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Oxazole-containing macrocycles, which include the natural product telomestatin, represent a promising class of anticancer agents that target G-quadruplex DNA. Two synthetic hexaoxazole-containing macrocyclic compounds (HXDV and HXLV-AC) have been characterized with regard to their cytotoxic activities versus human cancer cells, as well as the mode, thermodynamics, and specificity with which they bind to the intramolecular (3+1) G-quadruplex structural motif formed in the presence of K+ ions by human telomeric DNA. Both compounds exhibit cytotoxic activities versus human lymphoblast (RPMI 8402) and oral carcinoma (KB3-1) cells, with associated IC50 values ranging from 0.4 to 0.9microM. The compounds bind solely to the quadruplex nucleic acid form, but not to the duplex or triplex form. Binding to the quadruplex is associated with a stoichiometry of two ligand molecules per DNA molecule, with one ligand molecule binding to each end of the host quadruplex via a nonintercalative "terminal capping" mode of interaction. For both compounds, quadruplex binding is primarily entropy driven, while also being associated with a negative change in heat capacity. These thermodynamic properties reflect contributions from favorable ligand-induced alterations in the loop configurational entropies of the quadruplex, but not from changes in net hydration. The stoichiometry and mode of binding revealed by our studies have profound implications with regard to the number of ligand molecules that can potentially bind the 3-overhang region of human telomeric DNA.

The synthesis of a 24-membered macrocyclic hexaoxazole via ring-closing metathesis is described. The target compound selectively stabilizes G-quadruplex DNA with no detectable stabilization of duplex DNA. An MTT cytotoxicity assay indicated that this unsaturated macrocyclic hexaoxazole exhibits significant cytotoxicity toward P388, RPMI 8402, and KB3-1 cell lines with IC50 values of 45, 25, and 38 nM, respectively.

Macrocyclic hexaoxazoles having one or two lysinyl side chains in which the terminal nitrogen is either a primary amine, N,N-dimethylamine, or an acetamide have been synthesized. Sodium ion has been found to be beneficial to the macrocyclization step by acting as a template around which the linear polyoxazole can organize. Each of the targeted compounds selectivity stabilizes G-quadruplex versus duplex DNA. Compounds with one valine and one lysine residue display the best combination of G-quadruplex stabilizing ability with no detectable stabilization of duplex DNA.

Oxazole-containing macrocycles represent a promising class of anticancer agents that target G-quadruplex DNA. We report the results of spectroscopic studies aimed at defining the mode, energetics and specificity with which a hexaoxazole-containing macrocycle (HXDV) binds to the intramolecular quadruplex formed by the human telomeric DNA model oligonucleotide d(T2AG3)4 in the presence of potassium ions. HXDV binds solely to the quadruplex nucleic acid form, but not to the duplex or triplex form. HXDV binds d(T2AG3)4 with a stoichiometry of two drug molecules per quadruplex, with these binding reactions being coupled to the destacking of adenine residues from the terminal G-tetrads. HXDV binding to d(T2AG3)4 does not alter the length of the quadruplex. These collective observations are indicative of a nonintercalative 'terminal capping' mode of interaction in which one HXDV molecule binds to each end of the quadruplex. The binding of HXDV to d(T2AG3)4 is entropy driven, with this entropic driving force reflecting contributions from favorable drug-induced alterations in the configurational entropy of the host quadruplex as well as in net hydration. The 'terminal capping' mode of binding revealed by our studies may prove to be a general feature of the interactions between oxazole-containing macrocyclic ligands (including telomestatin) and intramolecular DNA quadruplexes.

The terbenzimidazoles are a class of anticancer agents that bind in the DNA minor groove. These compounds also exhibit a propensity for self-association, which can potentially impact their cellular bioavailabilities and activities. We have explored this possibility by using a broad range of biophysical and cytological techniques to characterize the self-association and cellular uptake properties of two terbenzimidazole analogues, 5-phenylterbenzimidazole (5PTB) and 5-phenyl-2'-(indolo-6-yl)bibenzimidazole (5P2'IBB). Concentration- and temperature-dependent fluorescence spectroscopy, dynamic light scattering, and transmission electron microscopy studies reveal that 5PTB and 5P2'IBB exhibit differing self-association properties. In this connection, 5PTB exhibits an enhanced propensity for self-association and forms larger and more stable aggregates than 5P2'IBB. In addition, the net uptake of 5PTB into human lymphoblast cells is diminished relative to that of 5P2'IBB. These observations suggest that the self-association properties of terbenzimidazoles modulate the cellular bioavailabilities of the compounds, with enhanced self-association propensity and aggregate size leading to reduced cellular bioavailability.

