Characteristics of triplex-directed photoadduct formation by psoralen-linked oligodeoxynucleotides.

A triplex-forming oligopyrimidine has been attached at its 5'-end to a photoreactive psoralen derivative and used to target a sequence which forms part of the coding region of the human aromatase gene. The 20 base pair sequence is not a perfect triplex target since it contains three pyrimidine interruptions within the purine-rich strand. Despite this, we have detected triplex-directed photoadduct formation at pH 7.0 between the psoralen-linked oligonucleotide and a 30mer duplex representing the aromatase target. Photoadduct formation was found to be sensitive to pH, temperature, cation concentration and the base composition of the third strand. By varying the base sequence of the target duplex around the psoralen intercalation site, we have characterised the site and mode of psoralen intercalation. The attached psoralen has been found to intercalate at the triplex-duplex junction with a strong preference for one orientation. We have shown that the psoralen will bind at the junction even when there is a preferred TpA step at an adjacent site. We have also compared the binding affinity and photoreactivity of oligodeoxyribonucleotides linked to two different psoralen derivatives and found differences in the rate of crosslinking and the extent of crosslink formation. Finally, we have examined oligodeoxyribonucleotides which are attached to psoralen by polymethylene linkers of different lengths.     

Long-range guanine oxidation in DNA restriction fragments by a triplex-directed naphthalene diimide intercalator

Naphthalene diimide (NDI), a powerful oxidant that binds avidly to DNA by intercalation, is seen to damage the 5' guanine of 5'-GG-3' sites by photoactivated charge transport through DNA. When covalently tethered to the center of a triplex-forming oligonucleotide and delivered by triplex formation within a pyrimidine.purine-pyrimidine motif to a specific site on a restriction fragment, NDI can photooxidize guanine over at least 25-38 bp in each direction from the site of binding. Charge migration occurs in both directions from the NDI intercalator and on both DNA strands of the target, but the oxidation is significantly more efficient to the 3' side of the triplex. NDI and octahedral rhodium intercalators, when tethered directly to the 5' terminus of the triplex-forming strand as opposed to the center, generate significant amounts of oxidative damage only in the immediate vicinity of the intercalation site. Given that long-range charge transport depends on DNA stacking, these results suggest that the base stack is distorted at the 5' end of the triplex region in the duplex-triplex junction. Targeting of photooxidative damage by triplex formation extends our previous studies of long-range charge transport to significantly longer DNA sequences through a strategy that does not require covalent attachment of the photooxidant to the DNA being probed. Moreover, triplex targeting of oxidative damage provides for the first time a typical distance distribution for genomic charge transport of approximately 200 A around the oxidant.     

Selective strand scission by intercalating drugs at DNA bulges

A bulge is an extra, unpaired nucleotide on one strand of a DNA double helix. This paper describes bulge-specific strand scission by the DNA intercalating/cleaving drugs neocarzinostatin chromophore (NCS-C), bleomycin (BLM), and methidiumpropyl-EDTA (MPE). For this study we have constructed a series of 5'-32P end labeled oligonucleotide duplexes that are identical except for the location of a bulge. In each successive duplex of the series, a bulge has been shifted stepwise up (from 5' to 3') one strand of the duplex. Similarly, in each successive duplex of the series, sites of bulge-specific scission and protection were observed to shift in a stepwise manner. The results show that throughout the series of bulged duplexes NCS-C causes specific scission at a site near a bulge, BLM causes specific scission at a site near a bulge, and MPE-Fe(II) causes specific scission centered around the bulge. In some sequences, NCS-C and BLM each cause bulge-specific scission at second sites. Further, bulged DNA shows sites of protection from NCS-C and BLM scission. The results are consistent with a model of bulged DNA with (1) a high-stability intercalation site at the bulge, (2) in some sequences, a second high-stability intercalation site adjacent to the first site, and (3) two sites of relatively unstable intercalation that flank the two stable intercalation sites. On the basis of our results, we propose a new model of the BLM/DNA complex with the site of intercalation on the 3' side (not in the center) of the dinucleotide that determines BLM binding specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intermolecular triplex formation distorts the DNA duplex in the regulatory region of human papillomavirus type-11.

