Drug binding to higher ordered DNA structures: netropsin complexation with a nucleic acid triple helix.

Abstract

We have used a combination of spectroscopic and calorimetric techniques to characterize how netropsin, a ligand that binds in the minor groove of DNA, influences the properties of a DNA triple helix. Specifically, our data allow us to reach the following conclusions: (i) netropsin binds to the triplex without displacing the major-groove-bound third strand; (ui) netropsin binding to the triplex exhibits a lower saturation binding density (7.0 base triplets per netropsin bound) than netropsin binding to the corresponding duplex (5.5 base pairs per netropsin bound); (iii) the netropsin-free and the netropsin-bound triplexes each melt in two well-resolved transitions, initial conversion of the triplex to the duplex state followed by duplex melting to the component single-stranded states; (iv) netropsin remains bound to DNA as the triplex melts to the duplex state; (v) netropsin binding thermally destabilizes the triplex = duplex equilibrium dramatically, while thermally stabilizing the duplex to single-strand equilibrium; (vi) netropsin binding to the triplex is enthalpically 4 times more favorable (more exothermic) than netropsin binding to the corresponding duplex; (vi) netropsin binding to the triplex decreases the cooperativity of the triplex --duplex melting event. These results demonstrate that occupancy of the minor groove of a triplex by a ligand such as netropsin can exert a profound impact on the properties of the host triplex, particularly with regard to the equilibrium in which the third strand is expelled from the major groove. Thus, our results reveal considerable major groove/minor groove crosstalk. Such knowledge may prove of practical importance by providing an approach for modulating the affinity and specificity of majorgroove-binding third strands in triplex-forming protocols designed to target specific duplex domains. Fundamentally, our results provide insights into the crosstalk that can result when ligands bind to the two major receptor sites of duplex DNAnamely, the major and minor grooves.Interest in triple helical DNA has been stimulated by the recognition of potential biological roles and/or applications for triple-stranded structures. For example, a structure called H-DNA, which includes a triple helical region, has been proposed to explain the enhanced sensitivity of polypurine-polypyrimidine sequences to chemical cutting agents and to single-strand-specific endonucleases (1-3). Triple strand formation also has been exploited to facilitate the delivery and to enhance the sequence specificity of DNA-cutting reagents (4-6) and drugs (7,8). In addition, triple strand formation has been used to modify enzyme cutting patterns by selectively blocking enzyme binding sites in the major groove (9, 10). In short, appropriately designed and constructed third-strand oligonucleotides that hybridize to targeted duplex domains can be used to control gene expression, to serve as artificial endonucleases in gene mapping strategies, to dictate or modulate the sequence specificity of DNA-binding ...

Drug Binding, Order-Order and Order-Disorder Transitions in DNA Triple Helices

“…Similar results were observed by Park et a!. 10 for dT · dA · dT triplex, each strand having 101 bases (202 base pairs). In their studies the influence ofnetropsin binding in the minor groove of DNA triplex ( Figure 2) was characterized by using spectroscopic and calorimetric techniques.…”

“…4 • 9 -11 • 18 -20 Recently, Plum et al 9 and Park et al 10 reported spectroscopic and calorimetric studies on the DNA triplexes made of Yl5 · U21 · Y21. Here U and Y stand for purine and pyrimidine strands, the subscripts stand for the number of bases (30 and 42 base pairs).…”

“…Our study is based on the model chosen by Plum et al 9 and Park et a!. 10 Their absorbance and heat capacity measurements have been used as experimental data. The Zimm and Bragg theory 26 for helix-coil transition has been suitably amended to explain order-order and order-disorder transitions in DNA triplexes.…”

PKD1 Unusual DNA Conformations Are Recognized by Nucleotide Excision Repair

“…Netropsin binds to the minor groove of A⅐T pairs, requiring four or more such bp for optimal contacts, introduces additional negative supercoils, destabilizes triplex DNA, and increases the stiffness of the double helix (26,(51)(52)(53)(54)(55)(56)(57)(58). We reasoned that netropsin would accentuate cell loss in two ways.…”

Structures of CUG Repeats in RNA

“…The 285 nm maximum is at too long a wavelength for the molecule to be adopting an A-DNA conformation, and the spectra do not contain a positive peak at around 205 nm, which would be expected if the tract were adopting duplex B-DNA structure (29 -31). The negative peak at 208 nm suggests that the tract has triplex character, as a negative peak between 200 and 220 nm is considered a hallmark of triplex DNA (29,30,32). Spectra for triplexes are usually able to be approximated as the sum of the CD for the component duplex and singlestranded DNA plus some extra intensity due to the more rigid structure (33).…”