Effect of the methyl group on DNA bending and curvature: structure of d(GA4U4C)2 in solution

NMR studies on d(GA4T4C)2 and d(GT4A4C)2 indicated two important factors that contribute to intrinsic DNA bending in polymers containing A/T tracts [Sarma, M.H., et al. (1988) Biochemistry 27, 3423; Gupta, G., et al. (1988) Biochemistry 27, 7909]. They are (i) propeller-twisted A.T pairs with associated bifurcated H bonds inside the A/T tract and (ii) the base sequence that joins the two neighboring A/T tracts. As an extension of our bending project, we carried out quantitative NMR studies on the decamer d(GA4U4C)2, a structural analogue of d(GA4T4C)2, to examine the effect of the methyl group on DNA bending. On the basis of quantitative NMR analysis, we arrive at the following results. (i) The decamer d(GA4U4C)2 adopts the gross morphology of a right-handed B-DNA duplex with A and U nucleotides belonging to C2'-endo,anti domain. (ii) A.U pairs are propeller twisted and hence can result in an array of interstrand bifurcated H bonds involving N6 of A and O4 of U (one base pair apart) inside the A/U tract. (iii) The orientations of A and U with respect to the long axis of the molecule are different; as a result, at the A5-U6 sequence that joins the two A/U tracts, two neighboring frames of reference do not exactly coincide in space and a junction is created at A5-U6. (iv) Inside the A/U tract, intrastrand stacking is more compact (average separation between secessive base planes being 3.2 A) than at the A5-U6 junction, where average separation between the base planes of A5 and U6 is 3.6 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of a bent DNA: two-dimensional NMR studies on d(GAAAATTTTC)2

Intrinsic DNA bending is caused by specific DNA sequences. The decamer d(GA4T4C)2, when it repeats in a synthetic polymer or in kinetoplast DNA, results in a macroscopic bending of the molecule as a whole. We employed high-resolution two-dimensional NMR methods to examine the intrinsic structural properties of the d(GA4T4C)2 duplex in solution. Examination of the NOESY data at 50- and 100-ms mixing times indicated that the kinds of observed NOEs can originate if each of the ten nucleotidyl residues belongs to the B-DNA family, i.e., C2'-endo,anti. However, the degree of observed NOE intensities from the A-T junction as well as the observed AH2-AH2 cross-peaks from adjacent AT pairs could not be rationalized on the basis of a straight B-DNA model but could be explained by only a B-DNA model with some structural discontinuity at the A-T junction--the site of 2-fold symmetry in the molecule. In view of the fact that the degree of observed NOE intensities can be complicated by spin diffusion and by fine structural distortion, we have resorted to the use of quantitative theoretical NOESY simulation (which takes into account primary, secondary, and higher orders of NOE) to delineate the structural discontinuity at the A-T junction and to arrive at a structure for the duplex d(GA4T4C)2. We propose a "junction B-DNA model" which can quantitatively explain the 2D NOESY data at 100- and 50-ms mixing times. In this model the two structural blocks in the molecule, i.e., d(GA4).d(T4C) and d(T4C).d(GA4), are conformationally equivalent and are connected at the A-T junction where the base pairs are stably stacked, but the two local structural frames do not coincide in space. This model can create an overall bending of 10 degrees with a center of curvature 50 A away from the center of the duplex. It is the thesis of this paper that the observed bending in polymers with a repeat of d(GA4T4C)2 and the bending in natural DNAs where AnTn.AnTn repeats are present originate at the oligonucleotide repeat level.     

Quantitative structure of a complex between a minor-groove-specific drug and a bent DNA decamer duplex: use of 2D NMR data and NOESY constrained energy minimization

