Enthalpy-entropy compensations in drug-DNA binding studies.

We present a comparative study of calorimetrically derived thermodynamic profiles for the binding of a series of drugs with selected DNA host duplexes. We use these data to demonstrate that comparisons between complete thermodynamic profiles (delta G zero, delta H zero, delta S zero, delta Cp) are required before drug binding can be used as a probe of DNA conformation, since enthalpy-entropy compensations can cause two drug-DNA binding events to exhibit similar binding free energies (delta G zero) despite being driven by entirely different thermodynamic forces (delta H zero, delta S zero). In this work, we employ a combination of spectroscopic and calorimetric techniques to characterize thermodynamically the DNA binding of netropsin and distamycin (two minor groove-directed ligands), ethidium (an intercalator), and daunomycin (a combined intercalator/groove binder). Our free energy data (delta G zero) show that each drug exhibits similar binding affinities at 25 degrees C for the alternating copolymer duplex poly[d(A-T)].poly[d(A-T)] and for the homopolymer duplex poly(dA).poly(dT). However, our calorimetric measurements reveal that the nature of the thermodynamic forces (delta H zero, delta S zero) that drive drug binding to these two host duplexes at 25 degrees C are entirely different, despite similar binding free energies (delta G zero) and similar salt dependencies (lnK/ln[Na+]). Specifically, the 25 degrees C binding of all four drugs to the alternating copolymer poly[d(A-T)].poly[d(A-T)] is overwhelmingly enthalpy driven, whereas the corresponding binding of each drug to the homopolymer duplex poly(dA).poly(dT) is overwhelmingly entropy driven. Thus, the similar binding free energies (delta G zero) we measure for complexation of each drug with poly[d(A-T)].poly[d(A-T)] and poly(dA).poly(dT) result from compensating changes in the enthalpy and entropy terms. Comparison with the thermodynamic profiles for the complexation of these drug molecules to other DNA host duplexes at 25 degrees C reveals that the binding of each is strongly enthalpy driven, except when the poly(dA).poly(dT) homopolymer serves as the host duplex. This comparison allows us to conclude that poly[d(A-T)].poly[d(A-T)] behaves thermodynamically as the more "normal" host duplex toward drug binding, whereas the entropy-driven binding to the poly(dA).poly(dT) duplex represents "aberrant" behavior. Furthermore, since each of the four drugs exhibits different modes of DNA binding, we conclude that the observed entropy-driven behavior for binding to poly(dA).poly(dT) reflects an intrinsic property of the homopolymer duplex that is perturbed in a common manner upon ligation rather than a common property of all four binding ligands. To rationalize the large positive entropy changes that drive drug complexation with poly(dA).poly(dT) duplex, we propose a model that emphasizes binding-induced perturbations of the more highly hydrated, altered B conformation of the homopolymer. Our results suggest that an aberrant thermodynamic binding profile may reflect an unusual DNA conformation in the host duplex. However, before such a conclusion can be reached, complete thermodynamic binding profiles must be examined, since enthalpy-entropy compensations can cause two binding events to exhibit similar binding constants even when they are driven by very different thermodynamic forces.     

Characterization of the minor groove environment in a drug-DNA complex: bisbenzimide bound to the poly[d(AT)].poly[d(AT)]duplex.

We compare the fluorescence properties of bisbenzimide (also known as Hoechst 33258) bound to the minor groove of the poly[d(AT)].poly[d(AT)] duplex with the corresponding fluorescence properties of bisbenzimide dissolved in neat organic solvents and mixed organic/aqueous solvents. Based on these comparisons, we conclude that the minor groove of the bisbenzimide-poly[d(AT)].poly[d(AT)] complex is quite nonpolar and exhibits a local dielectric constant of approximately 20 D. We discuss how this insight influences our understanding of the molecular forces that dictate and control the binding affinities and specificities of minor groove-directed DNA binding ligands.     

Drug binding to higher ordered DNA structures: netropsin complexation with a nucleic acid triple helix.

