Comparative thermodynamics for monomer and dimer sequence-dependent binding of a heterocyclic dication in the DNA minor groove

Phenylamidine cationic groups linked by a furan ring (furamidine) and related symmetric diamidine compounds bind as monomers in the minor groove of AT sequences of DNA. DB293, an unsymmetric derivative with one of the phenyl rings of furamidine replaced with a benzimidazole, can bind to AT sequences as a monomer but binds more strongly to GC-containing minor-groove DNA sites as a stacked dimer. The dimer-binding mode has high affinity, is highly cooperative and sequence selective. In order to develop a better understanding of the correlation between structural and thermodynamic aspects of DNA molecular recognition, DB293 was used as a model to compare the binding of minor-groove agents with AT and mixed sequence DNA sites. Isothermal titration calorimetry and surface plasmon resonance results clearly show that the binding of DB293 and other related compounds into the minor groove of AT sequences is largely entropy-driven while the binding of DB293 as a dimer into the minor groove of GC-containing sequences is largely enthalpy-driven. At 25 degrees C, for example, the AT binding has DeltaG degrees, DeltaH degrees and TDeltaS degrees values of -9.6, -3.6 and 6.0 kcal/mol while the values for dimer binding to a GC-containing site are -9.0, -10.9 and -1.9 kcal/mol (per mol of bound compound), respectively. These results show that the thermodynamic components for binding of compounds of this type to DNA are very dependent on the structure, solvation and sequence of the DNA binding site.     

Recognition of ATGA sequences by the unfused aromatic dication DB293 forming stacked dimers in the DNA minor groove

Furamidine and related diamidines represent a promising series of drugs active against widespread parasites, in particular the Pneumocystic carinii pathogen. In this series, the phenylfuranbenzimidazole diamidine derivative DB293 was recently identified as the first unfused aromatic dication capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Here we present a detailed biochemical and biophysical characterization of the DNA sequence recognition properties of DB293. Three complementary footprinting techniques using DNase I, Fe(II)-EDTA, and an anthraquinone photonuclease were employed to locate binding sites for DB293 in different DNA restriction fragments. Two categories of sites were identified by DNase I footprinting: (i) 4/5 bp sequences containing contiguous A.T pairs, such as 5'-AAAA and 5'-ATTA; and (ii) sequences including the motif 5'-ATGA.5'-TCAT. In particular, a 13-bp sequence including two contiguous ATGA motifs provided a highly preferential recognition site for DB293. Quantitative footprinting analysis revealed better occupancy of the 5'-ATGA site compared to the AT-rich sites. Preferential binding of DB293 to ATGA sites was also observed with other DNA fragments and was confirmed independently by means of hydroxyl radical footprinting generated by the Fe(II)-EDTA system, as well as by a photofootprinting approach using the probe anthraquinone-2-sulfonate (AQS). In addition, this photosensitive reagent revealed the presence of sites of enhanced cutting specific to DB293. This molecule, but not other minor groove binders such as netropsin, induces specific local structural changes in DNA near certain binding sites, as independently shown by DNase I and the AQS probe. Recognition of the ATGA sequence by DB293 was investigated further using melting temperature experiments and surface plasmon resonance (SPR). The use of different hairpin oligonucleotides showed that DB293 can interact with AT sites via the formation of 1:1 drug-DNA complexes but binds much more strongly, and cooperatively, to ATGA-containing sequences to form 2:1 drug-DNA complexes. DB293 binds strongly to ATGA sequences with no significant context dependence but is highly sensitive to the orientation of the target sequence. The formation of 2:1 DB293/DNA complexes is abolished by reversing the sequence 5'-ATGA-->3'-ATGA, indicating that directionality plays an important role in the drug-DNA recognition process. Similarly, a single mutation in the A[T-->G]GA sequence is very detrimental to the dimer interactions of DB293. From the complementary footprinting and SPR data, the 5'-ATGA sequence is identified as being a highly favored dimer binding site for DB293. The data provide clues for delineating a recognition code for diamidine-type minor groove binding agents, and ultimately to guide the rational design of gene regulatory molecules targeted to specific sites of the genetic material.     

