The magnitude and nature of lesion-induced energetic perturbations empirically correlate with mutagenicity/cytotoxicity profiles and can be predictive of lesion outcomes during polymerase-mediated replication in vitro. In this study, we assess the sequence and counterbase-dependent energetic impact of the Thymine glycol (T_g) lesion on a family of deoxyoligonucleotide duplexes. T_g damage arises from thymine and methyl-cytosine exposure to oxidizing agents or radiation-generated free-radicals. The T_g lesion blocks polymerase-mediated DNA replication in vitro and the unrepaired site elicits cytotoxic lethal consequences in vivo. Our combined calorimetric and spectroscopic characterization correlates T_g-induced energetic perturbations with biological and structural properties. Specifically, we incorporate a 5R-T_g isomer centered within the tridecanucleotide sequence 5′-GCGTACXCATGCG-3′ (X = T_g or T) which is hybridized with the corresponding complementary sequence 5′-CGCATGNGTACGC-3′ (N = A, G, T, C) to generate families of T_g-damaged (T_g·N) and lesion-free (T·N) duplexes. We demonstrate that the magnitude and nature of the T_g destabilizing impact is dependent on counterbase identity (i.e., A ∼ G < T < C). The observation that a T_g lesion is less destabilizing when positioned opposite purines suggests that favorable counterbase stacking interactions may partially compensate lesion-induced perturbations. Moreover, the destabilizing energies of T_g·N duplexes parallel their respective lesion-free T·N mismatch counterparts (i.e., G < T < C). Elucidation of T_g-induced destabilization relative to the corresponding undamaged mismatch energetics allows resolution of lesion-specific and sequence-dependent impacts. The T_g-induced energetic perturbations are consistent with its replication blocking properties and may serve as differential recognition elements for discrimination by the cellular repair machinery. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 491–508, 2015.

Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, noncanonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype–phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction-dependent destabilization of the underlying double helix, and a self-structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self-structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size-dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 1–12, 2014.

We describe a novel hybridization assay that employs a unique class of energy tunable, bulge loop-containing competitor strands (C*) that hybridize to a probe strand (P). Such initial "pre-binding" of a probe strand modulates its effective "availability" for hybridizing to a target site (T). More generally, the assay described here is based on competitive binding equilibria for a common probe strand (P) between such tunable competitor strands (C*) and a target strand (T).

We demonstrate that loop variable, energy tunable families of C*P complexes exhibit enhanced discrimination between targets and mismatched targets, thereby reducing false positives/negatives. We refer to a C*P complex between a C* competitor single strand and the probe strand as a “tuning fork,” since the C* strand exhibits branch points (forks) at the duplex-bulge interfaces within the complex. By varying the loop to create families of such “tuning forks,” one can construct C*P “energy ladders” capable of resolving small differences within the target that may be of biological/functional consequence. The methodology further allows quantification of target strand concentrations, a determination heretofore not readily available by conventional hybridization assays. The dual ability of this tunable assay to discriminate and quantitate targets provides the basis for developing a technology we refer to as a “DNA Meter.” Here we present data that establish proof-of-principle for an in solution version of such a DNA Meter. We envision future applications of this tunable assay that incorporate surface bound/spatially resolved DNA arrays to yield enhanced discrimination and sensitivity. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 408–417, 2013.

Acrolein is an α,β-unsaturated aldehyde that is a major environmental pollutant, as well as a product of cellular metabolism. DNA bases react with acrolein to form two regioisomeric exocyclic guanine adducts, namely γ-hydroxy-propanodeoxyguanosine (γ-OH-PdG) and its positional isomer α-hydroxy-propanodeoxyguanosine (α-OH-PdG). The γ-OH-PdG isomer adopts a ring-opened conformation with minimal structural perturbation of the DNA host duplex. Conversely, the α-OH-PdG isomer assumes a ring-closed conformation that significantly disrupts Watson-Crick base-pair alignments within the immediate vicinity of the damaged site. We have employed a combination of calorimetric and spectroscopic techniques to characterize the thermodynamic origins of these lesion-induced structural alterations. Specifically, we have assessed the energetic impact of α-OH-PdG centered within an 11-mer duplex by hybridizing the adduct-containing oligonucleotide with its complementary strand harboring a central base N [where N = C or A], yielding a pair of duplexes containing the nascent lesion (α-OH-PdG·C) or mismatched adduct (α-OH-PdG·A), respectively. Our data reveal that the nascent lesion is highly destabilizing, whereas its mismatched counterpart partially ameliorates α-OH-PdG-induced destabilization. Collectively, our data provide energetic characterizations of the driving forces that modulate error-free versus error-prone DNA translesion synthesis. The biological implications of our findings are discussed in terms of energetically probing acrolein-mediated mutagenicity versus adduct-induced genotoxicity. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 370–382, 2010.

This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Clusters of closely spaced oxidative DNA lesions present challenges to the cellular repair machinery. When located in opposing strands, base excision repair (BER) of such lesions can lead to double strand DNA breaks (DSB). Activation of BER and DSB repair pathways has been implicated in inducing enhanced expansion of triplet repeat sequences. We show here that energy coupling between distal lesions (8oxodG and/or abasic sites) in opposing DNA strands can be modulated by a triplet repeat bulge loop located between the lesion sites. We find this modulation to be dependent on the identity of the lesions (8oxodG vs. abasic site) and the positions of the lesions (upstream vs. downstream) relative to the intervening bulge loop domain. We discuss how such bulge loop-mediated lesion crosstalk might influence repair processes, while favoring DNA expansion, the genotype of triplet repeat diseases. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 355–369, 2010.

This article was originally published online as an acceptedpreprint. The “Published Online” date corresponds to the preprint version. You can reqest a copy of the preprint byemailing the Biopolymers editorial office at biopolymers@wiley.com