Characterization of DNA with an 8-oxoguanine modification

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2′-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2–8 kcal mol−1 over a 16–116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoG•C is fundamentally different than that found at G•C. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoG•C pairing and those base pairs that are 5′ of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding

Unfolding and Binding Thermodynamics of the PREQ1 DNA Analog

The PreQ1 riboswitch is a subset of riboswitches found mainly in eubacteria. It plays an essential role in the biosynthesis of preQ1, a precursor to queuosine (Q). Q is a hypermodified guanine nucleotide that is universally found at the wobble position of certain tRNAs. PreQ1 forms a pseudoknot structure upon binding to preQ1. We use a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry (DSC) to determine the unfolding thermodynamics of a DNA analog (PREQ1) and its control hairpin at two different salt concentrations, 16 mM and 116 mM Na+. Furthermore, we use DSC and fluorescence spectroscopy techniques to determine binding affinities, Kb, for the interaction of preq1 ligand with these DNA analogs. Both oligonucleotides unfold with TMs independent of strand concentration, indicating the formation of hairpin structures. The unfavorable unfolding free energy terms resulted from the typical compensation of an unfavorable enthalpy contributions (disruption of base-pair stacks) and favorable entropy contributions (release of ions and water molecules). The ligand preQ1 yielded a Kb of 5.7 x 105. Supported by NSF Grant (MCB-1912587).https://digitalcommons.unmc.edu/surp2021/1059/thumbnail.jp

Melting behavior and ligand binding of DNA intramolecular secondary structures

We use a variety of biophysical techniques to determine thermodynamic profiles, including hydration, for the unfolding of DNA stem-loop motifs (hairpin, a three-way junction and a pseudoknot) and their interaction with netropsin and random cationic copolymers. The unfolding thermodynamic data show that their helix–coil transition takes place according to their melting domains or sequences of their stems. All hairpins adopted the B-like conformation and their loop(s) contribute with an immobilization of structural water. The thermodynamic data of netropsin binding to the ^5′–AAATT–^3′/TTTAA site of each hairpin show affinities of ~ 10^6–7 M⁻¹, 1:1 stoichiometries, exothermic enthalpies of − 7 to − 12 kcal mol⁻¹ (− 22 kcal mol⁻¹ for the secondary site of the three-way junction), and water releases. Their interaction with random cationic copolymers yielded higher affinities of ~ 10⁶ M⁻¹ with the more hydrophobic hairpins. This information should improve our current picture of how sequence and loops control the stability and melting behavior of nucleic acid molecules

Energetics, Ion, and Water Binding of the Unfolding of AA/UU Base Pair Stacks and UAU/UAU Base Triplet Stacks in RNA

Triplex formation occurs via interaction of a third strand with the major groove of double-stranded nucleic acid, through Hoogsteen hydrogen bonding. In this work, we use a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry to determine complete thermodynamic profiles for the unfolding of polyadenylic acid (poly­(rA))·polyuridylic acid (poly­(rU)) (duplex) and poly­(rA)·2poly­(rU) (triplex). Our thermodynamic results are in good agreement with the much earlier work of Krakauer and Sturtevant using only UV melting techniques. The folding of these two helices yielded an uptake of ions, Δ<i>n</i>_Na⁺ = 0.15 mol Na⁺/mol base pair (duplex) and 0.30 mol Na⁺/mole base triplet (triplex), which are consistent with their polymer behavior and the higher charge density parameter of triple helices. The osmotic stress technique yielded a release of structural water, Δ<i>n</i>_W = 2 mol H₂O/mol base pair (duplex unfolding into single strands) and an uptake of structural water, Δ<i>n</i>_W = 2 mol H₂O/mole base pair (triplex unfolding into duplex and a single strand). However, an overall release of electrostricted waters is obtained for the unfolding of both complexes from pressure perturbation calorimetric experiments. In total, the Δ<i>V</i> values obtained for the unfolding of triplex into duplex and a single strand correspond to an immobilization of two structural waters and a release of three electrostricted waters. The Δ<i>V</i> values obtained for the unfolding of duplex into two single strands correspond to the release of two structural waters and the immobilization of four electrostricted water molecules

Loop Contributions to the Folding Thermodynamics of DNA Straight Hairpin Loops and Pseudoknots

