Host Duplex Research Articles

Impact of alpha-hydroxy-propanodeoxyguanine adducts on DNA duplex energetics: opposite base modulation and implications for mutagenicity and genotoxicity.

Acrolein is an alpha,beta-unsaturated aldehyde that is a major environmental pollutant, as well as a product of cellular metabolism. DNA bases react with acrolein to form two regioisomeric exocyclic guanine adducts, namely gamma-hydroxy-propanodeoxyguanosine (gamma-OH-PdG) and its positional isomer alpha-hydroxy-propanodeoxyguanosine (alpha-OH-PdG). The gamma-OH-PdG isomer adopts a ring-opened conformation with minimal structural perturbation of the DNA host duplex. Conversely, the alpha-OH-PdG isomer assumes a ring-closed conformation that significantly disrupts Watson-Crick base-pair alignments within the immediate vicinity of the damaged site. We have employed a combination of calorimetric and spectroscopic techniques to characterize the thermodynamic origins of these lesion-induced structural alterations. Specifically, we have assessed the energetic impact of alpha-OH-PdG centered within an 11-mer duplex by hybridizing the adduct-containing oligonucleotide with its complementary strand harboring a central base N [where N = C or A], yielding a pair of duplexes containing the nascent lesion (alpha-OH-PdG.C) or mismatched adduct (alpha-OH-PdG.A), respectively. Our data reveal that the nascent lesion is highly destabilizing, whereas its mismatched counterpart partially ameliorates alpha-OH-PdG-induced destabilization. Collectively, our data provide energetic characterizations of the driving forces that modulate error-free versus error-prone DNA translesion synthesis. The biological implications of our findings are discussed in terms of energetically probing acrolein-mediated mutagenicity versus adduct-induced genotoxicity.

Thermodynamics of Interactions of Water-Soluble Porphyrins with RNA Duplexes

We characterized the interactions of meso-tetrakis(4N-(2-hydroxyethyl)pyridinium-4-yl) porphyrin (TEtOHPyP4), meso-tetrakis(4N-allylpyridinium-4-yl) porphyrin (TAlPyP4), and meso-tetrakis(4N-metallylpyridinium-4-yl) porphyrin (TMetAlPyP4) with the poly(rA)poly(rU) and poly(rI)poly(rC) RNA duplexes between 18 and 45 degrees C by employing circular dichroism, light absorption, and fluorescence intensity spectroscopic measurements. Our results suggest that TEtOHPyP4 and TAlPyP4 intercalate into the poly(rA)poly(rU) and poly(rI)poly(rC) host duplexes, while TMetAlPyP4 associates with these RNA duplexes by forming outside-bound, self-stacked aggregates. We used our temperature-dependent absorption titration data to determine the binding constants and stoichiometry for each porphyrin-RNA binding event studied in this work. From the temperature dependences of the binding constants, we calculated the binding free energies, DeltaG(b), enthalpies, DeltaH(b), and entropies, DeltaS(b). For each RNA duplex, the binding enthalpy, DeltaH(b), is the most favorable for TEtOHPyP4 (an intercalator) followed by TAlPyP4 (an intercalator) and TMetAlPyP4 (an outside binder). On the other hand, for each duplex, external self-stacking of TMetAlPyP4 produces the most favorable change in entropy, DeltaS(b), followed by the intercalators TAlPyP4 and TEtOHPyP4. Thus, our results suggest that the thermodynamic profile of porphyrin-RNA binding may correlate with the binding mode. This correlation reflects the differential nature of molecular forces that stabilize/destabilize the two modes of binding-intercalation versus external self-stacking along the host duplex.

