RESUMO
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.
Assuntos
DNA/química , Animais , Sequência de Bases , Dano ao DNA , Guanina , Humanos , Dados de Sequência Molecular , Estrutura Molecular , TermodinâmicaRESUMO
We use a combination of spectroscopic, calorimetric, viscometric and computer modeling techniques to characterize the binding of the aminoglycoside antibiotic, tobramycin, to the polymeric RNA duplex, poly(rI).poly(rC), which exhibits the characteristic A-type conformation that is conserved among natural and synthetic double-helical RNA sequences. Our results reveal the following significant features: (i) CD-detected binding of tobramycin to poly(rI).poly(rC) reveals an apparent site size of four base-pairs per bound drug molecule; (ii) tobramycin binding enhances the thermal stability of the host poly(rI).poly(rC) duplex, the extent of which decreases upon increasing in Na(+) concentration and/or pH conditions; (iii) the enthalpy of tobramycin- poly(rI).poly(rC) complexation increases with increasing pH conditions, an observation consistent with binding-induced protonation of one or more drug amino groups; (iv) the affinity of tobramycin for poly(rI).poly(rC) is sensitive to both pH and Na(+) concentration, with increases in pH and/or Na(+) concentration resulting in a concomitant reduction in binding affinity. The salt dependence of the tobramycin binding affinity reveals that the drug binds to the host RNA duplex as trication. (v) The thermodynamic driving force for tobramycin- poly(rI).poly(rC) complexation depends on pH conditions. Specifically, at pH< or =6.0, tobramycin binding is entropy driven, but is enthalpy driven at pH > 6.0. (vi) Viscometric data reveal non-intercalative binding properties when tobramycin complexes with poly(rI).poly(rC), consistent with a major groove-directed mode of binding. These data also are consistent with a binding-induced reduction in the apparent molecular length of the host RNA duplex. (vii) Computer modeling studies reveal a tobramycin-poly(rI). poly(rC) complex in which the drug fits snugly at the base of the RNA major groove and is stabilized, at least in part, by an array of hydrogen bonding interactions with both base and backbone atoms of the host RNA. These studies also demonstrate an inability of tobramycin to form a stable low-energy complex with the minor groove of the poly(rI).poly(rC) duplex. In the aggregate, our results suggest that tobramycin-RNA recognition is dictated and controlled by a broad range of factors that include electrostatic interactions, hydrogen bonding interactions, drug protonation reactions, and binding-induced alterations in the structure of the host RNA. These modulatory effects on tobramycin-RNA complexation are discussed in terms of their potential importance for the selective recognition of specific RNA structural motifs, such as asymmetric internal loops or hairpin loop-stem junctions, by aminoglycoside antibiotics and their derivatives.
Assuntos
Antibacterianos/metabolismo , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Tobramicina/metabolismo , Antibacterianos/química , Pareamento de Bases/efeitos dos fármacos , Pareamento de Bases/genética , Sítios de Ligação , Calorimetria , Dicroísmo Circular , Simulação por Computador , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Modelos Moleculares , Peso Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Prótons , Estabilidade de RNA/efeitos dos fármacos , RNA de Cadeia Dupla/genética , Sódio/farmacologia , Espectrofotometria Ultravioleta , Eletricidade Estática , Especificidade por Substrato , Termodinâmica , Titulometria , Tobramicina/química , ViscosidadeRESUMO
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.
Assuntos
Cisplatino/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , DNA/química , DNA/metabolismo , Conformação de Ácido Nucleico , Pareamento de Bases/genética , Pareamento de Bases/efeitos da radiação , Sequência de Bases , Varredura Diferencial de Calorimetria , Dicroísmo Circular , DNA/genética , DNA/efeitos da radiação , Adutos de DNA/química , Adutos de DNA/genética , Adutos de DNA/metabolismo , Adutos de DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Conformação de Ácido Nucleico/efeitos da radiação , Desnaturação de Ácido Nucleico/efeitos da radiação , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Oligodesoxirribonucleotídeos/efeitos da radiação , Especificidade por Substrato , Temperatura , Termodinâmica , Raios UltravioletaRESUMO
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.
