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.     

DNA sequence preferences of several AT-selective minor groove binding ligands.

We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments containing different arrangements of AT base pairs by DNase I footprinting. For fragments which contain multiple blocks of (A/T)4 quantitative DNase I footprinting reveals that AATT and AAAA are much better binding sites than TTAA and TATA. Hoechst 33258 shows that greatest discrimination between these sites with a 50-fold difference in affinity between AATT and TATA. Alone amongst these ligands, Hoechst 33258 binds to AATT better than AAAA. These differences in binding to the various AT-tracts are interpreted in terms of variations in DNA minor groove width and suggest that TpA steps within an AT-tract decrease the affinity of these ligands. The behaviour of each site also depends on the flanking sequences; adjacent pyrimidine-purine steps cause a decrease in affinity. The precise ranking order for the various binding sites is not the same for each ligand.     

Designer DNA-binding drugs: the crystal structure of a meta-hydroxy analogue of Hoechst 33258 bound to d(CGCGAATTCGCG)2.

An analogue of the DNA binding compound Hoechst 33258, which has the para hydroxyl group altered to be at the meta position, together with the replacement of one benzimidazole group by pyridylimidazole, has been cocrystallized with the dodecanucleotide sequence d(CGCGAATTCGCG)2. The X-ray structure has been determined at 2.2 A resolution and refined to an R factor of 20.1%. The ligand binds in the minor groove at the sequence 5'-AATTC with the bulky piperazine group extending over the CxG base pair. This binding is stabilised by hydrogen bonding and numerous close van der Waals contacts to the surface of the groove walls. The meta-hydroxyl group was found in two distinct orientations, neither of which participates in direct hydrogen bonds to the exocyclic amino group of a guanine base. The conformation of the drug differs from that found previously in other X-ray structures of Hoechst 33258-DNA complexes. There is significant variation between the minor groove widths in the complexes of Hoechst 33258 and the meta-hydroxyl derivative as a result of these conformational differences. Reasons are discussed for the inability of this derivative to actively recognise guanine.     

Structure of a bis-amidinium derivative of hoechst 33258 complexed to dodecanucleotide d(CGCGAATTCGCG)2: the role of hydrogen bonding in minor groove drug-DNA recognition.

The crystal structure is reported of a complex between the dodecanucleotide sequence d(CGCGAATTCGCG)2and an analogue of the DNA binding drug Hoechst 33258, in which the piperazine ring has been replaced by an amidinium group and the phenol ring by a phenylamidinium group. The structure has been refined to an R factor of 19.5% at 2.2 A resolution. The drug is held in the minor groove by five strong hydrogen bonds, together with bridging water molecules at both ends. There are few other contacts with the floor of the groove, indicating a lack of isohelicity with the groove and suggesting (i) that the observed high DNA affinity of this drug is primarily due to the array of hydrogen bonds and (ii) that these more than compensate for its poor isohelicity.     

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.     

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.     

Low-temperature crystallographic analyses of the binding of Hoechst 33258 to the double-helical DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G.

The crystal structure of the complex of Hoechst 33258 and the DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G has been solved from X-ray data collected at three different low temperatures (0, -25, and -100 degrees C). Such temperatures have permitted collection of higher resolution data (2.0, 1.9, and 2.0 A, respectively) than with previous X-ray studies of the same complex. In all three cases, the drug is located in the narrow central A-A-T-T region of the minor groove. Data analyses at -25 and -100 degrees C (each with a 1:1 drug/DNA ratio in the crystallizing solution) suggest a unique orientation for the drug. In contrast, two orientations of the drug were found equally possible at 0 degrees C with a 2:1 drug/DNA ratio in solution. Dihedral angles between the rings of Hoechst 33258 appear to change in a temperature-dependent manner. The drug/DNA complex is stabilized by single or bifurcated hydrogen bonds between the two N-H hydrogen-bond donors in the benzimidazole rings of Hoechst and adenine N3 and thymine O2 acceptors in the minor groove. A general preference for AT regions is conferred by electrostatic potential and by narrowing of the walls of the groove. Local point-by-point AT specificity follows from close van der Waals contacts between ring hydrogen atoms in Hoechst 33258 and the C2 hydrogens of adenines. Replacement of one benzimidazole ring by purine in a longer chain analogue of Hoechst 33258 could make that particular site GC tolerant in the manner observed at imidazole substitution for pyrrole in lexitropsins.     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