The synthesis of 24-membered macrocycles containing four, six, and seven oxazole moieties is described. Selected compounds were evaluated for their ability to specifically bind and stabilize G-quadruplex DNA and for cytotoxic activity. An unexpected oxidative cleavage reaction afforded a macrocyclic imide that was also evaluated for G-quadruplex stabilizing and cytotoxic activity.

Spectroscopic and calorimetric techniques were employed to characterize and contrast the binding of the aminoglycoside paromomycin to three octamer nucleic acid duplexes of identical sequence but different strand composition (a DNA.RNA hybrid duplex and the corresponding DNA.DNA and RNA.RNA duplexes). In addition, the impact of paromomycin binding on both RNase H- and RNase A-mediated cleavage of the RNA strand in the DNA.RNA duplex was also determined. Our results reveal the following significant features: (i) Paromomycin binding enhances the thermal stabilities of the RNA.RNA and DNA.RNA duplexes to similar extents, with this thermal enhancement being substantially greater in magnitude than that of the DNA.DNA duplex. (ii) Paromomycin binding to the DNA.RNA hybrid duplex induces CD changes consistent with a shift from an A-like to a more canonical A-conformation. (iii) Paromomycin binding to all three octamer duplexes is linked to the uptake of a similar number of protons, with the magnitude of this number being dependent on pH. (iv) The affinity of paromomycin for the three host duplexes follows the hierarchy, RNA.RNA > DNA.RNA >> DNA.DNA. (v) The observed affinity of paromomycin for the RNA.RNA and DNA.RNA duplexes decreases with increasing pH. (vi) The binding of paromomycin to the DNA.RNA hybrid duplex inhibits both RNase H- and RNase A-mediated cleavage of the RNA strand. We discuss the implications of our combined results with regard to the specific targeting of DNA.RNA hybrid duplex domains and potential antiretroviral applications.

Using the X-ray crystal structure of the human topoisomerase I (TOP1)-DNA cleavable complex, we have developed a general model for the ternary drug-DNA-TOP1 cleavable complex formed with camptothecin (CPT) and its analogues. This model has the drug intercalated between the -1 and +1 base pairs, with the E-ring pointing into the minor groove and the A-ring directed toward the major groove. The ternary complex is stabilized by an array of hydrogen bonding and hydrophobic interactions between the drug and both the enzyme and the DNA. Significantly, the proposed model is consistent with the current body of experimental mutation, cross-linking, and structure-activity data. In addition, the model reveals potential sites of interaction that can provide a rational basis for the design of next generation compounds as well as for de novo drug design.

Many DNA binding ligands (e.g., nogalamycin, actinomycin D, terbenzimidazoles, indolocarbazoles, nitidine, and coralyne) and various types of DNA lesions (e.g., UV dimers, DNA mismatches, and abasic sites) are known to stimulate topoisomerase I-mediated DNA cleavage. However, the mechanism(s) by which these covalent and noncovalent DNA interactions stimulate topoisomerase I-mediated DNA cleavage remains unclear. Using nogalamycin as a model, we have studied the mechanism of ligand-induced topoisomerase I-mediated DNA cleavage. We show by both mutational and DNA footprinting analyses that the binding of nogalamycin to an upstream site (from position -6 to -3) can induce highly specific topoisomerase I-mediated DNA cleavage. Substitution of this nogalamycin binding site with a DNA bending sequence (A(5)) stimulated topoisomerase I-mediated DNA at the same site in the absence of nogalamycin. Replacement of the A(5) sequence with a disrupted DNA bending sequence (A(2)TA(2)) significantly reduced the level of topoisomerase I-mediated DNA cleavage. These results, together with the known DNA bending property of nogalamycin, suggest that the nogalamycin-DNA complex may provide a DNA structural bend to stimulate topoisomerase I-mediated DNA cleavage.

