The molecular structure of the complex of Hoechst 33258 and the DNA dodecamer d(CGCGAATTCGCG).

The crystal structure of the complex between the dodecamer d(CGCGAATTCGCG) and a synthetic dye molecule Hoechst 33258 was solved by X-ray diffraction analysis and refined to an R-factor of 15.7% at 2.25 A resolution. The crescent-shaped Hoechst compound is found to bind to the central four AATT base pairs in the narrow minor groove of the B-DNA double helix. The piperazine ring of the drug has its flat face almost parallel to the aromatic bisbenzimidazole ring and lies sideways in the minor groove. No evidence of disordered structure of the drug is seen in the complex. The binding of Hoechst to DNA is stabilized by a combination of hydrogen bonding, van der Waals interaction and electrostatic interactions. The binding preference for AT base pairs by the drug is the result of the close contact between the Hoechst molecule and the C2 hydrogen atoms of adenine. The nature of these contacts precludes the binding of the drug to G-C base pairs due to the presence of N2 amino groups of guanines. The present crystal structural information agrees well with the data obtained from chemical footprinting experiments.     

Flexibility of DNA in 2:1 drug-DNA complexes--simultaneous binding of two DAPI molecules to DNA.

Simultaneous binding of two DAPI molecules in the minor groove of (dA)15.(dT)15 B-DNA helix has been simulated by molecular mechanics calculations. The energy minimised structure shows some novel features in relation to binding of DAPI molecules as well as the flexibility of the grooves of DNA helices. The minor groove of the helix expands locally considerably (to 15 angstroms) to accommodate the two DAPI molecules and is achieved by positive propeller twisting of base pairs at the binding site concomitant with small variations in the local nucleotide stereochemistry. The expansion also brings forth simultaneously a contraction in the width of the major groove spread over to a few phosphates. These findings demonstrate another facet of the flexible stereochemistry of DNA helices in which the local features are significantly altered without being propagated beyond a few base pairs, and with the rest of the regions retaining the normal structure. Both the DAPI molecules are engaged in specific hydrogen bonds with the bases and non specific interactions with phosphates. Stacking interactions of DAPI molecules between themselves as well as with sugar-phosphate backbone contribute to the stability of the complex. The studies provide a stereochemical support to the experimental findings that under high drug-DNA ratio DAPI could bind in the 2:1 ratio.     

Solution structure of DAPI selectively bound in the minor groove of a DNA T.T mismatch-containing site: NMR and molecular dynamics studies.

The solution structure of the complex between 4', 6-diamidino-2-phenylindole (DAPI) and DNA oligomer [d(GCGATTCGC)]2, containing a central T.T mismatch, has been characterized by combined use of proton one- and two-dimensional NMR spectroscopy, molecular mechanics and molecular dynamics computations including relaxation matrix refinement. The results show that the DAPI molecule binds in the minor groove of the central region 5'-ATT-3' of the DNA oligomer, which predominantly adopts a duplex structure with a global right-handed B-like conformation. In the final models of the complex, the DAPI molecule is located nearly isohelical with its NH indole proton oriented towards the DNA helix axis and forming a bifurcated hydrogen bond with the carbonyl O2 groups of a mismatched T5 and the T6 residue of the opposite strand. Mismatched thymines adopt a wobble base pair conformation and are found stacked between the flanking base pairs, inducing only minor local conformational changes in global duplex structure. In addition, no other binding mechanisms were observed, showing that minor groove binding of DAPI to the mismatch-containing site is favoured in comparison with any other previously reported interaction with G.C sequences.     

DAPI binding to the DNA minor groove: a continuum solvent analysis

A continuum solvent model based on the generalized Born (GB) or finite-difference Poisson-Boltzmann (FDPB) approaches has been employed to compare the binding of 4'-6-diamidine-2-phenyl indole (DAPI) to the minor groove of various DNA sequences. Qualitative agreement between the results of GB and FDPB approaches as well as between calculated and experimentally observed trends regarding the sequence specificity of DAPI binding to B-DNA was obtained. Calculated binding energies were decomposed into various contributions to solvation and DNA-ligand interaction. DNA conformational adaptation was found to make a favorable contribution to the calculated total interaction energy but did not change the DAPI binding affinity ranking of different DNA sequences. The calculations indicate that closed complex formation is mainly driven by nonpolar contributions and was found to be disfavored electrostatically due to a desolvation penalty that outbalances the attractive Coulomb interaction. The calculated penalty was larger for DAPI binding to GC-rich sequences compared with AT-rich target sequences and generally larger for the FDPB vs the GB continuum model. A radial interaction profile for DAPI at different distances from the DNA minor groove revealed an electrostatic energy minimum a few Angstroms farther away from the closed binding geometry. The calculated electrostatic interaction up to this distance is attractive and it may stabilize a nonspecific binding arrangement.     