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.     

Out-of-shape DNA minor groove binders: induced fit interactions of heterocyclic dications with the DNA minor groove

DB921 and DB911 are benzimidazole-biphenyl isomers with terminal charged amidines. DB911 has a central meta-substituted phenyl that gives it a shape similar to those of known minor groove binding compounds. DB921 has a central para-substituted phenyl with a linear conformation that lacks the appropriate radius of curvature to match the groove shape. It is thus expected that DB911, but not DB921, should be an effective minor groove binder, but we find that DB921 not only binds in the groove but also has an unusually high binding constant in SPR experiments (2.9 x 10(8) M(-)(1), vs 2.1 x 10(7) M(-)(1) for DB911). ITC thermodynamic analysis with an AATT sequence shows that the stronger binding of DB921 is due to a more favorable binding enthalpy relative to that of DB911. CD results support minor groove binding for both compounds but do not provide an explanation for the binding of DB921. X-ray crystallographic analysis of DB921 bound to AATT shows that an induced fit structural change in DB921 reduces the twist of the biphenyl to complement the groove, and places the functional groups in position to interact with bases at the floor of the groove. The phenylamidine of DB921 forms indirect contacts with the bases through a bound water. The DB921-water pair forms a curved binding module that matches the shape of the minor groove and provides a number of strong interactions that are not possible with DB911. This result suggests that traditional views of compound curvature required for minor groove complex formation should be reevaluated.     

Influence of compound structure on affinity, sequence selectivity, and mode of binding to DNA for unfused aromatic dications related to furamidine

In the course of a program aimed at developing sequence-specific gene-regulatory small organic molecules, we have investigated the DNA interactions of a new series of nine diphenylfuran dications related to the antiparasitic drug furamidine (DB75). Two types of structural modifications were tested: the terminal amidine groups of DB75 were shifted from the para to the meta position, and the amidines were replaced with imidazoline or dimethyl-imidazoline groups, to test the importance of both the position and nature of positively charged groups on DNA interactions. The interactions of these compounds with DNA and oligonucleotides were studied by a combination of biochemical and biophysical techniques. Absorption and CD measurements suggested that the drugs bind differently to AT and GC sequences in DNA. The para-para dications, like DB75, bind into the minor groove of poly(dAT)(2) and intercalate between the base pairs of poly(dGC)(2), as revealed by electric linear dichroism experiments. In contrast, the meta-meta compounds exhibit a high tendency to intercalate into DNA whatever the target sequence. The lack of sequence selectivity of the meta-meta compounds containing amidines or dimethyl-imidazoline groups was also evident from DNase I footprinting and surface plasmon resonance (SPR) experiments. Accurate binding measurements using the BIAcore SPR method revealed that all nine compounds bind with similar affinity to an immobilized GC sequence DNA hairpin but exhibit very distinct affinities for the corresponding AT hairpin oligonucleotide. The minor groove-binding para-para compounds have a high specificity for AT sequences. The biophysical data clearly indicate that shifting the cationic substituents from the para to the meta position results in a loss of specificity and change in binding mode. The strong AT selectivity of the para-para compounds was independently confirmed by DNase I footprinting experiments performed with a range of DNA restrictions fragments. In terms of AT selectivity, the compounds rank in the order para-para > para-meta > meta-meta. The para dications bind preferentially to sequences containing four contiguous AT base pairs. Additional footprinting experiments with substrates containing the 16 possible [A.T](4) blocks indicated that the presence of a TpA step within an [A.T] (4) block generally reduces the extent of binding. The diverse methods, from footprinting to SPR to dichroism, provide a consistent model for the interactions of the diphenylfuran dications with DNA of different sequences. Altogether, the results attest unequivocally that the binding mode for unfused aromatic cations can change completely depending on substituent position and DNA sequence. These data provide a rationale to explain the relationships between sequence selectivity and mode of binding to DNA for unfused aromatic dications related to furamidine.     

