Biomedical subjects

D S Pilch

Publications and source records attributed to D S Pilch.

26 records · Page 2Linked to original sources

PubMed1995

Berenil [1,3-bis(4'-amidinophenyl)triazene] binding to DNA duplexes and to a RNA duplex: evidence for both intercalative and minor groove binding properties.

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.

Binding Sites↗

PubMed1995

The thermodynamics of DNA structures that contain lesions or guanine tetrads.

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.

Animals↗

PubMed1995

Nucleic acid hybridization: triplex stability and energetics.

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.

DNA↗

PubMed1994

Ligand-induced formation of nucleic acid triple helices.

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.

Circular Dichroism↗

PubMed1993

Characterization of a triple helix-specific ligand. BePI (3-methoxy-7H-8-methyl-11- [(3'-amino)propylamino]-benzo[e]pyrido[4,3-b]indole) intercalates into both double-helical and triple-helical DNA.

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.

Base Sequence↗

PubMed1991

Structure, stability, and thermodynamics of a short intermolecular purine-purine-pyrimidine triple helix.

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)

Base Composition↗

PubMed1990

Thermodynamics of triple helix formation: spectrophotometric studies on the d(A)10.2d(T)10 and d(C+3T4C+3).d(G3A4G3).d(C3T4C3) triple helices.

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.

Circular Dichroism↗

PubMed1990

Structural analysis of the (dA)10.2(dT)10 triple helix.

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.

Base Composition↗