A mechanistic study of the stability of triple-stranded DNA structures

Abstract

The DNA triple helix is a non-canonical nucleic acid structure with roles in cellular regulation. Formed when a single strand of nucleic acid, a triplex–forming oligomer (TFO), binds in the duplex major groove, its formation is known to be involved in the onset of the neurodegenerative disorder Friedreich’s ataxia (FRDA). Alternatively, it may have clinical applications in the development of novel antimicrobial agents. As a means to treat FRDA, methods to destabilise triplexes have been examined, with the binding of ligands in the minor groove a promising starting point. On the other hand, stabilisation of these structures may be achieved through modification of the backbone or the bases with the aim of suppressing bacterial genes as an interesting solution to the problem of multi-drug resistance. The origins of triplex stability are currently not well understood and a better comprehension of these properties is therefore an important step in the development of new medicinal technologies involving the creation or destruction of triplexes. In examining the effect of ligand binding on triplex stability, a single minor-groove binder, netropsin, a known triplex destabiliser, is considered. Free energy calculations on the binding of a 15-base TFO to a duplex give good agreement with experiment and it is found that netropsin destabilises this triplex by approximately 15 kcal/mol. This appears to be a highly localised effect, occurring only when netropsin is bound opposite the TFO, and associated with a decrease in the width of the minor groove. Structural distortions associated with netropsin and TFO binding appear, therefore, to play a large role in the ligand’s ability to destabilise triplexes. Although netropsin is thought to bind in the minor groove, binding in the other two grooves formed when a TFO binds, has not been examined. Here it is found that binding in both the minor and W–H, being the larger of the two grooves formed by triplex binding, grooves are similarly stable, with the minor groove potentially being more stable due to strong vdW interactions. To study the effect of TFO composition on triplex stability, the relative stabilities of purine and pyrimidine triplexes are examined. Significant distortion in the TFO backbone is observed in the case of the purine triplex, with Hoogsteen pairs between adenine bases failing to form, indicating it is likely less stable. However, pyrimidine TFO binding requires significantly more backbone rearrangement to bind to the duplex, potentially disfavouring its formation despite it likely being the more stable triplex. Examining the effect of changing the TFO backbone composition from DNA to RNA, for a purine triplex, the two structures are found to have similar stability, despite reports of the RNA purine triplex not forming experimentally. A significant change in the sugar pucker of the purine RNA backbone is found to be required for binding, potentially explaining this lack of formation. However, no large structural differences were found between DNA and RNA pyrimidine triplexes to explain the previously reported greater stability of the RNA structure, suggesting that conformational change may not entirely explain the relative stabilities of the triplexes.