An Unexpected Major Groove Binding of Netropsin and Distamycin A to tRNAphe

Abstract

Crystalline complexes of yeast tRNA^phe and the oligopeptide antibiotics netropsin and distamycin A were prepared by diffusing drugs into crystals of tRNA. X-ray structure analyses of these complexes reveal a single common binding site for both drugs which is located in the major or deep groove of the tRNA T-stem. The netropsin-tRNA complex is stabilized by specific hydrogen bonds between the amide groups of the drug and the tRNA bases G51 0(6), U52 0(4) and G53 N(7) on one strand, and is further stabilized by electrostatic interactions between the positively charges guanidino side chain of the drug and the tRNA phosphate P53 on the same strand and the positively charged amidino propyl side chain and the phosphates P61, P62 and P63 on the opposite strand of the double helix. These results are in contrast to the implicated minor groove binding of these drugs to non-guanine sequences in DNA. The binding to the GUG sequence in tRNA implies that major groove binding to certain DNA sequences is possible.     

Theoretical Design, Chemical Synthesis and Footprinting Analysis of a Novel Peptide Derivative of the Intercalator 7-H Pyridocarbazole Targeted Towards the Major Groove of DNA

Abstract In order to target the major groove of DNA, we have designed novel peptide derivatives of 7-H pyridocarbazole, which is the chromophoric ring of ditercalinium, a potent antitumor bisin- tercalator. We will present here the results obtained with a compound that has a D-Asn tethered to the pyridinium nitrogen of the ring by a protonated β-alanyl-ethyl chain. We have investigated two alternative means of intercalation of the chromophore: first, into the (pur-pur) sequences, d(CpG)(2) and d(CpA)·d(TpG); second, into the (pur-pyr) sequences, d(GpC)(2)and d(GpT)·d(ApC). For the first intercalative mode, the best bound triplet sequences are d(ACG)·d(CGT) and d(ACA) d(TGT), namely with an adenine immediately upstream from the intercalation site. In these complexes, the chromophore has its concave side in the major groove, its long axis nearly colinear with the mean long axis of the two base pairs of the intercalation site, and a bidentate H-bonded configuration occurs which involves the C=0 and NH groups of the D-Asn side chain and HN(6) and N(7) (resp.) of the adenine base upstream. One alkylammonium proton is H-bonded to N(7) of the guanine of the intercalation site, on the strand opposite to the one bearing the adenine. In the second intercalative mode, the chromophore's concave site now faces one DNA strand, and both alkylammonium protons are involved in H-bonds with N(7) and O(6) of the 3' guanine on the same strand. The peptide's complexes with sequences having A, G, or C upstream of this guanine were computed to be energetically competitive with those with the best (pyr-pur) triplets. This provides a rare example of energetically favourable drug intercalation in-between (pur-pyr) sequences as compared to the standard (pyr-pur) ones. The synthesis of this compound was performed, and a series of footprinting experiments undertaken on a total of approximately 300 nucleotides. These experiments were consistent with the inferences from the theoretical computations.     

