Stability of the Na+ Form of the Human Telomeric G-Quadruplex: Role of Adenines in Stabilizing G-Quadruplex Structure.

G-quadruplexes are higher order DNA structures that play significant roles in gene transcription and telomeric maintenance. The formation and stability of the G-quadruplex structures are under thermodynamic control and may be of biological significance for regulatory function of cellular processes. Here, we report the structural influence and energetic contributions of the adenine bases in the loop sequences that flank G-repeats in human telomeric DNA sequence. Spectroscopic and calorimetric techniques are used to measure the thermal stability and thermodynamic contributions to the stability of human telomeric G-quadruplexes that have been designed with systematic changes of A to T throughout the telomeric sequence. These studies demonstrate that the thermal stability of the G-quadruplex structure is directly related to the number and position of the adenines that are present in the telomeric sequence. The melting temperature (Tm) was reduced from 59 °C for the wild-type sequence to 47 °C for the sequence where all four adenines were replaced with thymines (0123TTT). Furthermore, the enthalpy required for transitioning from the folded to unfolded G-quadruplex structure was reduced by 15 kcal/mol when the adenines were replaced with thymines (37 kcal/mol for the wild-type telomeric sequence reduced to 22 kcal/mol for the sequence where all four adenines were replaced with thymines (0123TTT)). The circular dichroism melting studies for G-quadruplex sequences having a single A to T change showed significantly sloping pretransition baselines and their differential scanning calorimetry (DSC) thermograms revealed biphasic melting profiles. In contrast, the deoxyoligonucleotides having sequences with two or more A to T changes did not exhibit sloping baselines or biphasic DSC thermograms. We attribute the biphasic unfolding profile and reduction in the enthalpy of unfolding to the energetic contributions of adenine hydrogen bonding within the loops as well as the adenine stacking to the G-tetrads of the G-quadruplex structure.

Linking Temperature, Cation Concentration and Water Activity for the B to Z Conformational Transition in DNA.

High concentrations of Na⁺ or [Co(NH3)6]3+ can induce the B to Z conformational transition in alternating (dC-dG) oligo and polynucleotides. The use of short DNA oligomers (dC-dG)4 and (dm5C-dG)4 as models can allow a thermodynamic characterization of the transition. Both form right handed double helical structures (B-DNA) in standard phosphate buffer with 115 mM Na⁺ at 25 °C. However, at 2.0 M Na⁺ or 200 µM [Co(NH3)6]3+, (dm5C-dG)4 assumes a left handed double helical structure (Z-DNA) while the unmethylated (dC-dG)4 analogue remains right handed under those conditions. We have previously demonstrated that the enthalpy of the transition at 25 °C for either inducer can be determined using isothermal titration calorimetry (ITC). Here, ITC is used to investigate the linkages between temperature, water activity and DNA conformation. We found that the determined enthalpy for each titration varied linearly with temperature allowing determination of the heat capacity change (ΔCp) between the initial and final states. As expected, the ΔCp values were dependent upon the cation (i.e., Na⁺ vs. [Co(NH3)6]3+) as well as the sequence of the DNA oligomer (i.e., methylated vs. unmethylated). Osmotic stress experiments were carried out to determine the gain or loss of water by the oligomer induced by the titration. The results are discussed in terms of solvent accessible surface areas, electrostatic interactions and the role of water.

Linking pH, Temperature, and K+ Concentration for DNA i-Motif Formation.

The conformation a particular DNA segment assumes depends upon its sequence context and the environment under which it is prepared. To complement our findings with G-rich sequences related to the human telomere, we have been investigating the pH induced transition from single strand to i-motif for sequences related to the human telomere C-rich strand. We have carried out titrations of (CCCTAA)4 from pH 7.0 to pH 5.0 at temperatures ranging from 15 to 45 °C at 115 mM K+ and at K+ concentrations ranging from 15 to 215 mM at 25 °C. Circular dichroism (CD) spectra were determined to monitor the transition. The pH at the midpoint of the proton induced transition, pHmp, is dependent upon both temperature and [K+]. Wyman-type plots of log K vs pH yielded linear correlations and the slopes of those lines, ΔQ, were also linearly dependent on [K+] and T. For these studies, ΔQ represents the minimum number of protons that must be added to the oligomer to induce the initial folding. These results are consistent with Le Chatelier's principle. Optical melting studies were also carried out for (CCCTAA)4 at pH 5.0 and [K+] ranging from 15 to 315 mM. Linear correlations between the temperature at the midpoint of the transition, Tm, and log [K+] allowed determination of the differential ion binding term, ΔnK+. These linkages between pH, temperature, and [K+] can be utilized to design i-motif forming DNA oligomers with highly tunable properties.