Synthesis and DNA binding studies of a new asymmetric cyanine dye binding in the minor groove of [poly(dA-dT)]2

A new asymmetric cyanine dye has been synthesised and its interaction with different DNA has been investigated. In this dye, BEBO, the structure of the known intercalating cyanine dye BO has been extended with a benzothiazole substituent. The resulting crescent-shape of the molecule is similar to that of the well-known minor groove binder Hoechst 33258. Indeed, comparative studies of BO illustrate a considerable change in binding mode induced by this structural modification. Linear and circular dichroism studies indicate that BEBO binds in the minor groove to [poly (dA-dT)](2), but that the binding to calf thymus DNA is heterogeneous, although still with a significant contribution of minor groove binding. Similar to other DNA binding asymmetric cyanine dyes, BEBO has a large increase in fluorescence intensity upon binding and a relatively large quantum yield when bound. The minor groove binding of BEBO to [poly (dA-dT)](2) affords roughly a 180-fold increase in intensity, which is larger than to that of the commonly used minor groove binding probes DAPI and Hoechst 33258.     

The different binding modes of Hoechst 33258 to DNA studied by electric linear dichroism.

The binding mode of the bisbenzimidazole derivative Hoechst 33258 to a series of DNAs and polynucleotides has been investigated by electric linear dichroism. Positive reduced dichroisms were measured for the poly(dA-dT).poly(dA-dT)- and poly(dA).poly(dT)-Hoechst complexes in agreement with a deep penetration of the drug into the minor groove. Similarly, the drug displays positive reduced dichroism in the presence of the DNAs from calf thymus, Clostridium perfringens and Coliphage T4. Conversely, negative reduced dichroisms were obtained when Hoechst 33258 was bound to poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and poly(dG).poly(dC) as well as with the GC-rich DNA from Micrococcus lysodeikticus indicating that in this case minor groove binding cannot occur. Substitution of guanosines for inosines induces a reversal of the reduced dichroism from negative to positive. Therefore, as anticipated it is the 2-amino group of guanines protruding in this groove which prevents Hoechst 33258 from getting access to the minor groove of GC sequences. The ELD data obtained with the GC-rich biopolymers are consistent with an intercalative binding. Competition experiments performed with the intercalating drug proflavine lend credence to the involvement of an intercalative binding rather than to an external or major groove binding of Hoechst 33258 at GC sequences.     

Synthesis of a fluorescent microgonotropen (FMGT-1) and its interactions with the dodecamer d(CCGGAATTCCGG)

A new type of microgonotropen that fluoresces upon binding to dsDNA has been synthesized. FMGT-1, an analogue of the minor groove binder Hoechst 33258, is functionalized with a polyamine chain capable of interacting with the phosphate backbone of DNA. Binding studies indicate that FMGT-1 binds more tightly to dsDNA than the parent compound Hoechst 33258.     

Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex

The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT > TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the dissociation-rate parameters vary from 0.42 s(-1) to 96 s(-1). Thus the association constants are kinetically controlled and are close to their equilibrium-determined values. However, when obtained with increased signal-to-noise ratio, the kinetic traces for Hoechst 33258 binding at the AATT site reveal two components. The concentration dependencies of the two time constants and amplitudes are consistent with two different kinetically equivalent two-step models. In the first model, fast bimolecular binding is followed by an isomerization of the initial complex. In the second model, two single-step associations form two complexes that mutually exclude each other. For both models the four reaction-rate parameters are calculated. Finally, specific dissociation kinetics, using poly[d(A-5BrU)], show that the kinetics are even more complex than either two-step model. We correlate our results with the different binding orientations and locations of Hoechst 33258 in the DNA minor groove found in several structural studies in the literature.     