We have used a combination of spectroscopic and calorimetric techniques to characterize how netropsin, a ligand that binds in the minor groove of DNA, influences the properties of a DNA triple helix. Specifically, our data allow us to reach the following conclusions: (i) netropsin binds to the triplex without displacing the major-groove-bound third strand; (ii) netropsin binding to the triplex exhibits a lower saturation binding density (7.0 base triplets per netropsin bound) than netropsin binding to the corresponding duplex (5.5 base pairs per netropsin bound); (iii) the netropsin-free and the netropsin-bound triplexes each melt in two well-resolved transitions, initial conversion of the triplex to the duplex state followed by duplex melting to the component single-stranded states; (iv) netropsin remains bound to DNA as the triplex melts to the duplex state; (v) netropsin binding thermally destabilizes the triplex in equilibrium with duplex equilibrium dramatically, while thermally stabilizing the duplex to single-strand equilibrium; (vi) netropsin binding to the triplex is enthalpically 4 times more favorable (more exothermic) than netropsin binding to the corresponding duplex; (vii) netropsin binding to the triplex decreases the cooperativity of the triplex----duplex melting event. These results demonstrate that occupancy of the minor groove of a triplex by a ligand such as netropsin can exert a profound impact on the properties of the host triplex, particularly with regard to the equilibrium in which the third strand is expelled from the major groove. Thus, our results reveal considerable major groove/minor groove crosstalk. Such knowledge may prove of practical importance by providing an approach for modulating the affinity and specificity of major-groove-binding third strands in triplex-forming protocols designed to target specific duplex domains. Fundamentally, our results provide insights into the crosstalk that can result when ligands bind to the two major receptor sites of duplex DNA--namely, the major and minor grooves.     

Antibiotic-DNA interactions: intermolecular nuclear Overhauser effects in the netropsin-d(C-G-C-G-A-A-T-T-C-G-C-G) complex in solution.

The proton markers located in the minor groove of the d(C-G-C-G-A-A-T-T-C-G-C-G) duplex and its netropsin complex have been assigned from measurements of intramolecular nuclear Overhauser effects (NOEs) between exchangeable imino protons and nonexchangeable base protons on the same and adjacent base pairs. Several points of contact between the concave face of the antibiotic and the minor groove d(A-A-T-T) tetranucleotide segment of the dodecanucleotide duplex have been established based on intermolecular NOE effects between the pyrrole ring and side-chain methylene protons of netropsin and the adenosine H-2 protons of dA X dT base pairs in the center of the duplex. These NOE measurements provide a powerful method for differentiating between minor and major groove contacts in ligand-DNA complexes in solution. A model for netropsin interaction at dA X dT sites on duplex DNA is proposed.     

A mammalian high mobility group protein recognizes any stretch of six A.T base pairs in duplex DNA.

alpha-Protein is a high mobility group protein originally purified from African green monkey cells based on its affinity for the 172-base-pair repeat of monkey alpha-satellite DNA. We have used DNase I footprinting to identify 50 alpha-protein binding sites on simian virus 40 DNA and thereby to determine the DNA binding specificity of this mammalian nuclear protein. alpha-Protein binds with approximately equal affinity to any run of six or more A X T base pairs in duplex DNA, to many, if not all, runs of five A X T base pairs, and to a small number of other sequences within otherwise (A + T)-rich regions. Unlike well characterized sequence-specific DNA binding proteins such as bacterial repressors, alpha-protein makes extensive contacts within the minor groove of B-DNA. These and related findings indicate that, rather than binding to a few specific DNA sequences, alpha-protein recognizes a configuration of the minor groove characteristic of short runs of A X T base pairs. We discuss possible functions of alpha-protein and the similarities in DNA recognition by alpha-protein and the antibiotic netropsin.     

Mutual interaction between adjacent dG . dC actinomycin binding sites and dA . dT netropsin binding sites on the self-complementary d(C-G-C-G-A-A-T-T-C-G-C-G) duplex in solution.