Two-dimensional nuclear magnetic resonance (2D NMR) studies on d(GA4T4C)2 and d(GT4A4C)2 [Sarma, M.H., et al. (1988) Biochemistry 27, 3423-3432; Gupta G., et al. (1988) Biochemistry 27, 7909-7919] showed that A.T pairs are propeller twisted. As a result, A/T tracts form a straight rigid structural block with an array of bifurcated inter base pair H bonds in the major groove. It was demonstrated (previous paper) that replacement of methyl group by hydrogen (changing from T to U) in the major groove does not disrupt the array of bifurcated H bonds in the major groove. In this article, we summarize results of 2D NMR and molecular mechanic studies on the effect of a minor-groove-binding A.T-specific drug on the structure d(GA4T4C)2. A distamycin analogue (Dst2) was used for this study. It is shown that Dst2 binds to the minor groove of d(GA4T4C)2 mainly driven by van der Waals interaction between A.T pairs and the drug; as a consequence, an array of bifurcated H bonds can be formed in the minor groove between amide/amino protons of Dst2 and A.T pairs of DNA. NOESY data suggest that Dst2 predominantly binds at the central 5 A.T pairs. NOESY data also reveal that, upon drug binding, d(GA4T4C)2 does not undergo any significant change in conformation from the free state; i.e., propeller-twisted A.T pairs are still present in DNA and hence the array of bifurcated H bonds must be preserved in the major groove. NOESY data for the A5-T6 sequence also indicate that there is little change in junction stereochemistry upon drug binding.     

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.     

Cooperativity in A-tract structure and bending properties of composite TnAn blocks

The existence of intrinsically curved DNA molecules incorporating short runs of adenines is undisputed, but none of the current models can explain the entire experimental data set. Recently, Burkhoff and Tullius [Burkhoff, A. M., & Tullius, T. D. (1988) Nature 331, 455-457] offered an explanation for Hagerman's observations on A4T4N2 vs T4A4N2 polymers [Hagerman, P. J. (1986) Nature 321, 449-450], which showed that A4T4N2 multimers migrate anomalously slowly on polyacrylamide gels and T4A4N2 multimers migrate normally. In A4T4N2 multimers Burkhoff and Tullius observe a hydroxy-radical cutting pattern associated with bent DNA and a B-like cutting pattern in T4A4N2. They attribute this difference in cutting pattern to a clash in the TA step of T4A4N2 and suggest that TA4N5 might already adopt an unbent B-DNA conformation [Tullius T. D., & Burkhoff, A. M. (1988) in Structure and Expression. Vol. 3: DNA Bending and Curvature (Olson, W. K., Sarma, M. H., Sarma, R. H., & Sundaralingam, M., Eds.) pp 77-85, Adenine Press, Guilderland, NY]. We show that the conformation adopted by TnAn blocks is similar to that of AnTn blocks. Two A-tract structures of opposite polarity coexist in both blocks. Moreover, we demonstrate a cooperative buildup of a T-tract structure adjacent to an A-tract structure that cannot be predicted by any of the current models. We conclude that AA steps do not assume the same conformation in long tracts of A's as in isolated AA steps. Therefore, the assumption of nearest-neighbor models, that global curvature is an additive phenomenon of local effects, is invalid.     

Theoretical study of the geometrical arrangement of GT and GA wobble pairs in two short duplexes, Proton NMR chemical shifts and interaction energy calculations

NMR shielding constants are calculated for the base protons of duplexes formed by the dodecamer d(CGTGAATTCGCG) and the decamer d(CCAAGATTGG). A good agreement with experimental data is obtained for B-DNA helices in which the wobble GT and GA pairs are in the plane of the corresponding GC pairs of the parent duplexes formed by d(CGCGAATTCGCG) and d(CCAAGCTTGG), if the glycosyl bonds of T and G or A and G are symmetrical with respect to the dyad axis of the Watson-Crick GC pair. Interaction energy calculations show that this type of geometrical arrangement, which implies a distortion of the ribonphosphate backbone of both strands of the duplexes are more stable than those in which only one strand has its conformation modified by the presence of the wobble pair. For the duplex containing the GA pair, NMR chemical shifts as well as interaction energy computations favour the Watson-Crick hydrogen bonding scheme. The variation of the different contributions (intrastrand, interstrand, pair-pair) to the interaction energy between the bases of the duplexes, with the geometrical arrangement of the wobble pairs, is reported.     

DNA with adenine tracts contains poly(dA).poly(dT) conformational features in solution.