Protoberberines represent a structural class of organic cations that induce topoisomerase I-mediated DNA cleavage, a behavior termed topoisomerase I poisoning. We have employed a broad range of biophysical, biochemical, and computer modeling techniques to characterize and cross-correlate the DNA-binding and topoisomerase poisoning properties of four protoberberine analogues that differ with respect to the substituents on their A- and/or D-rings. Our data reveal the following significant features: (i) The binding of the four protoberberines unwinds duplex DNA by approximately 11 degrees, an observation consistent with an intercalative mode of interaction. (ii) Enthalpically favorable interactions, such as stacking interactions between the intercalated ligand and the neighboring base pairs, provide <50% of the thermodynamic driving force for the complexation of the protoberberines to duplex DNA. Computer modeling studies on protoberberine-DNA complexes suggest that only rings C and D intercalate into the host DNA helix, while rings A and B protrude out of the helix interior into the minor groove. (iii) All four protoberberine analogues are topoisomerase I-specific poisons, exhibiting little or no topoisomerase II poisoning activity. (iv) Modifications of the D-ring influence both DNA binding and topoisomerase I poisoning properties. Specifically, transference of a methoxy substituent from the 11- to the 9-position diminishes both DNA binding affinity and topoisomerase I poisoning activity, an observation suggesting that DNA binding is important in the poisoning of topoisomerase I by protoberberines. (v) Modifications of the A-ring have a negligible impact on DNA binding affinity, while exerting a profound influence on topoisomerase I poisoning activity. Specifically, protoberberine analogues containing either 2,3-dimethoxy; 3,4-dimethoxy; or 3, 4-methylenedioxy substituents all bind DNA with a similar affinity. By contrast, these analogues exhibit markedly different topoisomerase I poisoning activities, with these activities following the hierarchy: 3,4-methylenedioxy > 2,3-dimethoxy >> 3, 4-dimethoxy. These differences in topoisomerase I poisoning activity may reflect the differing abilities of the analogues to interact with specific functionalities on the enzyme, thereby stabilizing the enzyme in its cleavable state. In the aggregate, our results are consistent with a mechanistic model in which both ligand-DNA and ligand-enzyme interactions are important for the poisoning of topoisomerase I by protoberberines, with the DNA-directed interactions involving ring D and the enzyme-directed interactions involving ring A. It is reasonable to suggest that the poisoning of topoisomerase I by a broad range of other naturally occurring and synthetic ligands may entail a similar mechanism.

The anticancer activity of cisplatin derives from its ability to bind and cross-link DNA, with the major adduct being the 1,2-d(GpG) intrastrand cross-link. Here, the consequences of this adduct on the conformation, thermal stability, and energetics of duplex DNA are assessed, and the modulation of these parameters by the sequence context of the adduct is evaluated. The properties of a family of 15-mer DNA duplexes containing a single 1,2-d(GpG) cis-¿Pt(NH(3))(2)¿(2+) intrastrand cross-link are probed in different sequence contexts where the flanking base-pairs are systematically varied from T.A to C.G to A.T. By using a combination of spectroscopic and calorimetric techniques, the structural, thermal, and thermodynamic properties of each duplex, both with and without the cross-link, are characterized. Circular dichroism spectroscopic data reveal that the cross-link alters the structure of the host duplex in a manner consistent with a shift from a B-like to an A-like conformation. Thermal denaturation data reveal that the cross-link induces substantial thermal and thermodynamic destabilization of the host duplex. Significantly, the magnitudes of these cross-link-induced effects on duplex structure, thermal stability, and energetics are influenced by the bases that flank the adduct. The presence of flanking A.T base-pairs, relative to T.A or C.G base-pairs, enhances the extent of cross-link-induced alteration to an A-like conformation and dampens the extent of cross-link-induced duplex destabilization. These results are discussed in terms of available structural data, and in terms of the selective recognition of cisplatin-DNA adducts by HMG-domain proteins., (Copyright 2000 Academic Press.)