Some Properties of New DNA-Specific Bisbenzimidazole Fluorochromes without a Piperazine Ring

Three new bisbenzimidazole (BBI) compounds, which differ from Hoechst 33258 mainly by substitution of a N-dimethylaminopro-pylcarboxamide group in place of the N-methyl-piperazine ring, were studied for their DNA- and AT-base pair specificity as well as for their ability to be quenched by incorporated 5-bromodeoxy-uridine (BrdU). Each of them had DNA binding specificity comparable to or greater than that of Hoechst 33258 and each had a greater specificity for AT-rich regions than did Hoechst 33258. The dependence of fluorescence of new dyes on the BrdU-incorporation into DNA is different from that of Hoechst 33258 and related compounds with piperazine ring. The quenching effect is much weaker, and two of the new compounds (BBI-1 and BBI-2) even show somewhat enhanced binding (fluorescence) at lower concentrations. Certain BBI dyes without piperazine ring may have some advantage over Hoechst for accurate DNA [AT-specific] measurements. The piperazine ring appears to play an important role in the yet unknown mechanism of Hoechst quenching by incorporated BrdU.     

Effects of Polyamines on the Binding of Hoechst 33258 to Calf Thymus DNA

Abstract

The ability of polyamines to displace the minor groove-binding dye Hoechst 33258 from calf thymus DNA was investigated. Polyamines displace non-specific DNA phosphate bound Hoechst in a charge-dependent fashion, but show very little ability to displace the high affinity binding of Hoechst in the minor groove of DNA. This high affinity binding is, however, sensitive to ethidium bromide and the minor groove binding drug berenil. These studies suggest that polyamines probably bind DNA in the minor groove very weakly, if at all, relative to known minor groove binding agents.     

Sequence-dependent variation in DNA minor groove width dictates orientational preference of Hoechst 33258 in A-tract recognition: solution NMR structure of the 2:1 complex with d(CTTTTGCAAAAG)(2)

The solution structure of the dodecamer duplex d(CTTTTGCAAAAG)₂ and its 2:1 complex with the bis-benzimidazole Hoechst 33258 has been investigated by NMR and NOE-restrained molecular dynamics (rMD) simulations. Drug molecules are bound in each of the two A-tracts with the bulky N-methylpiperazine ring of each drug located close to the central TG (CA) step, binding essentially to the narrow minor groove of each A-tract. MD simulations over 1 ns, using an explicit solvation model, reveal time-averaged sequence-dependent narrowing of the minor groove from the 3′-end towards the 5′-end of each TTTT sequence. Distinct junctions at the TpG (CpA) steps, characterised by large positive roll, low helical and propeller twists and rapid AT base pair opening rates, add to the widening of the groove at these sites and appear to account for the bound orientation of the two drug molecules with the N-methylpiperazine ring binding in the wider part of the groove close to the junctions. Comparisons between the free DNA structure and the 2:1 complex (heavy atom RMSD 1.55 Å) reveal that these sequence-dependent features persist in both structures. NMR studies of the sequence d(GAAAAGCTTTTC)₂, in which the A-tracts have been inverted with the elimination of the TpG junctions, results in loss of orientational specificity of Hoechst 33258 and formation of multiple bound species in solution, consistent with the drug binding in a number of different orientations.     

Hydration changes in the association of Hoechst 33258 with DNA

The role of water in the interaction of Hoechst 33258 with the minor groove binding site of the (AATT)2 sequence was investigated using calorimetric and equilibrium constant measurements. Using isothermal titration calorimetry measurements, the heat capacity change for the reaction is -256 +/- 10 cal/(K mol of Hoechst). Comparison with the heat capacity changes based on area models supports the expulsion of water from the interface of the Hoechst-DNA complex. To further consider the role of water, the osmotic stress method was used to determine if the Hoechst association with DNA was coupled with hydration changes. Using four osmolytes with varying molecular weights and chemical properties, the Hoechst affinity for DNA decreases with increasing osmolyte concentration. From the dependence of the equilibrium constant on the solution osmolality, 60 +/- 13 waters are acquired in the complex relative to the reactants. It is proposed that the osmotic stress technique is measuring weakly bound waters that are not measured via the heat capacity changes.     