The conformation of DNA's with adenine-thymine tracts exhibiting retardation in electrophoretic migration and considered as curved were investigated in solution by CD and RAMAN spectroscopy. The following curved multimers with adenine tracts but of different flanking sequences d(CA5TGCC)n, d(TCTCTA6TATATA5)n, d(GA4T4C)n yield CD spectroscopic features indicating a non-B structure of the dA.dT tract with similarities to polyd(A).polyd(T). We suggest that adenine-thymine bases in these multimers contain some of the distinctive conformational features of poly(A).polyd(T) probably with large propeller twist found by NMR (Behling and Kearns, 1987) and by X-ray diffraction on oligonucleotides containing a tract of adenines (Nelson et al. 1987, Coll et al; 1987; DiGabriele et al. 1989). Some elements of distinctive CD features of the contiguous adenines run are also observed in the straight multi-9-mer d(CA5GCC)n which lacks in-phase relation to the helical repeat. Despite the presence of the TpA step in the straight multimer d(GT4A4)n, the altered dA.dT conformation is not completely destroyed. Interruption of adenine tract by a guanine in d(CAAGAATGCC)n leads to a B-like conformation and to a normal electrophoretic mobility. The Raman spectra reveal a rearrangement of the sugar-phosphate backbone of dA.dT tract in the multimer d(CA5TGCC)n with respect to that of polydA.polydT. This is reflected in the presence of an unique Raman band associated to C2'-endo sugar with a predominant contribution of C1'-exo puckering which is exhibited by the multimer whereas two distinct Raman bands characterize poly(dA).poly(dT) backbone conformation.     

Tracing the origin of HLA-DRB1 alleles by microsatellite polymorphism.

We analyzed the origin of allelic diversity at the class II HLA-DRB1 locus, using a complex microsatellite located in intron 2, close to the polymorphic second exon. A phylogenetic analysis of human, gorilla, and chimpanzee DRB1 sequences indicated that the structure of the microsatellite has evolved, primarily by point mutations, from a putative ancestral (GT)x(GA)y-complex-dinucleotide repeat. In all contemporary DRB1 allelic lineages, with the exception of the human *04 and the gorilla *08 lineages, the (GA)y repeat is interrupted, often by a G-->C substitution. In general, the length of the 3' (GA)y repeat correlates with the allelic lineage and thus evolves more slowly than a middle (GA)z repeat, whose length correlates with specific alleles within the lineage. Comparison of the microsatellite sequence from 30 human DRB1 alleles showed the longer 5' (GT)x to be more variable than the shorter middle (GA)z and 3' (GA)y repeats. Analysis of multiple samples with the same exon sequence, derived from different continents, showed that the 5' (GT)x repeat evolves more rapidly than the middle (GA)z and the 3' (GA)y repeats, which is consistent with findings of a higher mutation rate for longer tracts. The microsatellite-repeat-length variation was used to trace the origin of new DRB1 alleles, such as the new *08 alleles found in the Cayapa people of Ecuador and the Ticuna people of Brazil.     

Tracts of A.T base pairs retard the electrophoretic mobility of short DNA duplexes.

An investigation of the mobility of short duplexes of DNA -octamers and decamers-on polyacrylamide gels is presented, showing that molecules containing less than one helical turn exhibit sequence dependent mobilities. Analysis of chains with different sequences indicates that any arrangement of two or more adjacent A.T base pairs causes a duplex to move more slowly than does any combination of isolated A.T pairs This behavior appears to be an intrinsic property of these sequences, since the anomaly persists in the absence of magnesium or presence of spermine and is not due to strand dissociation. In two decamers we studied, the position of A.T tracts within a duplex can be shown to influence mobility: the sequence GA4T4C associated with bending or curvature of the helix axis when ligated into polymers migrates more slowly than the corresponding sequence GT4A4C, polymers of which migrate as linear B DNA.     