Characterization of the bisintercalative DNA binding mode of a bifunctional platinum-acridine agent

The DNA interactions of PT-BIS(ACRAMTU) ([Pt(en)(ACRAMTU)2](NO3)4; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, en = ethylenediamine), a bifunctional platinum–acridine conjugate, have been studied in native and synthetic double-stranded DNAs and model duplexes using various biophysical techniques. These include ethidium-DNA fluorescence quenching and thermal melting experiments, circular dichroism (CD) spectroscopy and plasmid unwinding assays. In addition, the binding mode was studied in a short octamer by NMR spectroscopy in conjunction with molecular modeling. In alternating copolymers, PT-BIS(ACRAMTU) shows a distinct preference for poly(dA-dT)2, which is ∼3-fold higher than that of ACRAMTU. In the ligand-oligomer complex, d(GCTATAGC)2·PT-BIS(ACRAMTU) (complex I*), PT-BIS(ACRAMTU) increases the thermal stability of the B-form host duplex by ΔTm > 30 K (CD and UV melting experiments). The agent unwinds pSP73 plasmid DNA by 44(±2)° per bound molecule, indicating bisintercalative binding. A 2-D NMR study unequivocally demonstrates that PT-BIS(ACRAMTU)'s chromophores deeply bisintercalate into the 5′-TA/TA base pair steps in I*, while the platinum linker lies in the minor groove. An AMBER model reflecting the NMR results shows that bracketing of the central AT base pairs in a classical nearest neighbor excluded fashion is feasible. PT-BIS(ACRAMTU) inhibits DNA hydrolysis by BstZ17 I at the enzyme's restriction site, GTA↓TAC. Possible consequences for other relevant DNA–protein interactions, such as those involved in TATA-box-mediated transcription initiation and the utility of the platinum-intercalator technology for the design of sequence-specific agents are discussed.

Aminoglycoside complexation with a DNA.RNA hybrid duplex: the thermodynamics of recognition and inhibition of RNA processing enzymes.

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.

Characterization of Parazoanthoxanthin A Binding to a Series of Natural and Synthetic Host DNA Duplexes

Parazoanthoxanthin A is a fluorescent yellow nitrogenous pigment of the group of zoanthoxanthins, which show a broad range of biological activity. These include, among others, the ability to bind to DNA. In this study we have used a variety of spectroscopic (intrinsic fluorescence emission and UV-spectroscopy) and hydrodynamic techniques (viscometry) to characterize in more detail the binding of parazoanthoxanthin A to a variety of natural and synthetic DNA duplexes in different buffer conditions. Our results reveal the following five significant features: (i) Parazoanthoxanthin A exhibits two modes of DNA binding: One binding mode exhibits properties of intercalation, while the second binding mode is predominantly electrostatic in origin. (ii) The apparent binding “site size” for parazoanthoxanthin A near physiological salt concentration (100 mM NaCl) is in the range of 7 ± 1 base pairs for natural genomic DNA duplexes (calf thymus and salmon testes DNA) and alternating synthetic polynucleotides (poly[d(AT)] · poly[d(AT)] and poly[d(GC)] · poly[d(GC)]). A slightly larger apparent binding site size of 9 ± 1 bp was obtained for parazoanthoxanthin A binding to the synthetic homopolymer poly[d(A)] · poly[d(T)]. (iii) Near physiological salt concentration (100 mM NaCl) parazoanthoxanthin A binds with the same approximate binding affinity of 2–5 × 105 M−1 to all DNA polymers studied. (iv) At low salt concentration, parazoanthoxanthin A preferentially binds alternating poly[d(AT)] · poly[d(AT)] and poly[d(GC)] · poly[d(GC)] host duplexes. (v) Parazoanthoxanthin A inhibits DNA polymerase in vitro.

DNA sequence context modulates the impact of a cisplatin 1,2-d(GpG) intrastrand cross-link on the conformational and thermodynamic properties of duplex DNA

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(N H 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.

Cellular uptake, cytotoxicity and DNA-binding studies of the novel imidazoacridinone antineoplastic agent C1311.