Loop Sequence Context Influences the Formation and Stability of the i-Motif for DNA Oligomers of Sequence (CCCXXX)4, where X = A and/or T, under Slightly Acidic Conditions.

The structure and stability of DNA is highly dependent upon the sequence context of the bases (A, G, C, and T) and the environment under which the DNA is prepared (e.g., buffer, temperature, pH, ionic strength). Understanding the factors that influence structure and stability of the i-motif conformation can lead to the design of DNA sequences with highly tunable properties. We have been investigating the influence of pH and temperature on the conformations and stabilities for all permutations of the DNA sequence (CCCXXX)4, where X = A and/or T, using spectroscopic approaches. All oligomers undergo transitions from single-stranded structures at pH 7.0 to i-motif conformations at pH 5.0 as evidenced by circular dichroism (CD) studies. These folded structures possess stacked C:CH(+) base pairs joined by loops of 5'-XXX-3'. Although the pH at the midpoint of the transition (pHmp) varies slightly with loop sequence, the linkage between pH and log K for the proton induced transition is highly loop sequence dependent. All oligomers also undergo the thermally induced i-motif to single-strand transition at pH 5.0 as the temperature is increased from 25 to 95 °C. The temperature at the midpoint of this transition (Tm) is also highly dependent on loop sequence context effects. For seven of eight possible permutations, the pH induced, and thermally induced transitions appear to be highly cooperative and two state. Analysis of the CD optical melting profiles via a van't Hoff approach reveals sequence-dependent thermodynamic parameters for the unfolding as well. Together, these data reveal that the i-motif conformation exhibits exquisite sensitivity to loop sequence context with respect to formation and stability.

The human telomere sequence, (TTAGGG)4, in the absence and presence of cosolutes: a spectroscopic investigation.

Historically, biophysical studies of nucleic acids have been carried out under near ideal conditions, i.e., low buffer concentration (e.g., 10 mM phosphate), pH 7, low ionic strength (e.g., 100 mM) and, for optical studies, low concentrations of DNA (e.g., 1×10⁻6 M). Although valuable structural and thermodynamic data have come out of these studies, the conditions, for the most, part, are inadequate to simulate realistic cellular conditions. The increasing interest in studying biomolecules under more cellular-like conditions prompted us to investigate the effect of osmotic stress on the structural and thermodynamic properties of DNA oligomers containing the human telomere sequence (TTAGGG). Here, we report the characterization of (TTAGGG)4 in potassium phosphate buffer with increasing percent PEG (polyethylene glycol) or acetonitrile. In general, the presence of these cosolutes induces a conformational change from a unimolecular hybrid structure to a multimolecular parallel stranded structure. Hence, the structural change is accompanied with a change in the molecularity of quadruplex formation.

Thermodynamics of forming a parallel DNA crossover.

The process of genetic recombination involves the formation of branched four-stranded DNA structures known as Holliday junctions. The Holliday junction is known to have an antiparallel orientation of its helices, i.e., the crossover occurs between strands of opposite polarity. Some intermediates in this process are known to involve two crossover sites, and these may involve crossovers between strands of identical polarity. Surprisingly, if a crossover occurs at every possible juxtaposition of backbones between parallel DNA double helices, the molecules form a paranemic structure with two helical domains, known as PX-DNA. Model PX-DNA molecules can be constructed from a variety of DNA molecules with five nucleotide pairs in the minor groove and six, seven or eight nucleotide pairs in the major groove. A topoisomer of the PX motif is the juxtaposed JX(1) molecule, wherein one crossover is missing between the two helical domains. The JX(1) molecule offers an outstanding baseline molecule with which to compare the PX molecule, so as to measure the thermodynamic cost of forming a crossover in a parallel molecule. We have made these measurements using calorimetric and ultraviolet hypochromicity methods, as well as denaturing gradient gel electrophoretic methods. The results suggest that in relaxed conditions, a system that meets the pairing requirements for PX-DNA would prefer to form the PX motif relative to juxtaposed molecules, particularly for the 6:5 structure.

Biophysical characterization of the human telomeric (TTAGGG)4 repeat in a potassium solution.