A conformational distortion in the DNA duplex at the regulatory region of human papillomavirus type-11 next to an intermolecular triplex, formed with a synthetic oligonucleotide, was investigated with several chemical probes. The sequence targeted for triplex formation borders on the binding sites for the regulatory proteins encoded by the viral E2 open reading frame. Dimethyl sulfate, diethyl pyrocarbonate, and OsO4 all react to a greater extent with nucleotides in the duplex that are immediately adjacent to the triplex as compared to other bases throughout the duplex. This hypermodification was observed on both the polypurine and polypyrimidine strands of the duplex DNA. Similar hyperreactivity of bases flanking a triplex also was seen when the contiguous target polypurine tract was effectively extended by mutating interrupting pyrimidines in the human papillomavirus type-11 sequence to purines. We propose that this hyperreactivity is due to a structural distortion caused by the junction between the triplex and the duplex tracts.     

Intramolecular recombination R-triplex in solution: stabilization by bis-intercalator YOYO

Recognition of double-stranded DNA with a mixed nucleotide sequence by oligonucleotide is a long-term challenge. This aim can be achieved via formation of the recombination R-triplex, accommodating two identical DNA strands in parallel orientation, and antiparallel complementary strand. In the absence of proteins the R-triplex stability is low, however, so that intermolecular R-triplex is not formed by three DNA strands in a ligand-free system. Recently, recognition of DNA with mixed base sequence by single-stranded oligonucleotide in the presence of bis-intercalator YOYO was reported. Here, we describe thermodynamic characteristics of YOYO complexes with the model oligonucleotides 5'-GT-2AP-GACTGAG TTTT CTCAGTCTACGC GAA GCGTAGACTGAG-3' (R(2AP)CW) bearing a single reporting 2-aminopurine (2AP) in place of adenine and 5'-CTCAGTCTACGC GAA GCGTAGACTGAG-3' (CW). We found that each oligonucleotide is able to bind two YOYO molecules via intercalation mode in 0.5 M LiCl. Fluorescence intensity of YOYO intercalated in triplex R(2AP)CW and in CW hairpin increased 40-fold compared to the free YOYO. Remarkably, the melting temperature of the triplex (determined using temperature dependence of the 2AP fluorescence) increased from 19 degrees C to 33 degrees C upon binding two YOYO molecules. Further increase in the YOYO concentration resulted in binding of up to five YOYO molecules to R(2AP)CW triplex and up to six YOYO molecules to CW hairpin.     

A structural analysis of the bent kinetoplast DNA from Crithidia fasciculata by high resolution chemical probing.

The chemical probes potassium permanganate (KMnO4) and diethylpyrocarbonate (DEPC) have been used to study the conformation of bent kinetoplast DNA from Crithidia fasciculata at different temperatures. Chemical reactivity data shows that the numerous short A-tracts of this bent DNA adopt a similar structure at 43 degrees C. This conformation appears to be very similar to the conformation of A-tracts in DNA exhibiting normal gel mobility. The A-tract structure detected by chemical probing is characterized by a high degree of base stacking on the thymine strand, and by an abrupt conformational change at the 3' end of the adenine strand. In general, no major alteration of this A-tract specific structure was detected between 4-53 degrees C. However, probing with KMnO4 revealed two unusual features of the C. fasciculata sequence that may contribute to the highly aberrant gel mobility of this DNA: 1) the B DNA/A-tract junction 5' dC/A3-6 3'. 5' dT3-6/G 3' is disproportionately represented and is conformationally distinct from other 5' end junctions, and 2) low temperature favors a novel strand-specific conformational distortion over a 20 base pair region of the bent kinetoplast DNA. Presence of the minor groove binding drug distamycin had little detectable effect on the A-tract conformation. However, distamycin did inhibit formation of the novel KMnO4 sensitive low temperature structure and partially eliminated the anomalous gel mobility of the kinetoplast DNA. Finally, we describe a simple and reproducible procedure for the production of an adenine-specific chemical DNA sequence ladder.     

Spectroscopic studies on the formation and thermal stability of DNA triplexes with a benzoannulated delta-carboline-oligonucleotide conjugate

A benzoannulated delta-carboline with a phenyl substituent has been covalently tethered to the 3'-end of a triplex-forming oligonucleotide and its ability to bind and stabilize DNA triple helices has been examined by various spectroscopic methods. UV thermal melting experiments were conducted with different hairpin duplexes and with a complementary single-stranded oligonucleotide as targets for the conjugate. The delta-carboline ligand preferentially binds triplexes over duplexes and leads to a temperature increase of the triplex-to-duplex transition by up to 23 degrees C. The results obtained from UV, CD and fluorescence measurements suggest that the delta-carboline ligand exhibits specific interactions with a triplex and favors binding by intercalation at the triplex-duplex junction.     

