Bistrand lesions embedded within a single helical turn of tridecameric deoxyoligonucleotide duplexes represent a model system for exploring the impact of clustered lesions that occur in vivo and pose a significant challenge to cellular repair machineries. Such investigations are essential for understanding the forces that dictate lesion‐induced mutagenesis, carcinogenesis, and cytotoxicity within a context that mimics local helical perturbations caused by an ionizing radiation event. This study characterizes the structural and energy profiles of DNA duplexes harboring synthetic abasic sites (tetrahydrofuran, F) as models of clustered bistrand abasic (AP) lesions. The standard tridecameric dGCGTACCCATGCG·dCGCATGGGTACGC duplex is employed to investigate the energetic impact of single and bistrand AP sites by strategically replacing one or two bases within the central CCC/GGG triplet. Our combined analysis of temperature‐dependent UV and circular dichroism (CD) profiles reveals that the proximity and relative orientation of AP sites within bistrand‐damaged duplexes imparts a significant thermodynamic impact. Specifically, 3′‐staggered lesions (CCF/GFG) exert a greater destabilizing effect when compared with their 5′‐counterpart (FCC/GFG). Moreover, a duplex harboring the central bistrand AP lesion (CFC/GFG) is moderately destabilized yet exhibits distinct properties relative to both the 3′ and 5′‐orientations. Collectively, our energetic data are consistent with structural studies on bistrand AP‐duplexes of similar sequence in which a 3′‐staggered lesion exerts the greatest perturbation, a finding that provides significant insight regarding the impact of orientation on lesion repair processing efficiency.

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