DNA sequence recognition by bispyrazinonaphthalimides antitumor agents

Bifunctional DNA intercalating agents have long attracted considerable attention as anticancer agents. One of the lead compounds in this category is the dimeric antitumor drug elinafide, composed of two tricyclic naphthalimide chromophores separated by an aminoalkyl linker chain optimally designed to permit bisintercalation of the drug into DNA. In an effort to optimize the DNA recognition capacity, different series of elinafide analogues have been prepared by extending the surface of the planar drug chromophore which is important for DNA sequence recognition. We report here a detailed investigation of the DNA sequence preference of three tetracyclic monomeric or dimeric pyrazinonaphthalimide derivatives. Melting temperature measurements and surface plasmon resonance (SPR) studies indicate that the dimerization of the tetracyclic planar chromophore considerably augments the affinity of the drug for DNA, polynucleotides, or hairpin oligonucleotides and promotes selective interaction with G.C sites. The (CH(2))(2)NH(CH(2))(3)NH(CH(2))(2) connector stabilizes the drug-DNA complexes. The methylation of the two nitrogen atoms of this linker chain reduces the binding affinity and increases the dissociation rates of the drug-DNA complexes by a factor of 10. DNase I footprinting experiments were used to investigate the sequence selectivity of the drugs, demonstrating highly preferential binding to G.C-rich sequences. It also served to select a high-affinity site encompassing the sequence 5'-GACGGCCAG which was then introduced into a biotin-labeled hairpin oligonucleotide to accurately measure the binding parameters by SPR. The affinity constant of the unmethylated dimer for this sequence is 500 times higher than that of the monomer compound and approximately 10 times higher than that of the methylated dimer. The DNA groove accessibility was also probed with three related oligonucleotides carrying G --> c(7)G, G --> I, and C --> M substitutions. The level of drug binding to the two hairpin oligonucleotides containing 7-deazaguanine (c(7)G) or 5-methylcytosine (M) residues is unchanged or only slightly reduced compared to that of the unmodified target. In contrast, incorporation of inosine (I) residues considerably decreases the extent of drug binding or even abolishes the interaction as is the case with the monomer. The pyrazinonaphthalimide derivatives are thus much more sensitive to the deletion of the exocyclic guanine 2-amino group exposed in the minor groove of the duplex than to the modification of the major groove elements. The complementary SPR footprinting methodology combining site selection and quantitative DNA affinity analysis constitutes a reliable method for dissecting the DNA sequence selectivity profile of reversible DNA binding small molecules.     

Base only binding of spermine in the deep groove of the A-DNA octamer d(GTGTACAC)

The crystal structure of a complex of spermine with the DNA octamer d(GTGTACAC) has been determined at 2.0-A resolution. The alternating sequence adopts an A-DNA conformation with a novel purine-purine extra-Watson-Crick hydrogen bond involving the central guanine G3 (G11) and adenine A13 (A5) in the deep groove. The oligocation spermine binds in the floor of the deep groove by interacting with the bases and assumes an S-shape. Its dyad is coincident with that of the DNA, reminiscent of repressor binding to B-DNA. The terminal and central ammonium groups of the top half of spermine form hydrogen-bonding interactions to the 5'-bases, GTG, of one strand; then the spermine winds across the groove to interact with the corresponding set of bases on the other strand. The methylene groups of spermine form a hydrophobic cluster with the methyl groups of the thymines and the O6 atoms of the guanines of the TGT sequences on either side of the dyad. The observed mode of binding of spermine to A-DNA can serve as a model for deep groove binding in RNA and DNA-RNA hybrids that show a propensity also for the A-conformation. It will be of interest to see if base binding of spermine to DNA is involved in the regulation of gene expression, since spermine and other oligocations are ubiquitous in cells and their concentration is coupled to stages in cell cycle.     

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.     

DNA sequence recognition by the antitumor drug ditercalinium

The antitumor drug ditercalinium is a rare example of a noncovalent DNA-binding ligand that forms bisintercalation complexes via the major groove of the double helix. Previous structural studies have revealed that the two connected pyridocarbazolium chromophores intercalate into DNA with the positively charged bis(ethylpiperidinium) linking chain oriented to the wide groove side of the helix. Although the interaction of ditercalinium with short oligonucleotides containing 4-6 contiguous GC base pairs has been examined in detail by biophysical and theoretical approaches, the sequence preference for ditercalinium binding to long DNA fragments that offer a wide variety of binding sites has been investigated only superficially. Here we have investigated both sequence preferences and possible molecular determinants of selectivity in the binding of ditercalinium to DNA, primarily using methods based upon DNase I footprinting. A range of multisite DNA substrates, including several natural restriction fragments and different PCR-generated fragments containing unconventional bases (2,6-diaminopurine, inosine, uridine, 5-fluoro- and 5-methylcytosine, 7-deazaguanine, 7-deazaadenine, and N(7)-cyanoboranoguanine), have been employed to show that ditercalinium selectively recognizes certain GC-rich sequences in DNA and to identify some of the factors which affect its DNA-binding sequence selectivity. Specifically, the footprinting data have revealed that the 2-amino group on the purines or the 5-methyl group on the pyrimidines is not essential for the formation of ditercalinium-DNA complexes whereas the major groove-oriented N(7) of guanine does appear as a key element in the molecular recognition process. The loss of N(7) at guanines but not adenines is sufficient to practically abolish sequence-selective binding of ditercalinium to DNA. Thus, as expected for a major groove binding drug, the N(7) of guanine is normally required for effective complex formation with GC base pairs, but interestingly the substitution of the N(7) with a relatively bulky cyanoborane group does not markedly affect the sequence recognition process. Therefore, the hydrogen bond accepting capability at N(7) of guanines is not sufficient to explain the GC-selective drug-DNA association, and the implications of these findings are considered.     