New wrinkles on polynucleotide duplexes

Most fibrous polynucleotides of general sequence exhibit secondary structures that are described adequately by regular helices with a repeated motif of only one nucleotide. Such helices exploit the fact that A:T, T:A, G:C, and C:G pairs are essentially isomorphous and have dyadically-related glycosylic bonds. Polynucleotides with regularly repeated base-sequences sometimes assume secondary structures with larger repeated motifs which reflect these base-sequences. The dinucleotide units of the Z-like forms of poly d(As4T):poly d(As4T), poly d(AC):poly d(GT) and poly d(GC):poly d(GC) are dramatic instances of this phenomenon. The wrinkled B and D forms of poly d(GC):poly d(GC) and poly d(AT):poly d(AT) are just as significant but more subtle examples. It is possible also to trap more exotic secondary structures in which the molecular asymmetric unit is even larger. There is, for example, a tetragonal form of poly d(AT):poly d(AT) which has unit cell dimensions a = b = 1.71nm, c = 7.40nm, gamma = 90 degrees. The c dimension corresponds to the pitch of a molecular helix which accommodates 24 successive nucleotide pairs arranged as a 4(3) helix of hexanucleotide duplexes. The great variety of nucleotide conformations which occur in these large asymmetric units has prompted us to describe them as pleiomeric, a term used in botany to describe whorls having more than the usual number of structures. Pleiomeric DNAs need not contain nucleotide conformations that are very different from one another. On the other hand, DNAs carrying nucleotides of very different conformation must be pleiomeric. This is because 4 nucleotides of different conformation are needed to join patches of secondary structure which are as different as A or B or Z. Differences in nucleotide structures may occur also between chains rather than within chains. In poly d(A):poly d(T), the purine nucleotides all contain C3'-endo furanose rings and the pyrimidine nucleotides C2'-endo rings. Analogous heteronomous structures may exist in DNA-RNA hybrids although these duplexes are also found to have symmetrical A-type conformations.     

Effect of competing self-structure on triplex formation with purine-rich oligodeoxynucleotides containing GA repeats.

Competition between triplex formation with double-stranded DNA and oligonucleotide self-association was investigated in 23mer GA and GT oligonucleotides containing d(GA)5 or d(GT)5 repeats. Whereas triplex formation with GT oligonucleotides was diminished when temperature increased from 4 to 37 degrees C, triplex formation with GA oligonucleotides was enhanced when temperature increased within the same range due to the presence of competing intermolecular GA oligonucleotide self-structure. This self-structure was determined to be a homoduplex stabilized by the internal GA repeats. UV spectroscopy of these homoduplexes demonstrated a single sharp transition with rapid kinetics (Tm = 38.5-43.5 degrees C over strand concentrations of 0.5-4 microM, respectively, with transition enthalpy, delta H = -89 +/- 7 kcal/mol) in 10 mM MgCl2, 100 mM NaCl, pH 7.0. Homoduplex formation was strongly stabilized by multivalent cations (spermine > Mg2+ = Ca2+) and destabilized by low concentrations of monovalent cations (K+ = Li+ = Na+) in the presence of divalent cations. However, unlike GA or GT oligonucleotide-containing triplexes, the homoduplex formed even in the absence of multivalent cations, stabilized by only moderate concentrations of monovalent cations (Li+ > Na+ > K+). Through the development of multiple equilibrium states and the resulting depletion of free oligonucleotide, it was found that the presence of competing self-structure could decrease triplex formation under a variety of experimental conditions.     

Relationship between curved DNA conformations and slow gel migration

We propose some specific DNA conformations that explain, in terms of molecular conformations, the anomalous gel electrophoretic behavior of the sequences (VA4T4X), and (V2A3T3X2)i where V and X are either G or C. Previously (J. Biomole. Struct. Dyn. 4, 41, 1986) we considered hydrophobic interactions among aliphatic hydrocarbon groups in A/T sequences. In the sequences (T)n.(A)n, the T's are slightly bent to yield structures with tightly stacked methyl groups along one side of the major groove. By folding together the two pairs of stacked methyls on the opposite sides of the major groove. TTAA might yield a relatively sharp bend. On this basis, we show below that the sequences (VT4A4X)i might form a very tightly coiled super-helix whereas the sequences (VA4T4X)i form a broad super-helix of radius approximately 120 A for i = 25. The sequence (V2A3T3X2)i forms a slightly smaller radius super-helix. The time of passage through the gel has been taken to be inversely proportional to the smallest dimension of the molecule. Specifically we are taking the ratio of the apparent molecular weight to the actual molecular weight to be related to the moment of inertia I1 about the smallest principal axis of the molecular conformation. We find a good fit to the experimental gel mobility data of Hagerman (2) if we assume this ratio to be proportional to (I1)1/5.     

Effects of the antitumor antibiotic mithramycin on the structure of repetitive DNA regions adjacent to its GC-rich binding site.