The Watson-Crick imino protons, the backbone phosphodiester resonances, and the antibiotic exchangeable protons have been used as markers to monitor the separate and simultaneous binding of actinomycin and netropsin to the d(C-G-C-G-A-A-T-T-C-G-C-G) self-complementary duplex in aqueous solution. We demonstrate that intercalation of actinomycin at dG(3'-5')dC sites at either end of the duplex results in a conformational perturbation at the dA . dT tetranucleotide core of the dodecanucleotide duplex. Parallel studies of the groove binding of netropsin at dA . dT sites in the interior of the duplex reveal a conformational perturbation which extends to adjacent dG . dC base pairs in the dodecanucleotide duplex. The NMR markers demonstrate that the d(C-G-C-G-A-A-T-T-C-G-C-G) duplex can accommodate actinomycin and netropsin simultaneously at adjacent dG . dC and dA . dT tetranucleotide blocks along its length with some mutual interaction between neighboring antibiotic binding sites.     

The molecular origin of DNA-drug specificity in netropsin and distamycin.

X-ray analysis of the complex of netropsin with the B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-BrC-G-C-G reveals that the antitumor antibiotic binds within the minor groove by displacing the water molecules of the spine of hydration. Netropsin amide NH furnish hydrogen bonds to bridge DNA adenine N-3 and thymine O-2 atoms occurring on adjacent base pairs and opposite helix strands, exactly as with the spine of hydration. The narrowness of the groove forces the netropsin molecule to sit symmetrically in the center, with its two pyrrole rings slightly non-coplanar so that each ring is parallel to the walls of its respective region of the groove. Drug binding neither unwinds nor elongates the double helix, but it does force open the minor groove by 0.5-2.0 A, and it bends back the helix axis by 8 degrees across the region of attachment. The netropsin molecule has an intrinsic twist that favors insertion into the minor groove of B-DNA, and it is given a small additional twist upon binding. The base specificity that makes netropsin bind preferentially to runs of four or more A X T base pairs is provided not by hydrogen bonding but by close van der Waals contacts between adenine C-2 hydrogens and CH groups on the pyrrole rings of the drug molecule. Substitution of one or more pyrroles by imidazole could permit recognition of G X C base pairs as well, and it could lead to a class of synthetic "lexitropsins," capable of reading any desired short sequence of DNA base pairs.     

Structure of an alternating-B DNA helix and its relationship to A-tract DNA.

The crystal structure of the synthetic DNA dodecamer CGCATATATGCG has been solved at 2.2-A resolution. Its central 6 base pairs adopt the alternating-B-DNA helix structure proposed nearly a decade ago. This alternating poly(AT) structure contrasts with the four known examples of what can be termed a poly(A) subfamily of B-DNA structures: CGCAAAAAAGCG, CGCAAATTTGCG, CGCGAATTCGCG, and CGCGAATTbrCGCG, their defining characteristic being a succession of two or more adenines along one strand, in a region of 4 or more A.T base pairs. All five helices show a characteristically narrow minor groove in their AT centers, but the mean propeller twist at A.T base pairs is lower in the alternating poly(AT) helix than in the poly(A) subfamily of helices. Three general principles emerge from x-ray analyses of B-DNA oligonucleotides: (i) GC and mixed-sequence B-DNA have a wide minor groove, whereas the minor groove is narrow in heteropolymer or homopolymer AT sequences. (ii) G.C base pairs have low propeller twist; A.T pairs can adopt a high propeller twist but need not do so. A high propeller twist can be stabilized by cross-strand hydrogen bonds in the major or minor groove, examples being the minor groove bonds seen in CCAAGATTGG and the major groove bonds that can accompany AA sequences in the poly(A) family. (iii) Homopolymer poly(A) tracts may be stiffer than are alternating AT or general-sequence DNA because of these cross-strand major groove hydrogen bonds. Poly(A) tracts appear internally unbent, but bends may occur at junctions with mixed-sequence DNA because of differences in propeller twist, base pair inclination, and base stacking on the two sides of the junction. Bending occurs most easily via base roll, favoring compression of the broad major groove.     

A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.

A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.     

Sequence-specific recognition and cleavage of DNA by metallobleomycin: minor groove binding and possible interaction mode.