Theoretical Design,Chemical Synthesis and Footprinting Analysis of a Novel Peptide Derivative of the Intercalator 7-H Pyridocarbazole Targeted Towards the Major Groove of DNA

Abstract

In order to target the major groove of DNA, we have designed novel peptide derivatives of 7-H pyridocarbazole, which is the chromophoric ring of ditercalinium, a potent antitumor bisin- tercalator. We will present here the results obtained with a compound that has a D-Asn tethered to the pyridinium nitrogen of the ring by a protonated β-alanyl-ethyl chain. We have investigated two alternative means of intercalation of the chromophore: first, into the (pur-pur) sequences, d(CpG)₂ and d(CpA)·d(TpG); second, into the (pur-pyr) sequences, d(GpC)₂and d(GpT)·d(ApC). For the first intercalative mode, the best bound triplet sequences are d(ACG)·d(CGT) and d(ACA) d(TGT), namely with an adenine immediately upstream from the intercalation site. In these complexes, the chromophore has its concave side in the major groove, its long axis nearly colinear with the mean long axis of the two base pairs of the intercalation site, and a bidentate H-bonded configuration occurs which involves the C=0 and NH groups of the D-Asn side chain and HN₆ and N₇ (resp.) of the adenine base upstream. One alkylammonium proton is H-bonded to N₇ of the guanine of the intercalation site, on the strand opposite to the one bearing the adenine. In the second intercalative mode, the chromophore's concave site now faces one DNA strand, and both alkylammonium protons are involved in H-bonds with N₇ and O₆ of the 3′ guanine on the same strand. The peptide's complexes with sequences having A, G, or C upstream of this guanine were computed to be energetically competitive with those with the best (pyr-pur) triplets. This provides a rare example of energetically favourable drug intercalation in-between (pur-pyr) sequences as compared to the standard (pyr-pur) ones. The synthesis of this compound was performed, and a series of footprinting experiments undertaken on a total of approximately 300 nucleotides. These experiments were consistent with the inferences from the theoretical computations.     

Variation in the inhibition of restriction enzyme cleavage of lambda phage DNA produced by two covalent binding antitumor agents: Anthramycin and mitomycin C

DNA-drug complexes containing various levels of covalently bound mitomycin C (MC) or anthramycin were subjected to the actions of a number of restriction enzymes. While MC presented only a partial block to the actions of a number of these enzymes, anthramycin, at high binding ratios, blocked enzymatic activity very well. The contrast seen in the restriction cleavage of these DNA-drug complexes may be related to the different points of attachment in DNA (minor groove vs. major groove) for these drugs. Although similarities in electrophoretic band patterns exist for both drug complexes, certain differences are indicative of preferences in binding sequences that these drugs may have for DNA. The results show that these sequences do not necessarily lie immediately within the restriction cut sites but may effect the cutting of these sites from a distance. The results also further support anthramycin's potential usage as a selective/reversible blocking agent for recombinant research.     