DNA sequence dependent binding modes of 4',6-diamidino-2-phenylindole (DAPI)

The interactions of DAPI with natural DNA and synthetic polymers have been investigated by hydrodynamic, DNase I footprinting, spectroscopic, binding, and kinetic methods. Footprinting results at low ratios (compound to base pair) are similar for DAPI and distamycin. At high ratios, however, GC regions are blocked from enzyme cleavage by DAPI but not by distamycin. Both poly[d(G-C)]2 and poly[d(A-T)]2 induce hypochromism and shifts of the DAPI absorption band to longer wavelengths, but the effects are larger with the GC polymer. NMR shifts of DAPI protons in the presence of excess AT and GC polymers are significantly different, upfield for GC and mixed small shifts for AT. The dissociation rate constants and effects of salt concentration on the rate constants are also quite different for the AT and the GC polymer complexes. The DAPI dissociation rate constant is larger with the GC polymer but is less sensitive to changes in salt concentration than with the AT complex. Binding of DAPI to the GC polymer and to poly[d(A-C)].poly[d(G-T)] exhibits slight negative cooperativity, characteristic of a neighbor-exclusion binding mode. DAPI binding to the AT polymer is unusually strong and exhibits significant positive cooperativity. DAPI has very different effects on the bleomycin-catalyzed cleavage of the AT and GC polymers, a strong inhibition with the AT polymer but enhanced cleavage with the GC polymer. All of these results are consistent with two totally different DNA binding modes for DAPI in regions containing consecutive AT base pairs versus regions containing GC or mixed GC and AT base pair sequences. The binding mode at AT sites has characteristics which are similar to those of the distamycin-AT complex, and all results are consistent with a cooperative, very strong minor groove binding mode. In GC and mixed-sequence regions the results are very similar to those observed with classical intercalators such as ethidium and indicate that DAPI intercalates in DNA sequences which do not contain at least three consecutive AT base pairs.     

Flexibility Of DNA In 2:1 Drug-DNA Complexes - Simultaneous Binding Of Two DAPI Molecules To DNA

Abstract

Simultaneous binding of two DAPI molecules in the minor groove of (dA)₁₅.(dT)₁₅ B-DNA helix has been simulated by molecular mechanics calculations. The energy minimised structure shows some novel features in relation to binding of DAPI molecules as well as the flexibility of the grooves of DNA helices. The minor groove of the helix expands locally considerably (to 15 Å) to accommodate the two DAPI molecules and is achieved by positive propeller twisting of base pairs at the binding site concomitant with small variations in the local nucleotide stereochemistry. The expansion also brings forth simultaneously a contraction in the width of the major groove spread over to a few phosphates. These findings demonstrate another facet of the flexible stereochemistry of DNA helices in which the local features are significantly altered without being propagated beyond a few base pairs, and with the rest of the regions retaining the normal structure. Both the DAPI molecules are engaged in specific hydrogen bonds with the bases and non specific interactions with phosphates. Stacking interactions of DAPI molecules between themselves as well as with sugar-phosphate backbone contribute to the stability of the complex. The studies provide a stereochemical support to the experimental findings that under high drug-DNA ratio DAPI could bind in the 2:1 ratio.     

Double helix conformation, groove dimensions and ligand binding potential of a G/C stretch in B-DNA.

The self-complementary DNA fragment CCGGCGCCGG crystallizes in the rhombohedral space group R3 with unit cell parameters a = 54.07 A and c = 44.59 A. The structure has been determined by X-ray diffraction methods at 2.2 A resolution and refined to an R value of 16.7%. In the crystal, the decamer forms B-DNA double helices with characteristic groove dimensions: compared with B-DNA of random sequence, the minor groove is wide and deep and the major groove is rather shallow. Local base pair geometries and stacking patterns are within the range commonly observed in B-DNA crystal structures. The duplex bears no resemblance to A-form DNA as might have been expected for a sequence with only GC base pairs. The shallow major groove permits an unusual crystal packing pattern with several direct intermolecular hydrogen bonds between phosphate oxygens and cytosine amino groups. In addition, decameric duplexes form quasi-infinite double helices in the crystal by end-to-end stacking. The groove geometries and accessibilities of this molecule as observed in the crystal may be important for the mode of binding of both proteins and drug molecules to G/C stretches in DNA.     