Regions of An.Tn, (GA)n.(TC)n, and (GT)n.(AC)n have been cloned into the SmaI (CCC/GGG) site of plasmid pUC19. HindIII-EcoRI restriction fragments containing these inserts have been used as substrates for footprinting experiments using DNase I, DNase II, and micrococcal nuclease as probes. These present good mithramycin binding sites (GGG) flanking repetitive regions to which the drug does not bind. In each case, mithramycin footprints are observed at the CCC/GGG sites, which are not affected by the nature of the surrounding sequences. Some weaker binding is detected at TCGA and ACCA sites and at regions of alternating GA. No binding is found to regions of alternating GT. An.Tn inserts (n = 23 or 69) are normally resistant to cleavage by all these probes; in the presence of mithramycin, a dramatic increase in DNase I cleavage is observed throughout the entire insert and is indicative of an alteration in DNA structure. Similar changes are seen with DNase II and micrococcal nuclease. These changes cannot be explained by invoking changes in the ratio of free substrate to cleavage agent. In contrast, cleavage of (GA)n.(CT)n and (GT)n.(AC)n inserts is not affected by drug binding. The results are consistent with a model in which mithramycin causes dramatic changes in the width of the DNA minor groove, generating a structure which has some properties of A-DNA, and suggest that this can be propagated into surrounding DNA regions in a sequence-dependent manner. The structural alterations with An.Tn are highly cooperative and can be transmitted over at least three turns of the DNA helix.     

Formation, stability and core histone positioning of nucleosomes reassembled on bent and other nucleosome-derived DNA

DNA originating from chicken erythrocyte mononucleosomes was cloned and sequenced. The properties of nucleosome reconstruction were compared for two cloned inserts, selected on account of their interesting sequence organization, length and difference in DNA bending. Cloned fragment 223 (182 base-pairs) carries alternatively (A)3-4 and (T)4-5 runs approximately every ten base-pairs and is bent; cloned fragment 213 (182 base-pairs) contains a repeated C4-5ATAAGG consensus sequence and is apparently not bent. Our experiments indicate the preference of the bent DNA fragment 223 over fragment 213 to associate in vitro with an octamer of histones under stringent conditions. We provide evidence that the in vitro nucleosome formation is hampered in the case of fragment 213, whereas the reconstituted nucleosomes were equally stable once formed. For the correct determination of the positioning of the histone octamer with regard to the two nucleosome-derived cloned DNA sequences, the complementary use of micrococcal nuclease, exonuclease III and DNase I is a prerequisite. No unique, but rotationally related, positions of the histone octamer were found on these nucleosome-derived DNA fragments. The sequence-dependent anisotropic flexibility, as well as intrinsic bending of the DNA, resulting in a rotational setting of the DNA fragments on the histone core, seems to be a strong determinant for the allowed octamer positions, Exonuclease III digestion indicates a different histone-DNA association when oligo(d(C.G)n) stretches are involved. The apparent stagger near oligo(d(A.T)n) stretches generated by DNase I digestion on reconstituted nucleosome 223 was found to be inverted from the normal two-base 3' overhang to a two-base 5' overhang. Two possibilities of the oligo(d(A.T)n) minor groove location relative to the histone core are envisaged to explain this anomaly in stagger.     

NMR studies of pH-dependent conformational polymorphism of alternating (C-T)n sequences.

Alternating (C-T)n sequences are involved in the H-DNA structure associated with (GA)n.(CT)n sequences. Low pH values facilitate H-DNA formation. We have undertaken a detailed analysis of the structural consequences of the (C-T)n sequence as a function of pH. The structures of three DNA oligonucleotides, d(CT)4, d(TC)4 and d(TC)15, have been studied by NMR. We found that their conformations are polymorphic and pH dependent. There are at least three major conformational species: an antiparallel-stranded (APS) duplex with entirely C:T base pairs at pH 7, an antiparallel-stranded (APS) duplex with entirely C+:T base pairs at pH 3, and a possible parallel-stranded (PS) duplex with C+:C and T:T base pairs near pH 5. In the intermediate pH range, the APS duplex may have varying numbers of C+:T and C:T base pairs, and there may be a fast exchange going on between APS duplex species involving these two kinds of base pairs. However, the transition between the APS and PS duplexes is slow. Structural refinement of the two octamers, d(TC)4 and d(CT)4, at pH = 6.9 and pH = 3 using 2D-NOE data suggests that the molecules are likely in the duplex form at 5 degrees C. We lack evidence that the structure at pH 3 is a PS structure with T nucleotides residing in the exterior of the helix. Titration of the longer oligonucleotide, d(TC)15, showed a prominent pKa of approximately 6, approaching the value of 7.0 obtained from the titration of poly-(dC).     