Crescent-shaped synthetic ligands containing aromatic amino acids have been designed for specific recognition of predetermined DNA sequences in the minor groove of DNA. Simple rules have been developed that relate the side-by-side pairings of Imidazole (Im) and Pyrrole (Py) amino acids to their predicted target DNA sequences. We report here thermodynamic characterization of the DNA-binding properties of the six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp (where gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide). Our data reveal that, at 20 degrees C, this ligand binds with a relatively modest 1.8-fold preference for the designated match site, 5'-TGGTA-3', over the single base pair mismatch site, 5'-TGTTA-3'. By contrast, we find that the ligand exhibits a 102-fold greater affinity for its designated match site relative to the double base pair mismatch site, 5'-TATTA-3'. These results demonstrate that the energetic cost of binding to a double mismatch site is not necessarily equal to twice the energetic cost of binding to a single mismatch site. Our calorimetrically measured binding enthalpies and calculated entropy data at 20 degrees C reveal the ligand sequence specificity to be enthalpic in origin. We have compared the DNA-binding properties of ImImPy-gamma-PyPyPy-beta-Dp with the hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (an Im --> Py "mutant"). Our data reveal that both ligands exhibit high affinities for their designated match sites, consistent with the Dervan pairing rules. Our data also reveal that, relative to their corresponding single mismatch sites, ImImPy-gamma-PyPyPy-beta-Dp is less selective than ImPyPy-gamma-PyPyPy-beta-Dp for its designated match site. This result suggests, at least in this case, that enhanced binding affinity can be accompanied by some loss in sequence specificity. Such systematic comparative studies allow us to begin to establish the thermodynamic database required for the rational design of synthetic polyamides with predictable DNA-binding affinities and specificities.

The terbenzimidazoles are a class of synthetic ligands that poison the human topoisomerase I (TOP1) enzyme and promote cancer cell death. It has been proposed that drugs of this class act as TOP1 poisons by binding to the minor groove of the DNA substrate of TOP1 and altering its structure in a manner that results in enzyme-mediated DNA cleavage. To test this hypothesis, we characterize and compare the binding properties of a 5-phenylterbenzimidazole derivative (5PTB) to the d(GA4T4C)2 and d(GT4A4C)2 duplexes. The d(GA4T4C)2 duplex contains an uninterrupted 8-bp A.T domain, which, on the basis of x-ray crystallographic data, should induce a highly hydrated "A-tract" conformation. This duplex also exhibits anomalously slow migration in a polyacrylamide gel, a feature characteristic of a noncanonical global conformational state frequently described as "bent." By contrast, the d(GT4A4C)2 duplex contains two 4-bp A.T tracts separated by a TpA dinucleotide step, which should induce a less hydrated "B-like" conformation. This duplex also migrates normally in a polyacrylamide gel, a feature further characteristic of a global, canonical B-form duplex. Our data reveal that, at 20 degrees C, 5PTB exhibits an approximately 2. 3 kcal/mol greater affinity for the d(GA4T4C)2 duplex than for the d(GT4A4C)2 duplex. Significantly, we find this sequence/conformational binding specificity of 5PTB to be entropic in origin, an observation consistent with a greater degree of drug binding-induced dehydration of the more solvated d(GA4T4C)2 duplex. By contrast with the differential duplex affinity exhibited by 5PTB, netropsin and 4',6-diamidino-2-phenylindole (DAPI), two AT-specific minor groove binding ligands that are inactive as human TOP1 poisons, bind to both duplexes with similar affinities. The electrophoretic behaviors of the ligand-free and ligand-bound duplexes are consistent with 5PTB-induced bending and/or unwinding of both duplexes, which, for the d(GA4T4C)2 duplex, is synergistic with the endogenous sequence-directed electrophoretic properties of the ligand-free duplex state. By contrast, the binding to either duplex of netropsin or DAPI induces little or no change in the electrophoretic mobilities of the duplexes. Our results demonstrate that the TOP1 poison 5PTB binds differentially to and alters the structures of the two duplexes, in contrast to netropsin and DAPI, which bind with similar affinities to the two duplexes and do not significantly alter their structures. These results are consistent with a mechanism for TOP1 poisoning in which drugs such as 5PTB differentially target conformationally distinct DNA sites and induce structural changes that promote enzyme-mediated DNA cleavage.

The effect of DNA binding on poisoning of human DNA TOP1 has been studied using a pair of related anthracyclines which differ only by a nogalose sugar ring. We show that the nogalose sugar ring of nogalamycin, which binds to the minor groove of DNA, plays an important role in affecting topoisomerase-specific poisoning. Using purified mammalian topoisomerases, menogaril is shown to poison topoisomerase II but not topoisomerase I. By contrast, nogalamycin poisons topoisomerase I but not topoisomerase II. Consistent with the biochemical studies, CEM/VM-1 cells which express drug-resistant TOP2alpha are cross-resistant to menogaril but not nogalamycin. The mechanism by which nogalamycin poisons topoisomerase I has been studied by analyzing a major topoisomerase I-mediated DNA cleavage site induced by nogalamycin. This site is mapped to a sequence embedded in an AT-rich region with four scattered GC base pairs (bps) (at -10, -6, +2, and +12 positions). GC bps embedded in AT-rich regions are known to be essential for nogalamycin binding. Surprisingly, DNase I footprinting analysis of nogalamycin-DNA complexes has revealed a drug-free region from -2 to +9 encompassing the major cleavage site. Our results suggest that nogalamycin, in contrast to camptothecin, may stimulate TOP1 cleavage by binding to a site(s) distal to the site of cleavage.