Use of Electric Linear Dichroism and Competition Experiments with Intercalating Drugs to Investigate the Mode of Binding of Hoechst 33258, Berenil and DAPI to GC Sequences

Abstract

The drugs Hoechst 33258, berenil and DAPI bind preferentially to the minor groove of AT sequences in DNA Despite a strong selectivity for AT sites, they can interact with GC sequences by a mechanism which remains so far controversial. The 2-amino group of guanosine represents a steric hindrance to the entry of the drugs in the minor groove of GC sequences. Intercalation and major groove binding to GC sites of GC-rich DNA and polynucleotides have been proposed for these drugs. To investigate further the mode of binding of Hoechst 33258, berenil and DAPI to GC sequences, we studied by electric linear dichroism the mutual interference in the DNA binding reaction between these compounds and a classical intercalator, proflavine, or a DNA-threading intercalating drug, the amsacrine-4-carboxamide derivative SN16713. The results of the competition experiments show that the two acridine intercalators markedly affect the binding of Hoechst 33258, berenil and DAPI to GC polynucleotides but not to DNA containing AT/GC mixed sequences such as calf thymus DNA Proflavine and SN16713 exert dissimilar effects on the binding of Hoechst 33258, berenil and DAPI to GC sites. The structural changes in DNA induced upon intercalation of the acridine drugs into GC sites are not identically perceived by the test compounds. The electric linear dichroism data support the hypothesis that Hoechst 33258, berenil and DAPI interact with GC sites via a non-classical intercalation process.     

The role of a minor groove spine of hydration in stabilizing poly(dA).poly(dT) against fluctuational interbase H-bond disruption in the premelting temperature regime.

Experimental estimates of the premelting Adenine-Thymine base pair opening probability for some B-DNA sequences are two orders of magnitude smaller than those of other B-DNA sequences. The AT pairs in the sequence with smaller open probability seem to be those that have a well defined spine of hydration in the minor groove. We show that this spine of hydration can significantly enhance the thermal stability of the base pairs to which they are attached. The effect of this spine of hydration coupled with the possible stabilization effect contributed from neighboring GC pairs can explain the differences in the observed AT pair opening probability for different AT containing B-DNA sequences.     

Molecular dynamics simulations of A. T-rich oligomers: sequence-specific binding of Na+ in the minor groove of B-DNA

Molecular dynamics simulations have been employed to probe the sequence-specific binding of sodium ions to the minor groove of B-DNA of three A. T-rich oligomers having identical compositions but different orders of the base pairs: C(AT)(4)G, CA(4)T(4)G, and CT(4)A(4)G. Recent experimental investigations, either in crystals or in solution, have shown that monovalent cations bind to DNA in a sequence-specific mode, preferentially in the narrow minor groove regions of uninterrupted sequences of four or more adenines (A-tracts), replacing a water molecule of the ordered hydration structure, the hydration spine. Following this evidence, it has been hypothesized that in A-tracts these events may be responsible for structural peculiarities such as a narrow minor groove and a curvature of the helix axis. The present simulations confirm a sequence specificity of the binding of sodium ions: Na(+) intrusions in the first layer of hydration of the minor groove, with long residence times, up to approximately 3 ns, are observed only in the minor groove of A-tracts but not in the alternating sequence. The effects of these intrusions on the structure of DNA depend on the ion coordination: when the ion replaces a water molecule of the spine, the minor groove becomes narrower. Ion intrusions may also disrupt the hydration spine modifying the oligomer structure to a large extent. However, in no case intrusions were observed to locally bend the axis toward the minor groove. The simulations also show that ions may reside for long time periods in the second layer of hydration, particularly in the wider regions of the groove, often leading to an opening of the groove.     

Molecular structure of the netropsin-d(CGCGATATCGCG) complex: DNA conformation in an alternating AT segment

The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step.     