Pseudoknots have diverse and important roles in many biological functions. We used a combination of UV spectroscopy and differential scanning calorimetry to investigate the effect of the loop length on the unfolding thermodynamics of three sets of DNA stem-loop motifs with the following sequences: (a) d­(GCGC<i><b>T</b></i><b><i>_n</i></b>­GCGC), where <b><i>n</i></b> = 3, 5, 7, 9; (b) d­(CGCG­CG<i><b>T</b></i>_<b>4</b>G­AAAT­TCGC­GCG<i><b>T_n</b></i>AAT­TTC), where <b><i>n</i></b> = 4, 6, and 8; and (c) d­(TCTCT­<i><b>T_n</b></i>AAA­AAAA­AGAGA­<i><b>T</b></i>_<b>5</b>TTT­TTTT), where <b><i>n</i></b> = 5, 7, 9, and 11. The increase in loop length of the first set of hairpins yielded decreasing <i>T</i>_M’s and constant unfolding enthalpies, resulting in an entropy driven decrease in the stability of the hairpin (Δ<i>G</i>° = −7.5 to −6.1 kcal/mol). In the second set, the increase in the length of the loops yielded similar <i>T</i>_M’s and slight increases in the unfolding enthalpies. This translated into more stable pseudoknots with an increasing Δ<i>G</i>° from −13.2 to −17.1 kcal/mol. This effect can be rationalized in terms of the increased flexibility of the pseudoknot with larger loops optimizing base-pair stacking interactions. In the last set of molecules, the increase in the length of one of the loops yielded an increase in the <i>T</i>_M’s and larger increases in the enthalpies, which stabilize the pseudoknot significantly increasing the Δ<i>G</i>° from −8.5 to −16.6 kcal/mol. In this set, the thymine loop is complementary to the stem of A·T base pairs and the longer loops are able to form T*A·T base triplets due to the partial folding of the thymine loop into the ceiling of the major groove of the duplex, thus yielding a net formation of 1–3 T*AT/T*AT base-triplet stacks at the middle of its stem, depending on the loop length

Effect of Loop Length and Sequence on the Stability of DNA Pyrimidine Triplexes with TAT Base Triplets

We report the thermodynamic contributions of loop length and loop sequence to the overall stability of DNA intramolecular pyrimidine triplexes. Two sets of triplexes were designed: in the first set, the C₅ loop closing the triplex stem was replaced with ^5′‑CT<i>_n</i>C loops (<i>n</i> = 1–5), whereas in the second set, both the duplex and triplex loops were replaced with a ^5′‑GCAA or ^5′‑AACG tetraloop. For the triplexes with a ^5′‑CT<i>_n</i>C loop, the triplex with five bases in the loop has the highest stability relative to the control. A loop length lower than five compromises the strength of the base-pair stacks without decreasing the thermal stability, leading to a decreased enthalpy, whereas an increase in the loop length leads to a decreased enthalpy and a higher entropic penalty. The incorporation of the GCAA loop yielded more stable triplexes, whereas the incorporation of AACG in the triplex loop yielded a less stable triplex due to an unfavorable enthalpy term. Thus, addition of the GCAA tetraloop can cause an increase in the thermodynamics of the triplex without affecting the sequence or melting behavior and may result in an additional layer of genetic regulation

Thermodynamic Profiles and Nuclear Magnetic Resonance Studies of Oligonucleotide Duplexes Containing Single Diastereomeric Spiroiminodihydantoin Lesions

The spiroiminodihydantoins (Sp) are highly mutagenic oxidation products of guanine and 8-oxo-7,8-dihydroguanine in DNA. The Sp lesions have recently been detected in the liver and colon of mice infected with <i>Helicobacter hepaticus</i> that induces inflammation and the development of liver and colon cancers in murine model systems [Mangerich, A., et al. (2012) <i>Proc. Natl. Acad. Sci. U.S.A. 109</i>, E1820–E1829]. The impact of Sp lesions on the thermodynamic characteristics and the effects of the diastereomeric Sp-<i>R</i> and Sp-<i>S</i> lesions on the conformational features of double-stranded 11-mer oligonucleotide duplexes have been studied by a combination of microcalorimetric methods, analysis of DNA melting curves, and two-dimensional nuclear magnetic resonance methods. The nonplanar, propeller-like shapes of the Sp residues strongly diminish the extent of local base stacking interactions that destabilize the DNA duplexes characterized by unfavorable enthalpy contributions. Relative to that of an unmodified duplex, the thermally induced unfolding of the duplexes with centrally positioned Sp-<i>R</i> and Sp-<i>S</i> lesions into single strands is accompanied by a smaller release of cationic counterions (Δ<i>n</i>_Na⁺ = 0.6 mol of Na⁺/mol of duplex) and water molecules (Δ<i>n</i>_w = 17 mol of H₂O/mol of duplex). The unfolding parameters are similar for the Sp-<i>R</i> and Sp-<i>S</i> lesions, although their orientations in the duplexes are different. The structural disturbances radiate one base pair beyond the flanking C:G pair, although Watson–Crick hydrogen bonding is maintained at all flanking base pairs. The observed relatively strong destabilization of B-form DNA by the physically small Sp lesions is expected to have a significant impact on the processing of these lesions in biological environments