Spectroscopic, calorimetric, DNA cleavage, electrophoretic, and computer modeling techniques have been employed to characterize the DNA binding and topoisomerase poisoning properties of three protoberberine analogs, 8-desmethylcoralyne (DMC), 5,6-dihydro-8-desmethylcoralyne (DHDMC), and palmatine, which differ in the chemical structures of their B- and/or D-rings. DNA topoisomerase-mediated cleavage assays revealed that these compounds were unable to poison mammalian type II topoisomerase. By contrast, the three protoberberine analogs poisoned human topoisomerase I according to the following hierarchy: DHDMC > DMC > palmatine. DNA binding by all three protoberberine analogs induced negative flow linear dichroism signals as well as unwinding of the host duplex. These two observations are consistent with an intercalative mode of protoberberine binding to duplex DNA. However, a comparison of the DNA binding properties for DMC and DHDMC, which differ only by the state of saturation at the 5,6 positions of the B-ring, revealed that the protoberberine analogs do not "behave" like classic DNA intercalators. Specifically, saturation of the 5-6 double bond in the B-ring of DMC, thereby converting it to the DHDMC molecule, was associated with enhanced DNA unwinding as well as a reversal of DNA binding preference from a DNA duplex with an inaccessible or occluded minor groove {poly[d(G-C)]2} to DNA duplexes with accessible or unobstructed minor grooves {poly[d(A-T)]2 and poly[d(I-C)]2}. In addition, a comparison of the DNA binding properties for DHDMC and palmatine revealed that transferring the 11-methoxy moiety on the D-ring of DHDMC to the 9 position, thereby converting it to palmatine, was associated with a reduction in binding affinity for both duplexes with unobstructed minor grooves as well as for duplexes with occluded minor grooves. These DNA binding properties are consistent with a "mixed-mode" DNA binding model for protoberberines in which a portion of the ligand molecule intercalates into the double helix, while the nonintercalated portion of the ligand molecule protrudes into the minor groove of the host duplex, where it is thereby available for interactions with atoms lining the floor and/or walls of the minor groove. Furthermore, saturation at the 5,6 positions of the B-ring, which causes the A-ring to be tilted relative to the plane formed by rings C and D, appears to stabilize the interaction between the host duplex and the minor groove-directed portion of the protoberberine ligand. Computer modeling studies on the DHDMC-poly[d(A-T)]2 complex suggest that this interaction may involve van der Waals contacts between the ligand A-ring and backbone sugar atoms lining the minor groove of the host duplex. The hierarchy of topoisomerase I poisoning noted above suggests that this minor groove-directed interaction may play an important role in topoisomerase I poisoning by protoberberine analogs. In the aggregate, our results presented here, coupled with the recent demonstration of topoisomerase I poisoning by minor groove-binding terbenzimidazoles [Sun, Q., Gatto, B., Yu, C., Liu, A. , Liu, L. F., & LaVoie, E. J. (1995) J. Med. Chem. 38, 3638-3644], suggest that minor groove-directed ligand-DNA interactions may be of general importance in the poisoning of topoisomerase I.