Binding of a Hoechst dye to d(CGCGATATCGCG) and its influence on the conformation of the DNA fragment

Hoechst dye 33258 is a planar drug molecule that binds to the minor groove of DNA, especially where there are a number of A.T base pairs. We have solved the structure of the Hoechst dye bound to the DNA dodecamer d(CGCGATATCGCG) at 2.3 A. This structure is compared to that of the same dodecamer with the minor-groove-binding drug netropsin bound to it, as well as to structures that have been solved for this Hoechst dye bound to a DNA dodecamer containing the central four base pairs with the sequence AATT. We find that the position of the Hoechst drug in this dodecamer is quite different from that found in the other dodecamer since it has an opposite orientation compared to the other two structures. The drug covers three of the four A.T base pairs and extends its piperazine ring to the first G.C base pair adjacent to the alternating AT segment. Furthermore, the drug binding has modified the structure of the DNA dodecamer. Other DNA dodecamers with alternating AT sequences show an alternation in the size of the helical twist between the ApT step (small twist) and the TpA step (large twist). In this structure the alternation is reversed with larger twists in the ApT steps than in the TpA step. In addition, there is a rotation of one of the thymine bases in the DNA dodecamer that is associated with hydrogen bonding to the Hoechst drug. This structure illustrates the considerable plasticity found in the DNA molecule when it binds to different planar molecules inserted into the minor groove.     

Interactions between a symmetrical minor groove binding compound and DNA oligonucleotides: 1H and 19F NMR studies

High-resolution NMR techniques (proton and 19F) have been used to study the interactions between several DNA oligonucleotides with varying length of AT base pairs and the synthetic pyrrole-containing compound (P1-F4S-P1), which has properties similar to the DNA minor groove binding drug distamycin A. When this two-fold symmetrical DNA binding molecule is added to the self-complementary DNA oligomers, the resulting complex exhibits an NMR spectrum without any doubling of individual resonances, consistent with a two-fold symmetry of the complex. This is in contrast to all other complexes studied so far. The minimum length of an AT stretch for specific ligand binding is judged to be greater than 4 base pairs. Inter-molecular proton nuclear Overhauser effects between the ligand molecule and a DNA dodecamer d(CGCAAATTTGCG) provide evidence that P1-F4S-P1 binds DNA in the minor groove and interacts with the middle AT base pairs. The presence of a specific interaction between P1-F4S-P1 and DNA is conclusively demonstrated by 19F NMR studies, in which four previously chemically equivalent fluorine nuclei in the free molecule become two non-equivalent pairs (yielding an AB quartet pattern) upon the binding of P1-F4S-P1 to DNA duplex. A sequence-dependent binding behavior of P1-F4S-P1 is evident by comparing the 19F NMR spectra of the complexes between P1-F4S-P1 and two different but related DNA dodecamers, d(CGCAAATTTGCG) and d(CGCTTTAAAGCG). P1-F4S-P1 binds more strongly to the former dodecamer with an association constant of approximately 1 X 10(3) M-1.     

Theoretical studies of the selective binding to DNA of two non-intercalating ligands: netropsin and SN 18071.

A theoretical study of the binding to DNA of netropsin and a bisquaternary ammonium heterocycle, SN 18071, is undertaken with an energy minimizing program based on empirical potential functions. The positioning of the ligand is achieved by force and torque calculations and its internal flexibility is taken into account. The binding preference of both drugs studied for the AT minor groove of B-DNA is shown to depend on both the electrostatic potential generated by the base sequence and the quality of the steric fit of the ligand in the groove. Ligand-DNA hydrogen bonds are shown to aid binding, but not to be essential in establishing binding preferences.     

Cationic porphyrins as probes of DNA structure.

The DNA binding specificity of a group of cationic manganese porphyrin complexes has been examined using DNase I footprinting methodology and by observing the sites of porphyrin-induced DNA strand scission in the presence of potassium superoxide. The compounds, which possess systematic changes in total charge, its distribution on the periphery on the macrocycle and ligand shape, bind in the minor groove of AT rich regions of DNA. While changes in total charge and charge arrangement do not significantly influence specificity, a shape change which blocks close ligand contact with the minor groove relaxes the original AT specificity causing the compound to cleave at both AT and GC sites. The observed changes in binding sequence specificity were interpreted in terms of electrostatic and steric factors associated with both the compounds and DNA.     

Groove width and depth of B-DNA structures depend on local variation in slide.

