The energetics of i-DNA tetraplex structures formed intermolecularly by d(TC5) and intramolecularly by d[(C5T3)3C5

Cytosine-rich DNA at low pH adopts an antiparallel tetraplex structure via the intercalation of two partially protonated, parallel stranded duplexes. This intriguing structural motif has been named i-DNA. We have used a combination of spectroscopic and calorimetric techniques to characterize the properties of an intermolecular i-DNA formed by d(TC(5)) and an intramolecular i-DNA formed by d[(C(5)T(3))(3)C(5)]. Our measurements reveal that both i-DNA complexes are enthalpically stabilized by 6.5-7.0 kcal/mol(base) and entropically destabilized by 20 cal/mol(base)/K. These values are about 50% larger than the corresponding enthalpy and entropy values per base for Watson and Crick duplexes and for Hoogsteen triplexes, while being similar to per base enthalpy and entropy values reported for G-quadruplexes. Our data also reveal a positive heat capacity change between 20 and 30 cal/mol(base)/K, values similar to that reported for polymeric Watson & Crick DNA duplexes. Solution-dependent studies reveal the overall thermal and thermodynamic stability of i-DNA complexes to be dictated by an interplay between pH and ionic strength. Based on the thermodynamic data measured, we discuss the feasibility of i-DNA formation in the context of conventional DNA sequences, while commenting on potential roles for this structural motif in biological regulatory mechanisms.     

Calorimetric and spectroscopic investigation of the helix-to-coil transition of the self-complementary deoxyribonucleotide ATGCAT

Differential scanning calorimetry and temperature-dependent uv spectroscopy are used to thermodynamically characterize the double-strand to single-strand transition of the self-complementary deoxyribo-oligonucleotide ATGCAT. The calorimetric experiments provide a value of 33.6 kcal (mol of double strand)^?1 for the transition between 10 and 90° C. In conjunction with available temperature-dependent nmr data (which reveals terminal base pair fraying), we attempt to define specifically those interactions to which the calorimetrically measured enthalpy change refers.Values of ΔH_V.H. (van 't Hoff enthalpy change) are derived from the spectroscopic and calorimetric data and compared with the ΔH obtained directly from the calorimetric experiment. This comparison reveals that the part of the thermally-induced transition that occurs between 10 and 90°C is well represented by a two-state process. It is noted that in assessing the applicability of the two-state model it is best to compare the ΔH_cal. with ΔH_V.H. obtained from the calorimetric rather than the spectroscopic data.     

Differential scanning calorimetry of Cu,Zn-superoxide dismutase, the apoprotein, and its zinc-substituted derivatives

We have employed differential scanning calorimetry (DSC) to investigate the thermally induced unfolding of native Cu,Zn-superoxide dismutase (SOD), the apoprotein derived from native SOD, and the zinc-substituted derivatives of the apoprotein. We observe two overlapping melting transitions for native bovine SOD with heat capacity maxima at temperatures (Tm) of 89 and 96 degrees C when a scanning rate of 0.82 deg/min is employed. By contrast, the dithionite-reduced native SOD (which contains Cu+ rather than Cu2+) exhibits only a single transition at 96 degrees C. Significantly, we find that the concentration of O2 present in native SOD samples influences the relative magnitudes of the 89 and 96 degrees C peaks. Specifically, the lower temperature transition becomes less pronounced as the concentration of O2 in the sample decreases. On the basis of these observations, we propose that the lower temperature peak corresponds to the melting of the oxidized native protein, while the higher temperature peak reflects the melting of the reduced native protein, which forms spontaneously during the heating process. Our interpretation profoundly differs from that of Lepock et al. [Lepock, J.R., Arnold, L.D., Torrie, B.H., Andrews, B., & Kruuv, J. (1985) Arch. Biochem. Biophys. 241, 243-251], who have proposed that the low-temperature transition corresponds to the reduced form of the protein. We present evidence that suggests that their experiments were complicated by the presence of potassium ferrocyanide, which, in addition to reducing the cupric center, also perturbs the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of sucrose on the structure, mechanical strength and thermal properties of corn extrudates

The effects of sucrose on extrusion process parameters and the structural, mechanical and thermal characteristics of the resultant extrudates have been investigated. To this end, sucrose in concentrations between 0 and 10% by weight was added to corn meal prior to extrusion at two levels of moisture (15 and 20%). The resulting data revealed the following significant features. (i) At the higher moisture level, sucrose progressively reduced the specific mechanical energy, while exhibiting little effect at the lower moisture level. (ii) At both moisture levels, sucrose increased the bulk density and reduced the cell size; this effect was progressive for high extrusion moisture samples and evident only at high sucrose contents for low extrusion moisture samples. (iii) In extrudate samples equilibrated to moisture contents between 12 and 17% wt, sucrose progressively plasticized the structures, as assessed by compression, dynamic mechanical spectrometry, and differential scanning calorimetry. In the aggregate, these results showed that the addition of sucrose requires a modification of the extrusion operating conditions to produce extrudates with optimal textural and storage properties.     

Novel post-synthetic generation, isomeric resolution, and characterization of Fapy-dG within oligodeoxynucleotides: differential anomeric impacts on DNA duplex properties

Accumulation of damaged guanine nucleobases within genomic DNA, including the imidazole ring opened N(6)-(2-Deoxy-α,β-D-erythro-pentafuranosyl)-2,6-diamino-4-hydroxy-5-formylamidopyrimidine (Fapy-dG), is associated with progression of age-related diseases and cancer. To evaluate the impact of this mutagenic lesion on DNA structure and energetics, we have developed a novel synthetic strategy to incorporate cognate Fapy-dG site-specifically within any oligodeoxynucleotide sequence. The scheme involves the synthesis of an oligonucleotide precursor containing a 5-nitropyrimidine moiety at the desired lesion site via standard solid-phase procedures. Following deprotection and isolation, the Fapy-dG lesion is generated by catalytic hydrogenation and subsequent formylation. NMR assignment of the Fapy-dG lesion (X) embedded within a TXT trimer reveals the presence of rotameric and anomeric species. The latter have been characterized by synthesizing the tridecamer oligodeoxynucleotide d(GCGTACXCATGCG) harboring Fapy-dG as the central residue and developing a protocol to resolve the isomeric components. Hybridization of the chromatographically isolated fractions with their complementary d(CGCATGCGTACGC) counterpart yields two Fapy-dG·C duplexes that are differentially destabilized relative to the canonical G·C parent. The resultant duplexes exhibit distinct thermal and thermodynamic profiles that are characteristic of α- and β-anomers, the former more destabilizing than the latter. These anomer-specific impacts are discussed in terms of differential repair enzyme recognition, processing and translesion synthesis.     

DNA meter: Energy tunable,quantitative hybridization assay

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.