DNA melting in the presence of fluorescent intercalating oxazole yellow dyes measured with a gel-based assay

We measured the effect of the intercalating oxazole yellow DNA dye quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide (YO-PRO) and its homodimer (YOYO) on the melting of self-complementary DNA duplexes using a gel-based assay. The assay, which requires a self-complementary DNA sequence, is independent of the optical properties of the molecules in solution. The melting temperature of the DNA is observed to increase in direct proportion to the number of occupied intercalation sites on the DNA, irrespective of whether the dye molecules are in monomer or dimer form. The increase is approximately 2.5 degrees C for each intercalation site occupied in the presence of 38 mM [Na(+)], for dye/duplex ratios in which less than 1/5 of the available intercalation sites are occupied.     

Anthracycline antibiotic arugomycin binds in both grooves of the DNA helix simultaneously: an NMR and molecular modelling study.

Perturbations to the 1H and 31P chemical shifts of DNA resonances together with twenty-four intermolecular nuclear Overhauser effects show that the anthracycline antibiotic arugomycin intercalates between the basepairs of the hexamer duplex d(5'-GCATGC)2 at the 5'-CpA and 5'-TpG binding sites. In the complex two drug molecules are bound per duplex with full retention of the dyad symmetry. Arugomycin adopts a threaded binding orientation with chains of sugars positioned in both the major and minor groove of the helix simultaneously. The complex is stabilized by hydrogen bonding, electrostatic and van der Waals interactions principally in the major groove and involving substituents on the rigidly oriented bicycloamino-glucose sugar of the antibiotic. A specific hydrogen bond is identified between the C2'-hydroxyl and the guanine N7 at the intercalation site. Together, interactions in the major groove appear to account for the intercalation specificity of arugomycin that requires both a guanine and thymine at the intercalation site. We are unable to identify any sequence specific interactions between the minor groove and the arugarose sugar (S1) which binds only weakly, through van der Walls contacts, over the d(GCA).d(TGC) trinucleotide sequence. The data indicate that the sugar chains of arugomycin are flexible and play little part in the interaction of the antibiotic with DNA. The intensity of sequential internucleotide NOEs identifies the intercalation site as being assymmetric. A family of conformers computed using restrained energy minimisation and molecular dynamics indicate that basepair buckling is a feature of the anthracycline intercalation site that may serve to maximise intermolecular van der Waals interactions by wrapping the basepairs around the antibiotic chromophore.     

Structural basis for the unusual properties of 2',5' nucleic acids and their complexes with RNA and DNA

To provide insights into the unusual properties of 2',5' nucleic acids (iso nucleic acids), that includes their rejection by Nature as information molecules, modeling studies have been carried out to examine if they indeed possess the stereochemical ability to form helical duplexes and triplexes, just as their 3',5' linked constitutional isomers. The results show that the formation of helical duplexes with 2',5' linkages demands a mandatory displacement of the Watson and Crick base pairs from the helical axis, as a direct consequence of the lateral shift of the sugar-phosphate backbone from the periphery towards the interior of the helix. Thus, both duplexes and triplexes formed with a 2',5'-sugar-phosphate backbone possess this intrinsic trait, manifested normally only in A type duplexes of DNA and RNA. It was found that only a 10-fold symmetric parallel triplex with isomorphous T.AT triplets is stereochemically favorable for isoDNA with 'extended' nucleotide repeats, unlike the 12-fold symmetric triplex favored by DNA. The wider nature of a 12-fold triplex, concomitant with mandatory slide requirement for helix formation in isoDNA, demands even larger displacement, especially with 'extended' nucleotide structural repeats, thereby violating symmetry. However, a symmetric triplex possessing higher twist, can be naturally formed for isoDNA with a 'compact' nucleotide repeat. Two nanosecond molecular dynamics simulation of a 2',5'-B DNA duplex, formed with an intrinsic base pair displacement of -3.3 A, does not seem to favor a total transition to a typical A type duplex, although enhanced slide, X-displacement, decrease in helical rise and narrowing of the major groove during simulation seem to indicate a trend. Modeling of the interaction between the chimeric isoDNA.RNA duplex and E. coli RNase H has provided a structural basis for the inhibitory action of the enzyme. Interaction of residues Gln 80, Trp 81, Asn 16 and Lys 99, of E. coli RNase H with DNA of the DNA.RNA hybrid, are lost when the DNA backbone is replaced by isoDNA. Based on modeling and experimental observations, it is argued that 2',5' nucleic acids possess restricted conformational flexibility for helical polymorphism. The inability of isoDNA to favor the biologically relevant B form duplex and the associated topological inadequacies related to nucleic acid compaction and interactions with regulatory proteins may be some of the factors that might have led to the rejection of 2',5' links.     