Assuntos
DNA/química , Indóis/química , Substâncias Intercalantes/química , Piridinas/química , Sequência de Bases , DNA/metabolismo , Fluorescência , Indóis/metabolismo , Substâncias Intercalantes/metabolismo , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Piridinas/metabolismo , Termodinâmica , ViscosidadeAssuntos
Antineoplásicos/metabolismo , DNA/metabolismo , Topotecan/metabolismo , Antineoplásicos/farmacologia , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Substâncias Intercalantes/metabolismo , Substâncias Intercalantes/farmacologia , Cinética , Poli dA-dT/metabolismo , Polidesoxirribonucleotídeos/metabolismo , Espectrofotometria/métodos , Topotecan/farmacologiaRESUMO
This unit describes the application of differential scanning and isothermal titration calorimetry (DSC and ITC) to the study of the thermodynamics of nucleic acid structure. DSC is used to study order-disorder transitions. A single DSC profile provides a wealth of thermodynamic and extrathermodynamic information: transition enthalpy, entropy, free energy, heat capacity, the state of the transition (two-state vs. multistate), and the size of the cooperative unit. ITC is used to study hybridization of nucleic acids at constant temperature. Results can be used to determine the stoichiometry of the association reaction, the enthalpy of association, the equilibrium association constant, and the free energy and entropy of association. A thorough discussion is presented of the details required to obtain meaningful results, as well as relevant methods for analyzing the data produced.
Assuntos
Calorimetria/métodos , Ácidos Nucleicos/análise , Varredura Diferencial de CalorimetriaRESUMO
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.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Poli A/química , Poli T/química , RNA/química , Dicroísmo Circular , Diminazena/análogos & derivados , Etídio , Corantes Fluorescentes , Indóis , Netropsina , Espectrofotometria , TermodinâmicaRESUMO
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.
Assuntos
Camptotecina/química , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , DNA/química , DNA/metabolismo , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Sequência de Bases , Camptotecina/farmacologia , Simulação por Computador , Reagentes de Ligações Cruzadas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação ProteicaRESUMO
We have stabilized the d(A)10.2d(T)10 and d(C+LT4C+3).d(G3A4G3).d(C3T4C3) triple helices with either NaCl or MgCl2 at pH 5.5. UV mixing curves demonstrate a 1:2 stoichiometry of purine to pyrimidine strands under the appropriate conditions of pH and ionic strength. Circular dichroic titrations suggest a possible sequence-independent spectral signature for triplex formation. Thermal denaturation profiles indicate the initial loss of the third strand followed by dissociation of the underlying duplex with increasing temperature. Depending on the base sequence and ionic conditions, the binding affinity of the third strand for the duplex at 25 degrees C is two to five orders of magnitude lower than that of the two strands forming the duplex. Thermodynamic parameters for triplex formation were determined for both sequences in the presence of 50 mM MgCl2 and/or 2.0 M NaCl. Hoogsteen base pairs are 0.22-0.64 kcal/mole less stable than Watson-Crick base pairs, depending on ionic conditions and base composition. C+.G and T.A Hoogsteen base pairs appear to have similar stability in the presence of Mg2+ ions at low pH.
Assuntos
Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Cloreto de Magnésio , Estrutura Molecular , Desnaturação de Ácido Nucleico , Cloreto de Sódio , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
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)
Assuntos
DNA/química , Purinas/química , Pirimidinas/química , Composição de Bases , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The existence of DNA triple helices in vitro has been known for some time. Recent evidence suggesting that DNA triplexes exist in vivo and showing their potential for chemotherapeutic applications has renewed interest in these triple-strand conformations. However, little structural information is currently known about these unusual nucleic acid forms. We have induced and stabilized triple-helical (dA)10.2(dT)10 with MgCl2 at neutral pH. UV mixing curves demonstrate a 1:2 (dA)10 to (dT)10 stoichiometry at suitable MgCl2 concentrations. Thermal denaturation profiles establish a melting mechanism characterized by the initial loss of the third strand, followed by dissociation of the remaining duplex. The circular dichroic spectrum of the triplex form is distinct from that of a duplex equimolar in (dA)10. NMR studies show that magnesium-induced triplex formation is accompanied by an upfield shift of several imino proton resonances present before stabilization of the triplex form with MgCl2 and the induction of new upfield imino proton resonances. Nuclear Overhauser effect spectroscopy measurements on both undeuterated and C8--H-deuterated (dA)10.2(dT)10 triplexes demonstrate dipolar contacts between resolvable imino proteins and both adenine C8--H and C2--H aromatic protons. Hence, MgCl2 stabilizes a triplex structure in which thymine N3--H imino protons are involved in both Watson-Crick and Hoogsteen base pairing.