Theoretical Exploration of Netropsin Binding to tRNAPhe

Abstract

Theoretical exploration of the possible interaction of netropsin with tRNA^Phe indicates that binding should occur preferentially with the major groove of the TψC 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.     

Missing-base and ethylation interference footprinting of P1 plasmid replication initiator.

RepA, the replication initiator protein of plasmid P1, binds to specific 19 bp sequences on the plasmid DNA. Earlier footprinting studies with dimethylsulfate identified the guanines that contact RepA through the major groove of DNA. In this study, base elimination was used to identify the contribution of all four bases to the binding reaction. Depurination and depyrimidation of any base in the neighborhood of the contacting guanines was found to decrease RepA binding. These results are consistent with the notion that RepA contacts bases of two consecutive major grooves on the same face of DNA. We also observed that depurination but not methylation of three guanines (G3, G8 and G9) affected binding. We identified the DNA phosphate groups (3 in the top strand, one of which mapped between G8 and G9, and 4 in the bottom strand, one of which was adjacent to C3) that strongly interfered with RepA binding upon ethylation. These results indicate that certain bases (e.g. G3, G8 and G9) may not contact RepA directly but contribute to base and backbone contacts by maintaining proper structure of the binding site.     

DNA nicking by HinP1I endonuclease: bending, base flipping and minor groove expansion

HinP1I recognizes and cleaves the palindromic tetranucleotide sequence G↓CGC in DNA. We report three structures of HinP1I–DNA complexes: in the presence of Ca²⁺ (pre-reactive complex), in the absence of metal ion (binary complex) and in the presence of Mg²⁺ (post-reactive complex). HinP1I forms a back-to-back dimer with two active sites and two DNA duplexes bound on the outer surfaces of the dimer facing away from each other. The 10 bp DNA duplexes undergo protein-induced distortions exhibiting features of A-, B- and Z-conformations: bending on one side (by intercalation of a phenylalanine side chain into the major groove), base flipping on the other side of the recognition site (by expanding the step rise distance of the local base pair to Z-form) and a local A-form conformation between the two central C:G base pairs of the recognition site (by binding of the N-terminal helix in the minor groove). In the pre- and post-reactive complexes, two metals (Ca²⁺ or Mg²⁺) are found in the active site. The enzyme appears to cleave DNA sequentially, hydrolyzing first one DNA strand, as seen in the post-reactive complex in the crystalline state, and then the other, as supported by the observation that, in solution, a nicked DNA intermediate accumulates before linearization.     

A study of the interaction of Cr(III) complexes and their selective binding with B-DNA: a molecular modeling approach

Molecular modeling and energy minimisation calculations have been used to investigate the interaction of chromium(III) complexes in different ligand environments with various sequences of B-DNA. The complexes are [Cr(salen)(H(2)O)(2)](+); salen denotes 1, 2 bis-salicylideneaminoethane, [Cr(salprn)(H(2)O)(2)](+); salprn denotes 1, 3 bis- salicylideneaminopropane, [Cr(phen)(3)](3+); phen denotes 1, 10 phenanthroline and [Cr(en)(3)](3+); en denotes ethylenediamine. All the chromium(III) complexes are interacted with the minor groove and major groove of d(AT)(12), d(CGCGAATTCGCG)(2) and d(GC)(12) sequences of DNA. The binding energy and hydrogen bond parameters of DNA-Cr complex adduct in both the groove have been determined using molecular mechanics approach. The binding energy and formation of hydrogen bonds between chromium(III) complex and DNA has shown that all complexes of chromium(III) prefer minor groove interaction as the favourable binding mode.     

DNA Sequence Determinants Controlling Affinity,Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.     