The DNase I cleavage-inhibition analysis shows binding sites of approximately 2 or 3 base pairs--in particular, 5' N-G-C sequences--for the green-colored CoIII and fully oxidized FeIII complexes of bleomycin. The apparent binding constant of the bleomycin-CoIII complex is quite similar for glucosylated and nonglucosylated phage T4 DNAs, whereas poly[d(I-C)] clearly gives a smaller binding constant than does poly[d(G-C)]. In contrast to the covalent attachment of the guanine N-7 with aflatoxin B1, the modification of the guanine 2-amino group with anthramycin remarkable inhibits the DNA cleavages at 5' G-C and 5' G-T sites by the FeIII and CoIII complex systems of bleomycin. These results strongly indicate that metallobleomycin binds in the minor groove of B-DNA and that the 2-amino group of guanine adjacent to the 5' side of the cleaved pyrimidine base is one key element of the specific 5' G-C or G-T recognition by the bleomycin-metal complex. A possible binding mode of metallobleomycin in the DNA helix has been proposed by computer-constructed model building.     

Map of distamycin, netropsin, and actinomycin binding sites on heterogeneous DNA: DNA cleavage-inhibition patterns with methidiumpropyl-EDTA.Fe(II).

We report a direct technique for determining the binding sites of small molecules on naturally occurring heterogeneous DNA. Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II) cleaves double helical DNA with low sequence specificity. Using a combination of MPE.Fe(II) cleavage of drug-protected DNA fragments and Maxam-Gilbert gel methods of sequence analysis, we have determined the preferred binding sites on a Rsa I-EcoRI restriction fragment from pBR322 for the intercalator actinomycin D and the minor groove binders netropsin and distamycin A. Netropsin and distamycin A gave identical DNA cleavage-inhibition patterns and bound preferentially to A+T-rich regions with a minimal protected site of four base pairs. We were able to observe the effect of increasing concentration on site selection by netropsin and distamycin A. Actinomycin D afforded a completely different cleavage-inhibition pattern, with 4- to 16-base-pair-long protected regions centered around one or more G.C base pairs.     

Sequence-specific binding of counterions to B-DNA

Recent studies by x-ray crystallography, NMR, and molecular simulations have suggested that monovalent counterions can penetrate deeply into the minor groove of B form DNA. Such groove-bound ions potentially could play an important role in AT-tract bending and groove narrowing, thereby modulating DNA function in vivo. To address this issue, we report here (23)Na magnetic relaxation dispersion measurements on oligonucleotides, including difference experiments with the groove-binding drug netropsin. The exquisite sensitivity of this method to ions in long-lived and intimate association with DNA allows us to detect sequence-specific sodium ion binding in the minor groove AT tract of three B-DNA dodecamers. The sodium ion occupancy is only a few percent, however, and therefore is not likely to contribute importantly to the ensemble of B-DNA structures. We also report results of ion competition experiments, indicating that potassium, rubidium, and cesium ions bind to the minor groove with similarly weak affinity as sodium ions, whereas ammonium ion binding is somewhat stronger. The present findings are discussed in the light of previous NMR and diffraction studies of sequence-specific counterion binding to DNA.     

Structural characterization of a 2:1 distamycin A.d(CGCAAATTGGC) complex by two-dimensional NMR.

Two-dimensional NMR has been used to study the 2:1 distamycin A.d(CGCAAATTGGC).d(GCCAATTTGCG) complex. The nuclear Overhauser effect spectroscopy (NOESY) experiment was used to assign the aromatic and C1'H DNA protons and to identify drug-DNA contacts. These data indicate that two drug molecules bind simultaneously in the minor groove of the central 5'-AAATT-3' segment and are in close contact with both the DNA and one another. One drug binds with the formyl end close to the second adenine base of the A-rich strand, while the other drug binds with the formyl end close to the second adenine of the complementary strand. With this binding orientation, the positively charged propylamidinium groups are directed toward opposite ends of the helix. Molecular modeling shows that the minor groove must expand relative to the 1:1 complex to accommodate both drugs. Energy calculations suggest that electrostatic interactions, hydrogen bonds, and van der Waals forces contribute to the stability of the complex.     

Crystal structure of a DNA decamer showing a novel pseudo four-way helix-helix junction.