Minor groove to major groove, an unusual DNA sequence-dependent change in bend directionality by a distamycin dimer

DNA sequence-dependent conformational changes induced by the minor groove binder, distamycin, have been evaluated by polyacrylamide gel electrophoresis. The distamycin binding affinity, cooperativity, and stoichiometry with three target DNA sequences that have different sizes of alternating AT sites, ATAT, ATATA, and ATATAT, have been determined by mass spectrometry and surface plasmon resonance to help explain the conformational changes. The results show that distamycin binds strongly to and bends five or six AT base pair minor groove sites as a dimer with positive cooperativity, while it binds to ATAT as a weak, slightly anticooperative dimer. The bending direction was evaluated with an in phase A-tract reference sequence. Unlike other similar monomer minor groove binding compounds, such as netropsin, the distamycin dimer changes the directionality of the overall curvature away from the minor groove to the major groove. This distinct structural effect may allow designed distamycin derivatives to have selective therapeutic effects.     

Evaluation of the influence of compound structure on stacked-dimer formation in the DNA minor groove

The Human Genome Project as well as sequencing of the genomes of other organisms offers a wealth of DNA targets for both therapeutic and diagnostic applications, and it is important to develop additional DNA binding motifs to fully exploit the potential of this new information. We have recently found that an aromatic dication, DB293, with an amidine-phenyl-furan-benzimidazole-amidine structure can recognize specific sequences of DNA by binding in the minor groove as a dimer [Wang, L., Bailly, C., Kumar, A., Ding, D., Bajic, M., Boykin, D. W., and Wilson, W. D. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 12-16]. The dimer binding is strong, highly cooperative and, in contrast to many closely related heterocyclic dications, has both GC and AT base pairs in the minor groove binding site. The aromatic heterocycle stacked dimer is quite different in structure from the polyamide-lexitropsin type compounds, and it is a dication while all lexitropsin dimers are monocations. The heterocyclic dimer represents only the second small molecule class that can recognize mixed sequences of DNA. To test the structural limits on the new type of complex, it is important to probe the influence of compound charge, chemical groups, and structural features. The effects of these compound molecular variations on DNA complex formation with several DNA sequences were evaluated by DNase I footprinting, CD and UV spectroscopy, thermal melting, and quantitative analysis with surface plasmon resonance biosensor methods. Conversion of the amidines to guanidinium groups does permit the cooperative dimer to form but removal of one amidine or addition of an alkyl group to the amidine strongly inhibited dimer formation. Changing the phenyl of DB293 to a benzimidazole or the benzimidazole to a phenyl or benzofuran also inhibited dimer formation. The results show that formation of the minor groove stacked-dimer complex is very sensitive to compound structure. The discovery of the aromatic dimer mode offers new opportunities to enhance the specificity and expand the range of applications of the compounds that target DNA.     

Cooperative dimerization of a heterocyclic diamidine determines sequence-specific DNA recognition

In the course of a program aimed at discovering novel DNA-targeted antiparasitic drugs, the phenylfuran-benzimidazole unfused aromatic dication DB293 was identified as the first diamidine capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Its preferred binding sequence encompasses the tetranucleotide 5'-ATGA.5'-TCAT to which DB293 binds tightly with a strong positive cooperativity. Here we have investigated the influence of the DNA sequence on drug binding using two complementary technical approaches: surface plasmon resonance and DNase I footprinting. The central dinucleotide of the primary ATGA motif was systematically varied to represent all of the eight possible combinations (AXGA and ATYA, where X or Y = A, T, G, or C). Binding affinities for each site were precisely measured by SPR, and the extent of cooperative drug binding was also determined. The sequence recognition process was found to be extremely dependent on the nature of the central dinucleotide pair. Modification of the central TG step decreases binding affinity by a factor varying from 2 to over 500 depending on the base substitution. However, the diminished binding affinity does not affect the unique binding mode. In nearly all cases, the SPR titrations revealed a positive cooperativity in complex formation which reflects the ease of the dication to form stacked dimeric motifs in the DNA minor groove. DNase I footprinting served to identify additional binding sites for DB293 in the context of long DNA sequences offering a large variety of randomly distributed or specifically designed sites. The ATGA motif provided the best receptor for the drug, but lower affinity sequences were also identified. The design of two DNA fragments composed of various targeted tetranucleotide binding sites separated by an "insulator" (nonbinding) sequence allowed us to delineate further the influence of DNA sequence on drug binding and to identify a novel high-affinity site: 5'-ACAA.5'-TTGT. Collectively, the SPR and footprinting results show that the consensus sequence 5'-(A/T)-TG-(A/T) represents the optimal site for cooperative dimerization of the heterocyclic diamidine DB293.     