Mode of binding of the cytotoxic alkaloid berberine with the double helix oligonucleotide d(AAGAATTCTT)(2)

Berberine, an isoquinoline plant alkaloid, belongs to the structural class of protoberberines. Recently, the ability of these compounds to act as Topoisomerase I or II poisons, was related to the antitumor activity. The binding of protoberberins to DNA has been studied and the partial intercalation into the double helix has been considered responsible for their activity. We have studied the interaction of berberine with the double helix oligonucleotides d(AAGAATTCTT)(2), d(GCGATCGC)(2), d(CGTATACG)(2), d(CGTACG)(2), 5'-d(ACCTTTTTGATGT)-3'/5(ACATCAAAAAGGT)-3' and with the single strand 5'-d(ACATCAAAAAGGT)-3', by 1H, 31P NMR and UV spectroscopy. Phosphorus resonance experiments were performed to detect small conformational changes of the phosphoribose backbone, in the case that an intercalation process occurs. Our data reveal that berberine does not intercalate into the duplexes studied, and binds preferentially to AT rich sequences. The structure of the complex with d(AAGAATTCTT)(2) was determined by using proton 2D NOESY spectra, which allowed to obtain several NOE contacts between the drug and the nucleotide. Structural models were built up by Molecular Mechanics (MM) and Molecular Dynamics (MD) calculations, by using the inter-proton distances derived from the NOE values. Berberine results to be located in the minor groove, lying with the convex side on the helix groove and presenting the positively charged nitrogen atom close to the negative ionic surface of the oligomer. The large 1H chemical shifts variation, observed for the drug when it is added to the above duplexes, as well as to the single strand oligomer, was interpreted with non-specific ionic interactions. The binding constants were measured by UV and NMR spectroscopy. They are strongly affected by the ionic strength and by the self-association process, which commonly occurs with this type of drugs. A dimerisation constant was measured and the value was included in the calculations of the binding constants. The results obtained show that the non-specific ionic interactions represent the major contribution to the values of the binding constants. These parameters, as well as the protons chemical shift variation of the ligand, are thus not diagnostic for the identification of a drug/DNA complex.     

Interactions between an anthracycline antibiotic and DNA: molecular structure of daunomycin complexed to d(CpGpTpApCpG) at 1.2-A resolution

The crystal structure of a daunomycin-d(CGTACG) complex has been solved by X-ray diffraction analysis and refined to a final R factor of 0.175 at 1.2-A resolution. The crystals are in a tetragonal crystal system with space group P4(1)2(1)2 and cell dimensions of a = b = 27.86 A and c = 52.72 A. The self-complementary DNA forms a six base pair right-handed double helix with two daunomycin molecules intercalated in the d(CpG) sequences at either end of the helix. Daunomycin in the complex has a conformation different from that of daunomycin alone. The daunomycin aglycon chromophore is oriented at right angles to the long dimension of the DNA base pairs, and the cyclohexene ring A rests in the minor groove of the double helix. Substituents on this ring have hydrogen-bonding interactions to the base pairs above and below the intercalation site. O9 hydroxyl group of the daunomycin forms two hydrogen bonds with N3 and N2 of an adjacent guanine base. Two bridging water molecules between the drug and DNA stabilize the complex in the minor groove. In the major groove, a hydrated sodium ion is coordinated to N7 of the terminal guanine and the O4 and O5 of daunomycin with a distorted octahedral geometry. The amino sugar lies in the minor groove without bonding to the DNA. The DNA double helix is distorted with an asymmetrical rearrangement of the backbone conformation surrounding the intercalator drug. The sugar puckers are C1,C2'-endo, G2,C1'-endo, C11,C1'-endo, and G12,C3'-exo. Only the C1 residue has a normal anti-glycosyl torsion angle (chi = -154 degrees), while the other three residues are all in the high anti range (average chi = -86 degrees). This structure allows us to identify three principal functional components of anthracycline antibiotics: the intercalator (rings B-D), the anchoring functions associated with ring A, and the amino sugar. The structure-function relationships of daunomycin binding to DNA as well as other related anticancer drugs are discussed.     