C1311 is a novel therapeutic agent with potent activity against experimental colorectal cancer that has been selected for entry into clinical trial. The compound has previously been shown to have DNA-binding properties and to inhibit the catalytic activity of topoisomerase II. In this study, cellular uptake and mechanisms by which C1311 interacts with DNA and exerts cytotoxic effects in intact colon carcinoma cells were investigated. The HT29 colon cancer cell line was chosen to follow cellular distribution of C1311 over a time course of 24 h at drug concentrations that just inhibited cell proliferation by 50% or 100%. Nuclear uptake of C1311 and co-localization with lysosomal or mitochondrial dyes was examined by fluorescence microscopy and effects on these cellular compartments were determined by measurement of acid phosphatase levels, rhodamine 123 release or DNA-binding behaviour. The strength and mode of DNA binding was established by thermal melting stabilization, direct titration and viscometric studies of host duplex length. The onset of apoptosis was followed using a TUNEL assay and DNA-fragmentation to determine a causal relationship of cell death. Growth inhibition of HT29 cells by C1311 was concomitant with rapid drug accumulation in nuclei and in this context we showed that the compound binds to duplex DNA by intercalation, with likely A/T sequence-preferential binding. Drug uptake was also seen in lysosomes, leading to lysosomal rupture and a marked increase of acid phosphatase activity 8 h after exposure to C1311 concentrations that effect total growth inhibition. Moreover, at these concentrations lysosomal swelling and breakdown preceded apoptosis, which was not evident up to 24 h after exposure to drug. Thus, the lysosomotropic effect of C1311 appears to be a novel feature of this anticancer agent. As it is unlikely that C1311-induced DNA damage alone would be sufficient for cytotoxic activity, lysosomal rupture may be a critical component for therapeutic efficacy. © 1999 Cancer Research Campaign

Thermodynamic, kinetic, and conformational properties of a parallel intermolecular DNA triplex containing 5' and 3' junctions.

The interaction of the 11-mer oligodeoxypyrimidine d(TCTTCTUTCCT) with the 17 bp duplex d(CGCTAGAAGAAAGGACG).d(CGTCCUTTCTTCTAGCG) in forming an intermolecular DNA triplex has been examined in solution by surface plasmon resonance (SPR), UV thermal denaturation, circular dichroism (CD), and NMR methods. Thermodynamic data were also acquired for the shorter 15 bp target duplex d(CGCTAGAAGAAAGGA). d(TCCUTTCTTCTAGCG), which forms a 3' flush-ended parallel triplex. CD titrations at pH 5 gave a triplex --> (duplex + strand) dissociation constant Kd of 0.5 microM at 15 degreesC and approximately 2 microM at 25 degreesC for both the 11-15.15 and 11-17.17 systems, in agreement with analysis of the UV melting data and a direct calorimetric measurement. In contrast, the "apparent" Kd value determined by SPR was 10-20-fold smaller. The rate constant for dissociation (kd) of the third strand from the triplex was found to be approximately 0.0002 s-1 at 25 degreesC by SPR. The rate constant for exchange between the triplex and duplex states determined by NMR was approximately 2 s-1 at 40 degreesC. The dissociation kinetics measured by SPR are considerably underestimated, which largely accounts for the poor estimation of Kd using this technique. Extensive 1H NMR assignments were obtained for both the 17 bp DNA duplex and the triplex. Large changes in chemical shifts were observed in the purine strand of the host duplex, but only small shift changes were induced in the complementary pyrimidine strand. Dramatic differences in shifts were observed for the G and A residues, especially in the minor groove, consistent with only small, localized conformational changes in the underlying duplex. The magnitude of the shift changes decreased to baseline within one base of the 3' triplex-duplex junction and over two to three bases at the 5' junction. Chemical shift changes at the 5' junction suggest small conformational anomalies at this site. COSY and NOESY spectra indicate that the nucleotides are in the "S" domain in both the triplex and duplex states. These data rule out major conformation changes at the triplex-duplex boundaries. NOEs between pyrimidines in the third strand and those in the duplex showed proximity for these bases in the major groove, which could be ascribed to buckling of the Hoogsteen bases out of the plane of the Watson-Crick base pairs.

Thermodynamic consequences of an abasic lesion in duplex DNA are strongly dependent on base sequence.

The abasic site in DNA may arise spontaneously, as a result of nucleotide base damage, or as an intermediate in glycosylase-mediated DNA-repair pathways. It is the most common damage found in DNA. We have examined the consequences of this lesion and its sequence context on DNA duplex structure, as well as the thermal and thermodynamic stability of the duplex, including the energetic origins of that stability. To this end, we incorporated a tetrahydrofuran abasic site analogue into a family of 13-mer DNA duplexes, wherein the base opposite the lesion (A, C, G, or T) and the base pairs neighboring the lesion (C.G or G.C) were systematically varied and characterized by a combination of spectroscopic and calorimetric techniques. The resulting data allowed us to reach the following conclusions: (i) the presence of the lesion in all sequence contexts studied does not alter the global B-form conformation characteristic of the parent undamaged duplex; (ii) the presence of the lesion induces a significant enthalpic destabilization of the duplex, with the magnitude of this effect being dependent on the sequence context; (iii) the thermodynamic impact of the lesion is dominated by the identity of the neighboring base pairs, with the cross strand partner base exerting only a secondary thermodynamic effect on duplex properties. In the aggregate, our data reveal that even in the absence of lesion-induced alterations in global structure, the abasic lesion can significantly alter the thermodynamic properties of the host duplex, with the magnitude of this impact being strongly dependent on sequence context.