Interaction of Hoechst 33258 with repeating synthetic DNA polymers and natural DNA

Fluorescence, circular dichroism and sedimentation through cesium chloride gradient techniques were performed to study the physical properties of the binding of the bisbenzimidazole dye Hoechst 33258 (H33258) to natural DNAs and synthetic polynucleotides of defined repeating units. These studies show that Hoechst 33258 exhibits at least two modes of interaction with duplex DNA: (1) a strong base pair specific mode which requires at least 4 consecutive AT base pairs and (2) a weaker mode of binding which is significantly reduced in the presence of high salt (0.4 M NaCl) and exhibits no apparent base specificity. The H33258 binding was found to be sensitive to the substitutions in the minor groove elements of a series of synthetic polynucleotides supporting the model of H33258 binding in the minor groove of the DNA with AT rich sequences. Similar mode of binding was predicted in natural DNAs by methylation of dye-DNA complexes. Footprint analysis of the complex of dye to a pBR322 fragment also supports that a minimum of 4 consecutive AT base pairs are required for H33258 binding to DNA.     

Influence of surface shape on DNA binding of bimetallo helicates

In order to probe the DNA-helicate interactions responsible for the DNA binding and remarkable changes of the DNA secondary structure induced by a tetracationic bi-metallo helicate [Fe(2)(L(1))(3)](4+) (L(1)=C(25)H(20)N(4)), we have designed and synthesised derivatives with hydrophobic methyl groups at different positions on the ligand backbone. Two dimetallo helicates [Fe(2)(L(i))(3)](4+) were prepared using ligands L(3) and L(5) with the methyl substituent on, respectively, the 3 and 5 positions of the pyridyl ring thus producing a wider or slightly longer tetracationic DNA binder. UV/visible absorbance, circular and linear dichroism spectroscopies have been used to characterize the interactions of the cylinders with DNA with the aim of investigating any sequence preference or selectivity upon binding. Competitive binding studies using fluorescent dyes Hoechst 33258 (a minor groove binder), ethidium bromide (an intercalator) and a major groove binding cation (cobalt (III) hexammine) which induces the B-->Z transition have been employed to determine the binding geometries of the enantiomers of two methylated helicates (L(3) and L(5)) to DNA and compare with the data obtained previously for the unmethylated analogue (L(1)). The results demonstrate that the racemic mixtures and the resolved enantiomers of all helicates bind to DNA inducing structural changes. The overall conclusion from the effect of adding these groups to the surface of the parent helicate is that increasing the width (L(3)) reduces the DNA binding strength, the bending and coiling effect and the groove selectivity of the enantiomers compared with the parent compound. There is limited evidence to suggest a slight GC sequence preference. Lengthening the helicate (L(5)) results in DNA interactions similar to those of the parent compounds, with an increased preference of the P enantiomer for the minor groove indicating an enhancement of mode selectivity.     

Determination of the NMR solution structure of the Hoechst 33258-d(GTGGAATTCCAC)2 complex and comparison with the X-ray crystal structure

BACKGROUND: The chromosomal stain, Hoechst 33258, binds to the minor groove of the DNA double helix and specifically recognizes a run of four A-T base pairs. Extensive biochemical and biophysical studies have been aimed at understanding the binding of the dye to DNA at the atomic level. Among these studies there have been several crystal structure determinations and some preliminary structural studies by NMR. RESULTS: On the basis of our own previously reported NMR data, we have now determined the three-dimensional solution structure of the 1:1 complex between Hoechst 33258 and the self-complementary DNA duplex d(GTGGAATTCCAC)2. Two coexisting families of con formers, which exhibit differences in their intermolecular hydrogen bonding pattern, were found and the two terminal rings of the dye displayed greater internal mobility than the rest of the molecule. CONCLUSIONS: The observed multiple ligand-binding modes in the complex between Hoechst 33258 and DNA and differential internal mobility along the bound ligand provide a novel, dynamic picture of the specific inter actions between ligands that bind in the minor groove and DNA. The dynamic state revealed by these studies may account for some of the significant differences previously observed between different crystal structures of Hoechst 33258 complexed with a different DNA duplex, d(CGCGAATTCGCG)2.