Probing the conformations of eight cloned DNA dodecamers; CGCGAATTCGCG, CGCGTTAACGCG, CGCGTATACGCG, CGCGATATCGCG, CGCAAATTTGCG, CGCTTTAAAGCG, CGCGGATCCGCG and CGCGGTACCGCG.

The self complementary DNA dodecamers d(CGCGAATTCGCG), d(CGCGTTAACGCG), d(CGCGTATACGCG), d(CGCGATATCGCG), d(CGCAAATTTGCG), d(CGCTTTAAAGCG), d(CGCGGATCCGCG) and d(CGCGGTACCGCG) have been cloned into the Smal site of plasmid pUC19. Radiolabelled polylinker fragments containing these inserts have been digested with nucleases and chemical agents, probing the structure of the central AT base pairs. The sequences AATT and AAATTT are relatively resistant to digestion by DNase I, micrococcal nuclease and hydroxyl radicals, consistent with the suggestion that they possess a narrow minor groove. Nuclease digestion of TTAA is much more even, and comparable to that at mixed sequence DNA. TpA steps in ATAT, TATA and GTAC are cut less well by DNAse I than in TTAA. DNasel cleavage of surrounding bases, especially CpG is strongly influenced by the nature of the central sequence.     

Molecular determinants for DNA minor groove recognition: design of a bis-guanidinium derivative of ethidium that is highly selective for AT-rich DNA sequences

The phenanthridinium dye ethidium bromide is a prototypical DNA intercalating agent. For decades, this anti-trypanosomal agent has been known to intercalate into nucleic acids, with little preference for particular sequences. Only polydA-polydT tracts are relatively refractory to ethidium intercalation. In an effort to tune the sequence selectivity of known DNA binding agents, we report here the synthesis and detailed characterization of the mode of binding to DNA of a novel ethidium derivative possessing two guanidinium groups at positions 3 and 8. This compound, DB950, binds to DNA much more tightly than ethidium and exhibits distinct DNA-dependent absorption and fluorescence properties. The study of the mode of binding to DNA by means of circular and electric linear dichroism revealed that, unlike ethidium, DB950 forms minor groove complexes with AT sequences. Accurate quantification of binding affinities by surface plasmon resonance using A(n)T(n) hairpin oligomer indicated that the interaction of DB950 is over 10-50 times stronger than that of ethidium and comparable to that of the known minor groove binder furamidine. DB950 interacts weakly with GC sites by intercalation. DNase I footprinting experiments performed with different DNA fragments established that DB950 presents a pronounced selectivity for AT-rich sites, identical with that of furamidine. The replacement of the amino groups of ethidium with guanidinium groups has resulted in a marked gain of both affinity and sequence selectivity. DB950 provides protection against DNase I cleavage at AT-containing sites which frequently correspond to regions of enhanced cleavage in the presence of ethidium. Although DB950 maintains a planar phenanthridinium chromophore, the compound no longer intercalates at AT sites. The guanidinium groups of DB950, just like the amidinium group of furamidine (DB75), are the critical determinants for recognition of AT binding sites in DNA. The chemical modulation of the ethidium exocyclic amines is a profitable option to tune the nucleic acid recognition properties of phenylphenanthridinium dyes.     

Structural comparison of the B-DNA dodecamers d(CGCGTTAACGCG) and d(CGCGAATTCGCG) with T2A2 and A2T2 tracts.