Minor groove-directed and intercalative ligand-DNA interactions in the poisoning of human DNA topoisomerase I by protoberberine analogs.

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.

Hydration and solution structure of d(CGCAAATTTGCG) 2 and its complex with propamidine from NMR and molecular modelling

The hydration of the d(CGCAAATTTGCG) 2 duplex and its complex with a propamidine reporter ligand has been examined in aqueous solution by two-dimensional NMR at two spectrometer frequencies and three temperatures. Quantitative analysis of ROESY and NOESY cross-peaks showed effective correlation times of ∼0.5 ns at 283 K for DNA-water interactions in the major groove. In some cases the sign of the NOE inverts on changing either the temperature or spectrometer frequency. Larger effective correlation times of ∼1 ns were observed for water interactions with A5(H2) and A6(H2) atoms located in the minor groove. Interproton NOEs and changes in chemical shifts showed that propamidine binds in the minor groove 5′-AATTT region of the host duplex, but does not displace waters adjacent to either A5(H2) or A6(H2). In the complex, the effective correlation times of these waters increase more than two-fold, possibly as a result of stabilisation due to H-bonded interaction with the amidine groups of the ligand. Hydration of the bound molecule was also found, suggesting that water may contribute to the DNA binding process for bis(amidine) drugs. Structure refinement by a NOE-restrained dynamic annealing procedure revealed that ligand binding is non-centrosymmetric with respect to the duplex, in accordance with the energetically favoured 5′-ATT (≡5′-AAT) sites predicted by analytical molecular modelling. In particular, the bound propamidine spans 3-4 base pairs in the A6-T7-T8 tract and makes close H-bonded contacts with A(N3/O4′) acceptors positioned close to the minor groove floor. The refined NMR structure for the DNA-propamidine complex is compared with that determined recently using X-ray crystallographic methods.

Influence of cisplatin intrastrand crosslinking on the conformation, thermal stability, and energetics of a 20-mer DNA duplex.

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.

Stabilization of DNA triple-helix formation by appended cationic peptides.

We have investigated the impact of appended cationic peptides on the triplex-forming and thermal-stabilizing abilities of an oligonucleotide "third strand" using a homopurine-homopyrimidine 37-mer as the host duplex target. The family of appended cationic peptides studied here contains arginine, lysine, ornithine, and diaminobutyric acid, with the residues being linked through either their alpha-amino or side chain amino groups. On the basis of optical melting profiles, we find that peptides with different amino acid compositions, but with four positive charges in common, are able to enhance, in a similar manner, the triplex-forming capability of an oligonucleotide. We note the implications of these results for the rational design of third-strand probes.

Thermodynamic characterization of daunomycin-DNA interactions: Comparison of complete binding profiles for a series of DNA host duplexes

Using a combination of spectroscopic and calorimetric techniques, we have determined complete thermodynamic binding profiles (delta G degree, delta H degree, and delta S degree) for the complexation of daunomycin to a series of 10 polymeric DNA duplexes. We find the resulting drug binding data to be sensitive to the base composition and sequence of the host duplex, with the binding free energies ranging from -7.5 to -10.8 kcal/mol of bound drug and the binding enthalpies ranging from +4.11 to -10.76 kcal/mol of bound drug at 25 degrees C. The smaller range in the free energy term reflects the impact of large enthalpy-entropy compensations. We observe that the three synthetic duplexes which exhibit the highest daunomycin binding affinities all contain GC (or IC) base pairs as part of alternating purine/pyrimidine sequence motifs, with these high binding affinities being strongly enthalpy driven at 25 degrees C. Specific comparisons between the binding profiles for daunomycin complexation with select pairs of host duplexes lead to the following observations: (1) The presence or absence of a major-groove methyl group does not alter daunomycin binding thermodynamics. (2) The presence or absence of a minor-groove amino group does alter daunomycin binding thermodynamics. (3) Duplexes with different base compositions but identical minor-groove functionality exhibit similar daunomycin binding thermodynamics. (4) Homopolymeric duplexes composed of either AT or AU base pairs, but not GC base pairs, exhibit large enthalpy-entropy compensations in their daunomycin binding profiles. We propose interpretations of these and other features of our thermodynamic data in terms of specific daunomycin-DNA interactions deduced from available structural data.