The aromatic diamidine, DAPI (4',6-diamidino-2-phenylindole), is used as an important biological and cytological tool since it forms highly fluorescent complexes with nucleic acid duplexes via minor groove-directed/intercalative modes of interaction. In this study, we find that DAPI binding can induce the formation of an RNA-DNA hybrid triplex that would not otherwise form. More specifically, through application of a broad range of spectroscopic, viscometric, and molecular modeling techniques, we demonstrate that DAPI intercalation induces the formation of the poly(dT).poly(rA).poly(dT) hybrid triple helix, a structure which does not form in the absence of the ligand. Using UV mixing studies, we demonstrate that, in the presence of DAPI, the poly(rA).poly(dT) duplex and the poly(dT) single strand form a 1:1 complex (a triplex) that does not form in the absence of DAPI. Through temperature-dependent absorbance measurements, we show that the poly(dT).poly(rA).poly(dT) triplex melts via two distinct transitions: initial conversion of the triplex to the duplex state, with the DAPI remaining bound, followed by denaturation of the duplex-DAPI complex to its component single strands and free DAPI. Using optical melting profiles, we show that DAPI binding enhances the thermal stability of the poly(dT).poly(rA).poly(dT) triplex, an observation consistent with the preferential binding of the ligand to the triplex versus the duplex and single-stranded states. Our differential scanning calorimetric measurements reveal melting of the DAPI-saturated poly(dT).poly(rA).poly(dT) triplex to be associated with a lower enthalpy but greater cooperativity than melting of the corresponding DAPI-saturated poly(rA).poly(dT) duplex. Our flow linear dichroism and viscometric data are consistent with an intercalative mode of binding when DAPI interacts with both the poly(dT).poly(rA).poly(dT) triplex and the poly(rA).poly(dT) duplex. Finally, computer modeling studies suggest that a combination of both stacking and electrostatic interactions between the intercalated ligand and the host nucleic acid play important roles in the DAPI-induced stabilization of the poly(dT).poly(rA).poly(dT) triplex. In the aggregate, our results demonstrate that ligand binding can be used to induce the formation of triplex structures that do not form in the absence of the ligand. This triplex-inducing capacity has potentially important implications in the design of novel antisense, antigene, antiviral, and diagnostic strategies.

Hairpin polyamides are synthetic ligands for sequence-specific recognition in the minor groove of double-helical DNA. A thermodynamic characterization of the DNA-binding properties exhibited by a six-ring hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (where Im = imidazole, Py = pyrrole, gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide), reveals an approximately 1-2 kcal/mol greater affinity for the designated match site, 5'-TGTTA-3', relative to the single base pair mismatch sites, 5'-TGGTA-3' and 5'-TATTA-3'. The enthalpy and entropy data at 20 degrees C reveal this sequence specificity to be entirely enthalpic in origin. Correlations between the thermodynamic driving forces underlying the sequence specificity exhibited by ImPyPy-gamma-PyPyPy-beta-Dp and the structural properties of the heterodimeric complex of PyPyPy and ImPyPy bound to the minor groove of DNA provide insight into the molecular forces that govern the affinity and specificity of pyrrole-imidazole polyamides.

cis-Diamminedichloroplatinum(II) (cisplatin) is a widely used anticancer drug that binds to and crosslinks DNA. The major DNA adduct of the drug results from coordination of two adjacent guanine bases to platinum to form the intrastrand crosslink cis-[Pt(NH3)2[d(GpG)-N7(1), -N7(2)]] (cis-Pt-GG). In the present study, spectroscopic and calorimetric techniques were employed to characterize the influence of this crosslink on the conformation, thermal stability, and energetics of a site-specifically platinated 20-mer DNA duplex. CD spectroscopic and thermal denaturation data revealed that the crosslink alters the structure of the host duplex, consistent with a shift from a B-like to an A-like conformation; lowers its thermal stability by approximately 9 degrees C; and reduces its thermodynamic stability by 6.3 kcal/mol at 25 degrees C, most of which is enthalpic in origin; but it does not alter the two-state melting behavior exhibited by the parent, unmodified duplex, despite the significant crosslink-induced changes noted above. The energetic consequences of the cis-Pt-GG crosslink are discussed in relation to the structural perturbations it induces in DNA and to how these crosslink-induced perturbations might modulate protein binding.