DNase I-tRNA interaction

Deoxyribonuclease I (DNase I) binds right-handed DNA duplex via a minor groove and the backbone phosphate group with no contact to the major groove. It hydrolyses double-stranded DNA predominantly by a single-stranded nicking mechanism under physiological conditions, in the presence of divalent Mg and Ca cations. Even though DNase-RNA interaction was observed, less is known about the protein-RNA binding mode and the effect of such complexation on both protein and RNA conformations. The aim of this study was to examine the effects of DNase I-tRNA interaction on tRNA and protein conformations. The interaction of DNase I with tRNA is monitored under physiological conditions, in the absence of Mg2+, using constant DNA concentration of 12.5 mM (phosphate) and various protein contents (10 microM to 250 microM). FTIR, UV-visible, and CD spectroscopic methods were used to analyze the protein binding mode, the binding constant, and the effects of polynucleotide-enzyme interaction on both tRNA and protein conformations. Spectroscopic evidence showed major DNase-PO2 and minor groove interactions with overall binding constant of K = 2.1 (+/-0.7) x 10(4) M(-1). The DNase I-tRNA interaction alters protein secondary structure with major reduction of the alpha-helix, and increases the random coil, beta-anti and turn structures, while tRNA remains in the A-conformation. No digestion of tRNA by DNase I was observed in the protein-tRNA complexes.     

The interaction of ellipticine derivatives with nucleic acids studied by optical and 1H-nmr spectroscopy: Effect of size of the heterocyclic ring system

The DNA interaction of derivatives of ellipticine with heterocyclic ring systems with three, four, or five rings and a dimethylaminoethyl side chain was studied. Optical spectroscopy of drug complexes with calf thymus DNA, poly [(dA-dT) · (dA-dT)], or poly [(dG-dC) · (dG-dC)] showed a 10 nm bathochromic shift of the light absorption bands of the pentacyclic and tetracyclic compounds upon binding to the nucleic acids, which indicates binding by intercalation. For the tricyclic compound a smaller shift of 1–3 nm was observed upon binding to the nucleic acids. Flow linear dichroism studies show that the geometry of all complexes is consistent with intercalation of the ring system, except for the DNA and poly [(dG-dC) · (dG-dC)] complexes of the tricyclic compound, where the average angle between the drug molecular plane and the DNA helix axis was found to be 65°. One-dimensional ¹H-nmr spectroscopy was used to study complexes between d(CGCGATCGCG)₂ and the tricyclic and pentacyclic compounds. The results on the pentacyclic compound show nonselective broadening due to intermediate chemical exchange of most oligonucleotide resonances upon drug binding. The imino proton resonances are in slow chemical exchange, and new resonances with upfield shifts approaching 1 ppm appear upon drug binding, which supports intercalative binding of the pentacyclic compound. The results on the tricyclic compound show more rapid binding kinetics and very selective broadening of resonances. The data suggest that the tricyclic compound is in an equilibrium between intercalation and minor groove binding, with a preference to bind close to the AT base pairs with the side chain residing in the minor groove. © 1994 John Wiley & Sons, Inc.     

The width of the minor groove affects the binding of the bisquaternary heterocycle SN-6999 to duplex DNA.

A complex between d(GGGAAAAACGG).d(CCGTTTTTCCC) and the minor groove binding drug SN-6999 has been studied by 1H nuclear magnetic resonance spectroscopy. The drug is found to bind in the d(A)5 tract, but with interactions extending one residue in the 3'-direction along each strand. Doubling of resonances in the complex indicates slow to intermediate exchange between two binding modes. An orientational preference (7:3) is found, the first such example in an SN-6999 complex. Furthermore, the upper limit of the lifetime for the major species is longer than was found for SN-6999 with other DNA duplexes. The preferred orientation of SN-6999 has the pyridinium ring near the 5'-end of the (+) strand; the minor binding mode has the reverse orientation. The orientational preference and slower exchange rate relative to other SN-6999 complexes is attributed to increased stabilization from van der Waals interactions due to better shape complementarity between the DNA duplex and ligand. The comparison of these results with studies of SN-6999 complexed to other DNA duplexes reveals the sensitivity of the binding properties to the delicate interplay between the molecular structure of the ligand and the specific characteristics of the DNA minor groove.