The x-ray structure of the deoxy oligonucleotide dodecamer d(CGCGTTAACGCG) recently determined in our laboratory shows that the helical parameters of the central TTAA segment are significantly different compared to the central AATT in d(CGCGAATTCGCG). The roll in the central TA step of the T2A2 dodecamer opens towards the minor groove while the AT step of the A2T2 dodecamer opens towards the major groove. Also, the roll angles at the steps 4 and 8 (GT and AC in T2A2) and (GA and TC in A2T2) are in opposite directions. The high cup and helical twist angles at the central base-pair of T2A2 decreases the base stacking interactions compared to A2T2. Tilt angles within the tetranucleotide segments TTAA and AATT have opposite signs. In spite of the local differences caused by the sequence inversion (TTAA----AATT), the two dodecamers exhibit similar overall bending. The top third is more bent than the bottom third relative to the central segment. This asymmetric bending in the two dodecamers is mainly due to crystal packing interactions.     

Unusually strong binding to the DNA minor groove by a highly twisted benzimidazole diphenylether: induced fit and bound water

RT29 is a dicationic diamidine derivative that does not obey the classical "rules" for shape and functional group placement that are expected to result in strong binding and specific recognition of the DNA minor groove. The compound contains a benzimidazole diphenyl ether core that is flanked by the amidine cations. The diphenyl ether is highly twisted and gives the entire compound too much curvature to fit well to the shape of the minor groove. DNase I footprinting, fluorescence intercalator displacement studies, and circular dichroism spectra, however, indicate that the compound is an AT specific minor groove binding agent. Even more surprisingly, quantitative biosensor-surface plasmon resonance and isothermal titration calorimetric results indicate that the compound binds with exceptional strength to certain AT sequences in DNA with a large negative enthalpy of binding. Crystallographic results for the DNA complex of RT29 compared to calculated results for the free compound show that the compound undergoes significant conformational changes to enhance its minor groove interactions. In addition, a water molecule is incorporated directly into the complex to complete the compound-DNA interface, and it forms an essential link between the compound and base pair edges at the floor of the minor groove. The calculated DeltaCp value for complex formation is substantially less than the experimentally observed value, which supports the idea of water being an intrinsic part of the complex with a major contribution to the DeltaCp value. Both the induced fit conformational changes of the compound and the bound water are essential for strong binding to DNA by RT29.     

DNA binding of the nonintercalative ligands SN-6132, SN-6131 and SN-6113: minor variations of the ligand structure may cause changes in the base pair preference.

The DNA binding selectivity of three ligands of a series of antitumor agents of bisquaternary ammonium heterocycles has been investigated by means of CD spectroscopy and melting measurements. From the spectroscopic results and binding data it is concluded that the agents SN-6132, SN-6131 and SN-6113 have relatively high affinity to AT base pair sequences whereas the binding to GC pairs is very low. The binding selectivity to AT base pair sequences decreases in the order netropsin > SN-6132 > SN-6113 > SN-6131. Poly(dA).poly(dT) has the highest binding preference for SN-6132 relative to that of SN-6131. The different binding behavior of the ligands is related to their distinct changes in the chemical structure and to the DNA minor groove properties which determines the adaptability of the ligands in the groove.     

Sequence and length dependent thermodynamic differences in heterocyclic diamidine interactions at AT base pairs in the DNA minor groove

With the goal of developing a better understanding of the antiparasitic biological action of DB75, we have evaluated its interaction with duplex alternating and nonalternating sequence AT polymers and oligomers. These DNAs provide an important pair of sequences in a detailed thermodynamic analysis of variations in interaction of DB75 with AT sites. The results for DB75 binding to the alternating and nonalternating AT sequences are quite different at the fundamental thermodynamic level. Although the Gibbs energies are similar, the enthalpies for DB75 binding with poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) are +3.1 and -4.5 kcal/mol, respectively, while the binding entropies are 41.7 and 15.2 cal/mol.K, respectively. The underlying thermodynamics of binding to AT sites in the minor groove plays a key role in the recognition process. It was also observed that DB75 binding with poly(dA).poly(dT) can induce T.A.T triplet formation and the compound binds strongly to the dT.dA.dT triplex.