Multi-drug resistance-1 gene polymorphisms in nephrotic syndrome: Impact on susceptibility and response to steroids

Background

Role of multidrug resistance-1 (MDR-1) gene polymorphisms has not been clarified in nephrotic syndrome (NS). Additionally, researchers studied several genetic polymorphisms to explain their influence on different patients' responses to steroid; however the data were inconsistent. Therefore, we aimed to investigate the association of MDR-1 gene polymorphisms [C1236T, G2677T/A, C3435T] and haplotypes with susceptibility to childhood nephrotic syndrome, and whether they influence steroid response.

Methods

We detected MDR-1 gene polymorphisms using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in 138 NS patients and 140 age and sex matched healthy children.

Results

The frequencies of MDR1 G2677T/A GT, GA, TT + AA genotypes or T allele, MDR1 C3435T TT genotype, and T allele genotype frequencies were significantly increased in NS group. While no significant differences were observed in distributions of C1236T genotypes or allele between NS patients and healthy children. Moreover, steroid non-responder NS patients had significantly higher frequencies of MDR1 G2677T/A GT, GA, and TT + AA genotypes than steroid responsive NS patients. We observed also that NS patients with age less than 6 years old had increased frequencies of MDR1 G2677T/A GT, GA, TT + AA genotypes or T allele MDR1 C3435T CT, TT genotypes and T allele. Interestingly the frequency of the TGC haplotype of MDR1 was lower in the initial steroid responders than in non-responders NS patients. On the contrary, there were no any association between the MDR1 haplotypes with NS susceptibility and they did not influence renal pathological findings.

Conclusion

Our data suggested that MDR1 C3435T or G2677T/A gene polymorphisms are risk factors of increased susceptibility, earlier onset of NS, and steroid resistance.     

Minor groove binding DNA ligands with expanded A/T sequence length recognition, selective binding to bent DNA regions and enhanced fluorescent properties

DNA minor groove ligands provide a paradigm for double-stranded DNA recognition, where common structural motifs provide a crescent shape that matches the helix turn. Since minor groove ligands are useful in medicine, new ligands with improved binding properties based on the structural information about DNA-ligand complexes could be useful in developing new drugs. Here, two new synthetic analogues of AT specific Hoechst 33258 5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl] benzimidazole (DMA) and 5-(4-methylpiperazin-1-yl)-2-[2'[2'-(4-hydroxy-3-methoxyphenyl)-5' '-benzimidazolyl]-5'-benzimidazolyl] benzimidazole (TBZ) were evaluated for their DNA binding properties. Both analogues are bisubstituted on the phenyl ring. DMA contains two ortho positioned methoxy groups, and TBZ contains a phenolic group at C-4 and a methoxy group at C-3. Fluorescence yield upon DNA binding increased 100-fold for TBZ and 16-fold for DMA. Like the parent compound, the new ligands showed low affinity to GC-rich (K approximately 4 x 10(7) M(-1)) relative to AT-rich sequences (K approximately 5 x 10(8) M(-1)), and fluorescence lifetime and anisotropy studies suggest two distinct DNA-ligand complexes. Binding studies indicate expanded sequence recognition for TBZ (8-10 AT base pairs) and tighter binding (DeltaT(m) of 23 degrees C for d (GA(5)T(5)C). Finally, EMSA and equilibrium binding titration studies indicate that TBZ preferentially binds highly hydrated duplex domains with altered A-tract conformations d (GA(4)T(4)C)(2) (K= 3.55 x 10(9) M(-1)) and alters its structure over d (GT(4)A(4)C)(2) (K = 3.3 x 10(8) M(-1)) sequences. Altered DNA structure and higher fluorescence output for the bound fluorophore are consistent with adaptive binding and a constrained final complex. Therefore, the new ligands provide increased sequence and structure selective recognition and enhanced fluorescence upon minor groove binding, features that can be useful for further development as probes for chromatin structure stability.