The groove widths of DNA helix, especially minor groove width, are generally believed to be important for recognition of DNA by various types of ligands. It has been postulated earlier that large negative propeller twist, in the AT rich regions compresses the minor groove of duplex DNA. A systematic study has now been carried out by generating models with different values of local doublet and intra-basepair parameters and calculating their minor groove widths. It is found that several local doublet parameters affect the minor groove width but it depends most strongly on the local step parameters roll and slide when each parameter is considered individually. However, a detailed analysis of the various local parameters within the B-DNA family of crystal structures indicates that propeller twist and slide are most strongly correlated with the observed values of minor groove width. The groove depth is also strongly correlated with slide. Thus the local base sequence dependent variations in slide can modify both the groove width and depth and consequently determine the ligand binding properties of DNA.     

Groove Width and Depth of B-DNA Structures Depend on Local Variation in Slide

Abstract

The groove widths of DNA helix, especially minor groove width, are generally believed to be important for recognition of DNA by various types of ligands. It has been postulated earlier that large negative propeller twist, in the AT rich regions compresses the minor groove of duplex DNA A systematic study has now been carried out by generating models with different values of local doublet and intra-basepair parameters and calculating their minor groove widths. It is found that several local doublet parameters affect the minor groove width but it depends most strongly on the local step parameters roll and slide when each parameter is considered individually. However, a detailed analysis of the various local parameters within the B-DNA family of crystal structures indicates that propeller twist and slide are most strongly correlated with the observed values of minor groove width. The groove depth is also strongly correlated with slide. Thus the local base sequence dependent variations in slide can modify both the groove width and depth and consequently determine the ligand binding properties of DNA.     

DNA binding specificity of a series of cationic metalloporphyrin complexes

The sequence specificities of a series of cationic metalloporphyrins toward a 139 base pair restriction fragment of pBR-322 DNA have been studied by DNase I footprinting methodology. Analysis using controlled digests and quantitative autoradiography/microdensitometry revealed that the 5- and 6-coordinate complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine, MT4MPyP, where M is Mn, Fe, Co, and Zn, were found to bind to AT regions of DNA. Footprinting analysis involving the radiolabel on the opposing strand of restriction fragment showed site skewing in the direction of the 3' end of the fragment, indicating that the porphyrins bind in the minor groove of DNA. The significant increase in DNase I catalyzed hydrolysis observed in various regions of the fragment appeared to be primarily due to a decrease in available substrate DNA upon porphyrin binding with possible contributions from structural changes in DNA caused by ligand binding. The complexes NiT4MPyP and CuT4MPyP were found to bind to both AT and GC regions of the fragment, producing different degrees of inhibition in the two regions. Since the outside-binding porphyrins can neither intercalate or effectively hydrogen bond to DNA, they appear to read sequence by responding to steric and/or electrostatic potential effects located in the minor groove of DNA.     

Structural change in a B-DNA helix with hydrostatic pressure

Study of the effects of pressure on macromolecular structure improves our understanding of the forces governing structure, provides details on the relevance of cavities and packing in structure, increases our understanding of hydration and provides a basis to understand the biology of high-pressure organisms. A study of DNA, in particular, helps us to understand how pressure can affect gene activity. Here we present the first high-resolution experimental study of B-DNA structure at high pressure, using NMR data acquired at pressures up to 200 MPa (2 kbar). The structure of DNA compresses very little, but is distorted so as to widen the minor groove, and to compress hydrogen bonds, with AT pairs compressing more than GC pairs. The minor groove changes are suggested to lead to a compression of the hydration water in the minor groove.     

Theoretical exploration of netropsin binding to tRNA(Phe)

Theoretical exploration of the possible interaction of netropsin with tRNAPhe indicates that binding should occur preferentially with the major groove of the T psi C stem of the macromolecule, specifically with the bases G51, U52, G53 and phosphates 52, 53, 61 and 62. This agrees with the recent crystallographic result of Rubin and Sundaralingam. It is demonstrated that the difference with respect to netropsin binding with B-DNA, where it occurs specifically in the minor groove of AT sequences, is due to the differences in the distribution of the electrostatic molecular potential generated by these different types of DNA: this potential is sequence dependent in B-DNA (located in the minor groove of AT sequences and the major groove of GC sequences), while it is sequence independent and always located in the major groove in A-RNA. The result demonstrates the major role of electrostatics in determining the location of the binding site.