Relative specificities in binding of Watson-Crick base pairs by third strand residues in a DNA pyrimidine triplex motif.

The specificity of binding of Watson-Crick base pairs by third strand nucleic acid residues via triple helix formation was investigated in a DNA pyrimidine triplex motif by thermal melting experiments. The host duplex was of the type A10-X-A10: T10-Y-T10, and the third strand T10-Z-T10, giving rise to 16 possible triplexes with Z:XY inserts, 4 duplexes with the Watson-Crick base pairs (XY) and 12 duplexes with mismatch pairs (XZ), all of whose stabilities were compared. Two Z:XY combinations confirm the primary binding of AT and GC target pairs in homopurine.homopyrimidine sequences by T and C residues, respectively. All other Z:XY combinations in the T:AT environment result in triplex destabilization. While some related observations have been reported, the present experiments differ importantly in that they were performed in a T:AT nearest neighbor environment and at physiological ionic strength and pH, all of which were previously untested. The conclusions now drawn also differ substantially from those in previous studies. Thus, by evaluating the depression in Tm due to base triplet mismatches strictly in terms of third strand residue affinity and specificity for the target base pair, it is shown that none of the triplet combinations that destabilize qualify for inclusion in the third strand binding code for the pyrimidine triplex motif. Hence, none of the mismatch triplets afford a general way of circumventing the requirement for homopurine.homopyrimidine targets when third strands are predominated by pyrimidines, as others have suggested. At the same time, the applicability of third strand binding is emphasized by the finding that triplexes are equally or much more sensitive to base triplet mismatches than are Watson-Crick duplexes to base pair mismatches.

Thermodynamic characterization of daunomycin-DNA interactions: microcalorimetric measurements of daunomycin-DNA binding enthalpies

We report the first direct determination of binding enthalpies for the complexation of monomeric daunomycin with a series of 10 polymeric DNA duplexes. These measurements were accomplished by using a recently developed stopped-flow microcalorimeter capable of detecting reaction heats on the microjoule level. This enhanced sensitivity allowed us to measure daunomycin-DNA binding enthalpies at monomeric drug concentrations (e.g., 10-20 microM), thereby precluding the need to correct for daunomycin self-association, as has been required in previous batch calorimetric studies [Remeta, D. P., Marky, L. A., & Breslauer, K. J. (1984) Abstracts of Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy, 838a; Breslauer, K. J., Remeta, D. P., Chou, W. Y., Ferrante, R., Curry, J., Zaunczkowski, D., Snyder, J. G., & Marky, L. A. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8922-8926]. We correct the published daunomycin-DNA binding enthalpies measured by batch calorimetry at higher drug concentrations (e.g., 0.5-1.0 M) for the enthalpy contribution associated with the binding-induced disruption of drug aggregates. The requisite correction term was obtained from a van't Hoff analysis of temperature-dependent NMR measurements on daunomycin solutions. We find remarkable agreement between the net binding enthalpies derived from these corrected batch calorimetric data and the corresponding binding enthalpies measured directly by stopped-flow microcalorimetry. The enhanced sensitivity of the stopped-flow instrument also allowed us to evaluate the influence of drug binding density on the daunomycin-DNA binding enthalpies. This assessment was accomplished by conducting stopped-flow calorimetric measurements over a range of seven different drug-to-phosphate ratios (r). For most of the 10 DNA host duplexes studied, we find that the daunomycin binding enthalpies exhibit small but significant r dependencies. The sensitivity of the stopped-flow instrument also enabled us to detect significant dilution enthalpies for several of the drug-free DNA duplexes, a quantity generally assumed to be negligible in previous studies. We discuss the binding enthalpies, their dependence on binding density, and the duplex dilution enthalpies in terms of the influence of base composition, sequence, conformation/hydration, and binding cooperativity on the sign and the magnitudes of the daunomycin-DNA binding enthalpy data reported here.