Berenil is an antitrypanosomal agent that binds to nucleic acid duplexes. Recently, we reported that this drug can bind to both DNA and RNA duplexes, while exhibiting properties characteristic of both intercalation and groove binding [Pilch, D. S., Kirolos, M. A., Liu, X., Plum, G. E., & Breslauer, K. J. (1995) Biochemistry 34, 9962-9976]. In this work, we use spectroscopic, calorimetric, and hydrodynamic techniques to demonstrate that berenil also can bind to DNA and RNA triplexes. Our results reveal the following significant features: (i) Berenil binds to the poly(dA).2poly(dT) DNA triplex and to the poly(rA).2poly(rU) RNA triplex without displacing the major groove-bound third strands. (ii) Both berenil-bound triplexes melt via two distinct transitions: initial conversion of the triplex to the duplex state, with the berenil remaining bound, followed by denaturation of the duplex to its component single strands. (iii) The magnitude and even the direction of the impact of berenil binding on the thermal stability of the DNA triplex depends on both the Na+ concentration and the drug binding density (the [base triplet]/[total berenil] ratio). Specifically, at Na+ concentrations < or = 0.08 M, the DNA triplex to duplex transition is thermally stabilized, while at Na+ concentrations > or = 0.125 M it is thermally destabilized. Between these two salt concentrations, berenil binding either enhances or diminishes the thermal stability of the DNA triplex in a manner that depends on the [base triplet]/[total berenil] ratio. (iv) The effect of berenil binding on the thermal stability of the RNA triplex to duplex equilibrium also depends on the [base triplet]/[total berenil] ratio, having a weakly destabilizing effect on this equilibrium at [base triplet]/[total berenil] ratios > 5, while thermally stabilizing this equilibrium at [base triplet]/[total berenil] ratios < 5. (v) The apparent "site sizes" associated with berenil binding to the triplexes range from approximately 1 to 12 base triplets per bound berenil and depend, in part, on the host triplex. One of the site sizes common to both triplexes is consistent with berenil binding to the minor groove. (vi) Berenil exhibits a higher apparent binding affinity for the DNA triplex relative to the RNA triplex. (vii) Viscometric data reveal nonintercalative binding properties when berenil complexes with both triplexes, consistent with a minor groove binding mode. (viii) Berenil binding to either the DNA or the RNA triplex is enthalpically more favorable than berenil binding to the corresponding duplex. (ix) Berenil binding to both triplexes decreases the cooperativity of the triplex to duplex melting event.(ABSTRACT TRUNCATED AT 400 WORDS)

Berenil is an antitrypanosomal agent that binds to nucleic acid duplexes. The generally accepted mode of berenil binding is via complexation into the minor groove of AT-rich domains of DNA double helices. We find that berenil can bind to RNA as well as DNA duplexes, while exhibiting properties characteristic of both intercalation as well as minor groove binding. More specifically, we use spectroscopic, calorimetric, and hydrodynamic techniques to characterize berenil binding to four DNA duplexes and to one RNA duplex. Our results reveal the following features: (i) Berenil binding to the poly[d(A-T)]2, poly(dA).poly(dT), poly[d(I-C)]2, poly[d(G-C)]2, and poly(rA).poly(rU) duplexes exhibits intercalative as well as minor groove binding characteristics. (ii) The apparent "site sizes" associated with berenil binding to these five duplexes range from 1 to 13 base pairs per bound berenil and depend, in part, on the host duplex. One of the site sizes common to all five duplexes is consistent with berenil binding to the minor groove. (iii) The apparent berenil binding affinity follows the hierarchy: poly(dA).poly(dT) > poly-[d(A-T)]2 approximately poly[d(I-C)]2 >> poly(rA).poly(rU) > poly[d(G-C)]2. (iv) Viscometric data reveal properties characteristic of a significant contribution from an intercalative mode of binding when berenil interacts with the poly[d(A-T)]2, poly[d(I-C)]2, poly[d(G-C)]2, and poly(rA).poly(rU) duplexes, while revealing an apparent nonintercalative mode when the drug binds to the poly(dA).poly(dT) duplex. (v) Berenil binding unwinds negative supercoils in the pBR322 plasmid, an observation consistent with an intercalative mode of binding to duplex DNA. (vi) Salt-dependent melting data suggest that both positively charged amidino groups of berenil participate in the complexation of the drug to the poly[d(I-C)]2, poly[d(A-T)]2, poly(dA).poly(dT), and poly(rA).poly(rU) duplexes, while also suggesting that the binding event is site-specific. In the aggregate, our results suggest that, in contrast to the conventional wisdom, berenil can exhibit intercalative as well as minor groove binding properties when it binds to both DNA and RNA duplexes, while also exhibiting a preference for DNA duplexes with unobstructed minor grooves. We comment on the potential correlation between drugs, such as berenil, that exhibit "mixed" binding motifs and those that express anticancer activity via inhibition of topoisomerase I activity.

It is becoming increasingly apparent that energetic as well as structural information is required to develop a complete appreciation of the critical interrelationships between structure, energetics, and biological function. Motivated by this recognition, we have reviewed in this article the current state of the thermodynamic databases associated with lesion-containing DNA duplexes and DNA quadruplexes, while highlighting important considerations concerning the methods used to obtain the requisite data.