Assuntos
Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , Poli dA-dT , Polidesoxirribonucleotídeos , Composição de Bases , Dicroísmo Circular , Deutério , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Desnaturação de Ácido Nucleico , Espectrofotometria Ultravioleta/métodos , TermodinâmicaRESUMO
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.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA/efeitos dos fármacos , DNA/metabolismo , Nogalamicina/farmacologia , Animais , Pareamento de Bases , Sítios de Ligação , Bovinos , DNA/química , Pegada de DNA , Análise Mutacional de DNA , Ligantes , Conformação de Ácido Nucleico , Especificidade por Substrato , Timo/enzimologiaRESUMO
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)
Assuntos
DNA/química , Diminazena/análogos & derivados , RNA/química , Tripanossomicidas/química , Composição de Bases , Calorimetria , Diminazena/química , Cinética , Substâncias Macromoleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Poli A-U/química , Poli dA-dT/química , Relação Estrutura-Atividade , TermodinâmicaRESUMO
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.
Assuntos
DNA/metabolismo , Diminazena/análogos & derivados , Substâncias Intercalantes/metabolismo , RNA de Cadeia Dupla/metabolismo , Tripanossomicidas/metabolismo , Sítios de Ligação , Calorimetria , Dicroísmo Circular , Diminazena/metabolismo , Temperatura Alta , Espectroscopia de Ressonância Magnética , Modelos Químicos , Desnaturação de Ácido Nucleico , Espectrofotometria Ultravioleta , ViscosidadeRESUMO
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.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , TermodinâmicaRESUMO
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.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Nylons/química , Pareamento Incorreto de Bases , Sítios de Ligação , Dicroísmo Circular , Imidazóis/química , Ligantes , Pirróis/química , Espectrofotometria Ultravioleta , Termodinâmica , beta-Alanina/química , Ácido gama-Aminobutírico/químicaRESUMO
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.
Assuntos
DNA/química , DNA/efeitos dos fármacos , Indóis/farmacologia , Substâncias Intercalantes/farmacologia , Hibridização de Ácido Nucleico/efeitos dos fármacos , RNA/química , RNA/efeitos dos fármacos , Varredura Diferencial de Calorimetria , Fenômenos Químicos , Físico-Química , DNA/metabolismo , Estabilidade de Medicamentos , Corantes Fluorescentes/farmacologia , Calefação , Ligantes , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Poli A/química , Poli A/metabolismo , RNA/metabolismo , Espectrofotometria UltravioletaRESUMO
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.
Assuntos
Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , DNA Topoisomerases Tipo I/metabolismo , DNA/efeitos dos fármacos , DNA/metabolismo , Sequência de Bases , Benzimidazóis/química , Sítios de Ligação , Dicroísmo Circular , DNA/química , Humanos , Técnicas In Vitro , Indóis/metabolismo , Ligantes , Netropsina/metabolismo , Conformação de Ácido Nucleico/efeitos dos fármacos , Especificidade por Substrato , TermodinâmicaRESUMO
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.
Assuntos
Cisplatino/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , DNA/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Desoxiguanosina/análogos & derivados , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , TermodinâmicaRESUMO
5-Phenylterbenzimidazole (1) is active as a topoisomerase I poison (topo I) and is cytotoxic to human tumor cells. No cross-resistance was observed for 1 when it was evaluated against the camptothecin-resistant cell line, CPT-K5. Derivatives of 1 substituted at the 2"-position, however, did exhibit cross-resistance to this cell line. The basis for the resistance of this cell line towards CPT is that it possesses a mutant form of topo I. These results suggest that substituents at the 2"-position may be in proximity to the wild-type enzyme. Therefore, we hypothesized that terbenzimidazoles with 2"-substituents could be capable of interacting with the enzyme and thereby influence activity within this class of topo I poisons. 5-Phenylterbenzimidazoles with a hydroxy, hydroxymethyl, mercapto, amino, N-benzoylaminomethyl, chloro, and trifluoromethyl group at the 2"-position were synthesized. In addition, several 2"-ethyl-5-phenylterbenzimidazoles were prepared containing either a methoxy, hydroxy, amino, or N-acetylamino group at the 2-position of the ethyl side-chain. These 2"-substituted 5-phenylterbenzimidazoles were evaluated as topo I poisons and for cytotoxic activity. The presence of a strong electron-withdrawing group at the 2"-position, such as a chloro or trifluoromethyl group, did enhance both topo I poisoning activity and cytotoxicity. Studies on the relative DNA binding affinity of 1 to its 2"-amino and 2"-trifluoromethyl derivatives did exhibit a correlation with their relative differences in biological activity.