Physical studies of DNA premelting equilibria in duplexes with and without homo dA.cntdot.dT tracts: correlations with DNA bending

We have employed a variety of physical methods to study the equilibrium melting and temperature-dependent conformational dynamics of dA.dT tracts in fractionated synthetic DNA polymers and in well-defined fragments of kinetoplast DNA (kDNA). Using circular dichroism (CD), we have detected a temperature-dependent, "premelting" event in poly(dA).poly(dT) which exhibits a midpoint near 37 degrees C. Significantly, we also detect this CD "premelting" behavior in a fragment of kDNA. By contrast, we do not observe this "premelting" behavior in the temperature-dependent CD spectra of poly[d(AT)].poly[d(AT)], poly(dG).poly(dC), poly[d(GC)].poly[d(GC)], or calf thymus DNA. Thus, poly(dA).poly(dT) and kDNA exhibit a common CD-detected "premelting" event which is absent in the other duplex systems studied in this work. Furthermore, we find that the anomalous electrophoretic retardation of the kDNA fragments we have investigated disappears at temperatures above approximately 37 degrees C. We also observe that the rotational dynamics of poly(dA).poly(dT) and kDNA as assessed by singlet depletion anisotropy decay (SDAD) and electric birefringence decay (EBD) also display a discontinuity near 37 degrees C, which is not observed for the other duplex systems studied. Thus, in the aggregate, our static and dynamic measurements suggest that the homo dA.dT sequence element [common to both poly(dA).poly(dT) and kDNA] is capable of a temperature-dependent equilibrium between at least two helical states in a temperature range well below that required to induce global melting of the host duplex. We suggest that this "preglobal" melting event may correspond to the thermally induced "disruption" of "bent" DNA.

Enthalpy-entropy compensations in drug-DNA binding studies.

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

The thermodynamics of drug-DNA interactions: ethidium bromide and propidium iodide.

We report the first calorimetrically-derived characterization of the thermodynamics of ethidium bromide (EB) and propidium iodide (PI) binding to a series of nucleic acid host duplexes. Our spectroscopic and calorimetric measurements yield the following results: 1) At low salt (16mM Na+) and 25 degrees C. PI binds more strongly than EB to a given host duplex. The magnitude of this PI preference depends only marginally on base sequence, with AT base pairs showing a greater PI preference than GC base pairs. 2) The enhanced binding of PI relative to EB at low salt and 25 degrees C reflects a more favorable entropic driving force for PI binding. 3) The PI binding preference diminishes at higher salt concentrations (216mM). In other words, the binding preference is electrostatic in origin. 4) The salt dependence of the binding constants (delta lnKb/delta ln[Na+]) reveal that PI binds as a dication while EB binds as a monocation. 5) PI and EB both exhibit impressive enthalpy-entropy compensations when they bind to the deoxy homopolymers poly dA.poly dT and poly dA.poly dU. We have observed a similar enthalpy-entropy compensation for netropsin binding to the poly dA.poly dT homopolymer duplex. We therefore conclude that the compensation phenomenon is an intrinsic property of the host duplex rather than reflecting a property of the binding ligand. 6) When either PI or EB bind to the corresponding ribo homopolymer (poly rA.poy rU) we do not observe the enthalpy-entropy compensation that characterizes the binding to the deoxy homopolymer. 7) EB and PI both bind more strongly to poly d(AT).poly d(AT) than to poly d(AU).poly d(AU). Specifically, the absence of the thymine methyl group in poly d(AU).poly d(AU) reduces the binding constant of both drugs by a factor of four. This reduction in binding is due to a less favorable entropy change. In this paper we present and discuss possible molecular origins for our observed thermodynamic and extra-thermodynamic data. In particular, we evoke solvent effects involving both the drugs and the host duplexes when we propose molecular interpretations which are consistent with our thermodynamic data.