Unwinding of a DNA triple helix by the Werner and Bloom syndrome helicases

Bloom syndrome and Werner syndrome are genome instability disorders, which result from mutations in two different genes encoding helicases. Both enzymes are members of the RecQ family of helicases, have a 3' --> 5' polarity, and require a 3' single strand tail. In addition to their activity in unwinding duplex substrates, recent studies show that the two enzymes are able to unwind G2 and G4 tetraplexes, prompting speculation that failure to resolve these structures in Bloom syndrome and Werner syndrome cells may contribute to genome instability. The triple helix is another alternate DNA structure that can be formed by sequences that are widely distributed throughout the human genome. Here we show that purified Bloom and Werner helicases can unwind a DNA triple helix. The reactions are dependent on nucleoside triphosphate hydrolysis and require a free 3' tail attached to the third strand. The two enzymes unwound triplexes without requirement for a duplex extension that would form a fork at the junction of the tail and the triplex. In contrast, a duplex formed by the third strand and a complement to the triplex region was a poor substrate for both enzymes. However, the same duplex was readily unwound when a noncomplementary 5' tail was added to form a forked structure. It seems likely that structural features of the triplex mimic those of a fork and thus support efficient unwinding by the two helicases.     

Sequence-specific recognition of the major groove of DNA by oligodeoxynucleotides via triple helix formation. Footprinting studies.

Homopyrimidine oligodeoxynucleotides recognize the major groove of the DNA double helix at homopurine.homopyrimidine sequences by forming local triple helices. The oligonucleotide is bound parallel to the homopurine strand of the duplex. This binding can be revealed by a footprinting technique using copper-phenanthroline as a cleaving reagent. Oligonucleotide binding in the major groove prevents cleavage by copper-phenanthroline. The cleavage patterns on opposite strands of the duplex at the boundaries of the triple helix are asymmetric. They are shifted to the 3'-side, indicating that the copper-phenanthroline chelate binds in the minor groove of the duplex structure. Binding of the chelate at the junction between the triple and the double helix is not perturbed on the 5'-side of the bound homopyrimidine oligonucleotide. In contrast, a strong enhancement of cleavage is observed on the purine-containing strand at the triplex-duplex junction on the 3'-side of the homopyrimidine oligonucleotide.     

Conformation and dynamics of drug-DNA intercalation.

Molecular dynamics simulations have been undertaken for a B-form dodecanucleotide duplex in solution with and without an intercalated proflavine molecule between the central C.G base pairs. The introduction of this simple intercalator affects both the conformational features and dynamic properties of the oligonucleotide double helix. Changes are seen in the rms atomic fluctuations and anisotropy of phosphate, sugar and base atoms. The backbone conformation is slightly changed on average and more sugars adopt the C3' endo conformation in the simulation of the complex compared with the simulation of the oligonucleotide alone. Both major and minor grooves becomes wider on average with the addition of the intercalating drug. Flanking A.T base pairs on both sides of the intercalation site have undergone an increase in flexibility, with the base pairs, especially at the 5' side, having the N1...N3 hydrogen bonds being broken.     

The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I

Various branched DNA structures were created from synthetic, partly complementary oligonucleotides combined under annealing conditions. Appropriate mixtures of oligonucleotides generated three specific branched duplex DNA molecules: (i) a Holliday junction analog having a fixed (immobile) crossover bounded by four duplex DNA branches, (ii) a similar Holliday junction analog which is capable of limited branch migration and, (iii) a Y-junction, with three duplex branches and fixed branch point. Each of these novel structures was specifically cleaved by bacteriophage T7 gene 3 product, endonuclease I. The cleavage reaction "resolved" the two Holliday structure analogs into pairs of duplex DNA products half the size of the original molecules. The point of cleavage in the fixed-junction molecules was predominantly one nucleotide removed to the 5' side of the expected crossover position. Multiple cleavage positions were mapped on the Holliday junction with the mobile, or variable, branch point, to sites consistent with the unrestricted movement of the phosphodiester crossover within the region of limited dyad symmetry which characterizes this molecule. Based on the cleavage pattern observed with this latter substrate, the enzyme displayed a modest degree of sequence specificity, preferring a pyrimidine on the 3' side of the cleavage site. Branched molecules that were partial duplexes (lower order complexes which possessed single-stranded as well as duplex DNA branches) were also substrates for the enzyme. In these molecules, the cleaved phosphodiester bonds were in duplex regions only and predominantly one nucleotide to the 5' side of the branch point. The phosphodiester positions 5' of the branch point in single-stranded arms were not cleaved. Under identical reaction conditions, individually treated oligonucleotides were completely refractory. Thus, cleavage by T7 endonuclease I displays great structural specificity with an efficiency that can vary slightly according to the DNA sequence.     

Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation.     

Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.

Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5'-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5'-ApT sequence at the triplex-duplex junction and at a target site with 5'-ApT and 5'-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide "linkers" from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen-TFO conjugates formed DNA cross-links with high efficiency (56-65%) at 5'-ApT sequences located at triplex junctions. At a 5'-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5'-TpA and 5'-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO-linker-psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.     

Sequence specific conformation of a DNA decamer containing an adenine tract studied in solution by H-NMR spectroscopy

The decanucleotide duplex d(AAAACGTTTT)2 and a variety of phase-sensitive two-dimensional (2D) NMR experiments have been used to investigate the solution conformation of an adenine-tract and its junction with another DNA sequence. 2D nuclear Overhauser effect data confirm that the oligonucleotide has a general B-type DNA morphology but an array of unusual correlations implies that the adenine tract and the 5'-ApC junction have conformations more compatible with the modified X-ray structures recently reported for DNAs of similar sequence (Nelson, H.C.M., Finch, J.T., Luisi, B.F. and Klug, A. (1987) Nature 330, 221-226). The pattern and magnitude of interstrand NOEs from the adenine H2s to the sugar H1's of the complementary base to the 5'-neighbouring residue indicate that the A-T basepairs are highly propeller twisted and that the minor groove is narrowed, showing its greatest compression at the 3'-end of the tract at the 5'-ApC step. Quantifying spin-coupling interactions within the deoxyribose rings by analysing both 1D and high-resolution 2D DQF-COSY data reveals that the conformation of the purines is predominantly C2'-endo, with the pseudorotation phase angle P lying in the range 140-180 degrees. For the pyrimidines, however, there are distortions away from this standard B-type geometry with the data being best described by P values lying in the range 90-130 degrees (i.e., O4'-endo, C1'-exo). The sugar puckers of A1, T9 and T10 are dynamically distorted no doubt as a consequence of their positions at, or close to, the ends of the duplex. Thus the conformation of the adenine and thymine sugars within the oligo(dA) and oligo(dT) strands are different with an abrupt change in sugar puckering occurring at the 5'-ApC (5'-GpT) step. Peculiar chemical shifts values for A4H2, T7CH3 and sugar C5 H1', H2' and H2", together with a number of interresidue NOEs with unusual intensities, imply that there are also substantial modifications to basepair stacking interactions at this step. Taken as a whole, our data are consistent with the view that the conformational dislocation at the 5'-ApC dinucleotide results from a combination of slide and roll manoeuvres and that the junction between the AAAA and CG sequences is a potential nucleation site for DNA bending.     

Sequence-specific interaction of Hoechst 33258 with the minor groove of an adenine-tract DNA duplex studied in solution by 1H NMR spectroscopy.

The interaction of Hoechst 33258 with the minor groove of the adenine-tract DNA duplex d(CTTTTGCAAAAG)2 has been studied in both D2O and H2O solutions by 1D and 2D 1H NMR spectroscopy. Thirty-one nuclear Overhauser effects between drug and nucleotide protons within the minor groove of the duplex, together with ring-current induced perturbations to the chemical shifts of basepair and deoxyribose protons, define the position and orientation of the bound dye molecules. Two drug molecules bind cooperatively and in symmetry related orientations at the centre of the 5'-TTTT and 5'-AAAA sequences with the binding interactions spanning only the four A-T basepairs. The positively charged N-methylpiperazine moieties point towards the centre of the duplex while the phenol groups are disposed towards the 3'-ends of the sequence. Resonance averaging is apparent for both the D2/D6 and D3/D5 phenol protons and D2"'/D6"' and D3"'/D5"' of the N-methylpiperazine ring and is consistent with these groups being involved in rapid rotation or ring-flipping motions in the bound state. Interstrand NOEs between adenine H2s and deoxyribose H1' are consistent with a high degree of propeller twisting of the A-T basepairs at the binding site of the aromatic benzimidazole and phenol rings of Hoechst. The data imply that the minor groove is particularly narrow with many contacts between the complementary curved surfaces of the drug and DNA indicating that strong van der Waals interactions, involving the floor and the walls of the minor groove, stabilize the complex. In our model the NH groups of the benzimidazole rings are positioned to make a pair of bifurcated hydrogen bonds with the adenine N3 and thymine O2 on the floor of the minor groove.