In this chapter, we review the current state of the thermodynamic database for triple helical oligonucleotide hybridization reactions and present a critical assessment of the methods used to obtain the relevant data. The thermodynamic stability of triple-helix oligonucleotide constructs is discussed in terms of its dependence on temperature, chain length, pH, salt, base sequence, base and backbone modifications, and ligand binding. In particular, we examine the coupling of hybridization equilibria to proton, cation, and drug-binding equilibria. Throughout the chapter, we emphasize that a detailed understanding of the endogenous and exogenous variables that control triplex stability is required for the rational design of oligonucleotides for specific therapeutic, diagnostic, and/or biotechnological applications, as well as for elucidating the potential cellular roles of these higher-order nucleic acid complexes.

We demonstrate that ligand binding can be used to induce the formation of triplex structures that would not otherwise form. Specifically, we show that binding of berenil or 4',6-diamidino-2-phenylindole DAPI) induces formation of the poly(rA).poly(rA).poly(dT) triplex, providing an example of an RNA(purine).RNA(purine).DNA(pyrimidine) triplex. We also show that binding of berenil, DAPI, ethidium, or netropsin can induce formation of the poly(dT).poly(rA).poly(dT) triplex, thereby overcoming a practical limitation to the formation of DNA.RNA.DNA triplexes with a purine RNA strand. Based on the enhanced thermal stabilities of the drug-bound poly(dT).poly(rA).poly(dT) complexes at 18 mM Na+, we define the relative triplex-inducing efficiencies of these four ligands to be: berenil > DAPI > ethidium > netropsin. Our results demonstrate that ligand binding can be used to induce the formation of triplex structures that do not form in the absence of the ligand. This triplex-inducing capacity has potentially important implications in the design of novel antisense, antigene, and diagnostic strategies.

A benzo[e]pyridoindole derivative, 3-methoxy-7H-8-methyl-11-[(3'-amino)propylamino] -benzo[e]pyrido[4,3-b]indole (BePI), and its interactions with double and triple-helical DNA have been investigated by a variety of fluorescence, spectrophotometric, hydrodynamic and molecular modeling techniques. Binding to DNA stabilizes the doubly charged (+2) form of BePI, increasing the apparent pKa of the 10-NH proton by approximately 1 pH unit. Binding to DNA also quenches the fluorescence of BePI, with a greater extent of quenching upon binding triplex relative to duplex DNA. BePI preferentially binds (and stabilizes) triple-helical relative to double-helical DNA. This preferential binding is not restricted to triplexes containing solely T x A.T base triplets. In addition, BePI preferentially stabilizes the poly(dA).poly(dT) relative to the poly[d(A-T)].poly[d(A-T)] duplex. Viscosity studies demonstrate that, upon binding, BePI induces the unwinding of negative supercoils in the pBR322 plasmid, and increases the relative contour lengths of double and triple-helical polydeoxynucleotides. Fluorescence studies reveal that energy transfer occurs from polynucleotide bases to bound BePI molecules in both BePI/duplex and BePI/triplex complexes. In a BePI/triplex complex, an average of 4.8 bases appear to transfer excitation energy totally to a bound BePI molecule, while in various BePI/duplex complexes an average of only 2.5 bases appear to do so, indicating that energy transfer is more efficient in the former complex. Measurements of fluorescence quenching indicate that BePI is protected from quenching by acrylamide when bound to either double or triple-helical polynucleotides. The viscosity and fluorescence behavior of BePI are fully consistent with the conclusion that BePI intercalates into both double and triple-helical DNA. Molecular modeling studies suggest that stronger stacking interactions between intercalated BePI and adjacent bases in BePI/triplex relative to BePI/duplex complexes may account for the enhanced thermal stability of the former complex.

We have investigated the structure and physical chemistry of the d(C3T4C3).2[d(G3A4G3)] triple helix by polyacrylamide gel electrophoresis (PAGE), 1H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl2 at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur.pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the purine strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 degrees C, depending on the DNA concentration. The free energy of triplex formation (-26.0 +/- 0.5 kcal/mol) is approximately twice that of duplex formation (-12.6 +/- 0.7 kcal/mol), suggesting that the overall stability of the pur.pur base pairs is similar to that of the W-C base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)