The crystal structure of the decanucleotide d(CGCAATTGCG)2 has been solved by a combination of molecular replacement and heavy-atom procedures and has been refined to an R factor of 20.2% at 2.7 A. It is not a fully base-paired duplex but has a central core of eight Watson-Crick base pairs flanked by unpaired terminal guanosines and cytosines. These participate in hydrogen-bonding arrangements with adjacent decamer duplexes in the crystal lattice. The unpaired guanosines are bound in the G+C regions of duplex minor grooves. The cytosines have relatively high mobility, even though they are constrained to be in one region where they are involved in base-paired triplets with G.C base pairs. The 5'-AATT sequence in the duplex region has a narrow minor groove, providing further confirmation of the sequence-dependent nature of groove width.     

Sequence dependence of base-pair stacking in right-handed DNA in solution: proton nuclear Overhauser effect NMR measurements.

Single-crystal x-ray studies of d(C-G-C-G-A-A-T-T-C-G-C-G) exhibit base-pair propeller twisting [Dickerson, R. E. & Drew, H. R. (1981) J. Mol. Biol. 149, 761-786] that results in close contacts between adjacent purines in the minor groove in pyrimidine (3'-5')-purine steps and in the major groove in purine (3'-5')-pyrimidine steps [Calladine, C. R. (1982) J. Mol. Biol. 161, 343-362]. These observations require an approximately 3.4 A separation between the minor groove edges of adenosines on adjacent base pairs for the dA-dA step but predict a smaller separation for the dT-dA step and a larger separation for the dA-dT step in a D(A-T-T-A).d(T-A-A-T) fragment. We have confirmed these predictions from steady-state nuclear Overhauser effect measurements between assigned minor groove adenosine H-2 protons on adjacent base pairs in the proton NMR spectrum of the d(C1-G2-A3-T4-T5-A6-T6-A5-A4-T3-C2-G1) self-complementary dodecanucleotide duplex (henceforth called the Pribnow 12-mer) in solution. The measured cross-relaxation rates (product of steady-state nuclear Overhauser effect and selective spin- lattice relaxation rates) translate to interproton separations between adjacent adenosine H-2 protons of 4.22 A in the (dA3-dT4).(dA4-dT3) step, of 3.56 A in the (dT4-dT5).dA5-dA4) step, and of 3.17 A in the (dT5-dA6).(dT6-dA5) step for the Pribnow 12-mer duplex with an isotropic rotational correlation time of 9 ns at 5 degrees C. These proton NMR results show that the sequence-dependent base-pair stacking resulting from base-pair propeller twisting of defined handedness for right-handed DNA in the solid state is maintained in aqueous solution.     

A structural snapshot of base-pair opening in DNA

The response of double-helical DNA to torsional stress may be a driving force for many processes acting on DNA. The 1.55-A crystal structure of a duplex DNA oligonucleotide d(CCAGGCCTGG)(2) with an engineered crosslink in the minor groove between the central guanine bases depicts how the duplex can accommodate such torsional stress. We have captured in the same crystal two rather different conformational states. One duplex contains a strained crosslink that is stabilized by calcium ion binding in the major groove, directly opposite the crosslink. For the other duplex, the strain in the crosslink is relieved through partial rupture of a base pair and partial extrusion of a cytosine accompanied by helix bending. The sequence used is the target sequence for the HaeIII methylase, and this partially flipped cytosine is the same nucleotide targeted for extrusion by the enzyme. Molecular dynamics simulations of these structures show an increased mobility for the partially flipped-out cytosine.     

Netropsin-poly(dA-dT) complex in solution: structure and dynamics of antibiotic-free base pair regions and those centered on bound netropsin.

The biphasic duplex-to-strand transition for the netropsin.poly(dA-dT) complex, phosphate/drug mole ratio (P/D) = 50, has been investigated by high-resolution proton nuclear magnetic resonance (NMR) spectroscopy at the nonexchangeable base and sugar protons in 0.1 M cacodylate solution. The NMR spectral parameters monitor the structure and dynamics of the opening of antibiotic-free base pair regions (55 degrees-65 degrees) and the opening of base regions centered on bound netropsin (90 degrees-100 degrees). The gradual addition of netropsin to poly(dA-dT) results in structural perturbations extending into the antibiotic-free base pair regions that begin to level off above 0.02 antibiotic molecules per polynucleotide phosphate (P/D = 50). The NMR chemical shift parameters at the antibiotic-free base pair regions in the P/D = 50 complex suggest changes in the glycosidic torsion angles of the deoxyadenosine and thymidine residues and less pronounced changes in the base pair overlap geometries. The dissociation rates of the antibiotic-free base pair regions are at least an order of magnitude slower in the P/D = 50 netropsin.poly(dA-dT) complex compared to related parameters for poly(dA-dT) and the P/D = 50 ethidium bromide-poly(dA-dT) complex. There is decreased segmental mobility at the antibiotic-free strand regions in the temperature range (65 degrees-90 degrees) between the two transitions in the biphasic melting curve of the P/D = 50 netropsin-poly(dA-dT) complex. Netropsin stabilizes at least five base pairs, with their center at its binding site.     

