful structures are found only when the helical axes of successive base pairs are disjointed.