Solution structural studies of the A and Z forms of DNA.

We report transient electric dichroism studies of short double-helical DNA and poly[d(G-C)] fragments in alcohol/water mixtures. The limiting reduced dichroism and the rotational correlation time changed abruptly in the alcohol concentration range expected for the DNA B-to-A and poly[d(G-C)] B-to-Z transitions. The Z form of poly[d(G-C)] was also induced by mitomycin C crosslinking in aqueous solution. The rotational correlation times observed for A- and Z-DNA were approximately consistent with dimensions determined by crystallographic and fiber diffraction analysis: the estimated rise per base pair was 2.8 A for A-DNA and 3.7 A for Z-DNA in solution. In addition, the observed limiting reduced dichroism values for A- and Z-DNA were close to the theoretical limit of -1.5, requiring a structure in which the base transition moments are effectively perpendicular to the double-helix axis. This is the result expected for any DNA double helix having dyad symmetry in which the base pairs are flat and the base transition moments lie predominantly in the short axis of the base pair and therefore close to a helix dyad axis. Only B-DNA deviates from this rule, strongly reinforcing our earlier conclusion that the base pairs in B-DNA are not flat but are propeller twisted, either statically or as a dynamic average.     

Molecular structure of an anticancer drug-DNA complex: daunomycin plus d(CpGpTpApCpG).

The structure of the crystalline daunomycin-d(CpGpTpApCpG) complex has been solved by x-ray diffraction analysis. The DNA forms a six-base-pair right-handed double helix with two daunomycin molecules intercalated in the d(CpG) sequences. The daunomycin aglycone chromophore is oriented at right angles to the long dimension of the DNA base pairs and the cyclohexene ring rests in the minor groove. Substituents on this ring have hydrogen bonding interactions to the base pairs above and below the intercalation site. These appear to be specific for anthracycline antibiotics. The amino sugar lies in the minor groove of the double helix without bonding to the DNA. The DNA double helix is distorted in a novel manner in accommodating the drug.     

Calicheamicin-DNA complexes: warhead alignment and saccharide recognition of the minor groove.

The solution structures of calicheamicin gamma 1I, its cycloaromatized analog (calicheamicin epsilon), and its aryl tetrasaccharide complexed to a common DNA hairpin duplex have been determined by NMR and distance-refined molecular dynamics computations. Sequence specificity is associated with carbohydrate-DNA recognition that places the aryl tetrasaccharide component of all three ligands in similar orientations in the minor groove at the d(T-C-C-T).d(A-G-G-A) segment. The complementary fit of the ligands and the DNA minor groove binding site creates numerous van der Waals contacts as well as hydrogen bonding interactions. Notable are the iodine and sulfur atoms of calicheamicin that hydrogen bond with the exposed amino proton of the 5'- and 3'-guanines, respectively, of the d(A-G-G-A) segment. The sequence-specific carbohydrate binding orients the enediyne aglycone of calicheamicin gamma 1I such that its C3 and C6 proradical centers are adjacent to the cleavage sites. While the enediyne aglycone of calicheamicin gamma 1I is tilted relative to the helix axis and spans the minor groove, the cycloaromatized aglycone is aligned approximately parallel to the helix axis in the respective complexes. Specific localized conformational perturbations in the DNA have been identified from imino proton complexation shifts and changes in specific sugar pucker patterns on complex formation. The helical parameters for the carbohydrate binding site are comparable with corresponding values in B-DNA fibers while a widening of the groove is observed at the adjacent aglycone binding site.