Quadruplex structures arise from four coplanar G bases arranged in a Hoogsteen base pairing motif to create a central pore that can coordinate cations. The termini of eukaryotic chromosomes contain structures, known as telomeres, which are capable of forming quadruplex structures. Quadruplexes have been implicated in a variety of disease states, including cancer. The literature seems to agree that the human telomeric repeat containing four stretches of three guanines displays conformational states that are different in the presence of Na+ and K+ and an unknown number of species involved in the quadruplex to single strand transition. Using circular dichroism spectroscopy, differential scanning calorimetry, and singular-value decomposition, the number of species present in the dissociation process is assessed. The results indicate that three species exist in equilibria during the melting process. We present a model for the heat-induced denaturation from the folded to the unfolded state, whereby the hybrid parallel-antiparallel quadruplex undergoes a transition to an unknown intramolecular intermediate followed by a transition to a single strand.

Enthalpy of the B-to-Z conformational transition of a DNA oligonucleotide determined by isothermal titration calorimetry.

The influence of high concentrations of Na(+) or [Co(NH(3))(6)](3+) on the conformation of two related DNA oligomers was investigated by circular dichroism spectropolarimetry (CD), isothermal titration calorimetry (ITC), and differential scanning calorimetry (DSC). As revealed by CD, DNA oligomers, (dC-dG)(4) and (dm(5)C-dG)(4), both form right-handed double helical structures (B-DNA) in standard phosphate buffer with 115 mM Na(+) at 25 degrees C. However, at 2.0 M Na(+) or 200 microM [Co(NH(3))(6)](3+), (dm(5)C-dG)(4) assumes a left-handed double helical structure (Z-DNA), whereas the unmethylated (dC-dG)(4) analog remains right-handed under those conditions. ITC was then used to determine the enthalpy change upon increasing the concentration of either Na(+) or [Co(NH(3))(6)](3+) for both DNA oligomers at 25 degrees C. The titration with Na(+) resulted in endothermic isotherms with (dm(5)C-dG)(4) being more endothermic than (dC-dG)(4) by 700 cal/mol basepair. In contrast, titration with [Co(NH(3))(6)](3+) resulted in exothermic isotherms with (dC-dG)(4) being more exothermic than (dm(5)C-dG)(4) by 720 cal/mol basepair. We attribute the enthalpy difference to the conformational transition from B-form DNA to Z-form DNA for (dm(5)C-dG)(4), a transition which does not occur for the unmethylated (dC-dG)(4). The value of approximately 700 cal/mol basepair for the enthalpy of the B-Z transition compares favorably with previously published results obtained by different techniques. DSC was used to monitor the duplex to single strand transitions for both oligomers under the different concentrations. These results indicated that methylation of the cytidine destabilizes (dm(5)C-dG)(4) relative to (dC-dG)(4). Coupling the DSC data with the ITC data allowed construction of a thermodynamic cycle which gives insight into the influence of both temperature and ionic strength on the heat content of the two DNA systems studied. Further, this study reveals the utility of using ITC for determinations of transition enthalpies with the appropriate choice of control.

Characterization of the secondary structure and stability of an RNA aptamer that binds vascular endothelial growth factor.

Thermal denaturation studies and spectroscopic studies were employed to investigate the secondary structure and stability of an RNA-PEG conjugate commercially called Macugen. The RNA aptamer is conjugated to a pegylated moiety, and the majority of its 2'-hydroxyl groups are methylated or otherwise modified. UV optical melting studies and differential scanning calorimetry (DSC) were carried out under different conditions to evaluate the effects of Na+ and oligomer concentrations on the stability of the secondary structure of the RNA oligomer. The results of these studies indicated that the T(m) of the RNA is independent of oligomer concentration but dependent on the salt concentration, in a predictable fashion. Further, the DSC melting profiles obtained under all conditions were highly reversible. Circular dichroism (CD) studies were determined under different salt concentrations, various RNA concentrations, and temperatures as well. Together, the thermal denaturation and CD studies provide evidence that the secondary structure of the RNA oligonucleotide is a stable hairpin at 25 degrees C and that the thermally induced hairpin to single strand transition is highly reversible.

The influence of sequence context and length on the kinetics of DNA duplex formation from complementary hairpins possessing (CNG) repeats.

The formation of unusual structures during DNA replication has been invoked for gene expansion in genomes possessing triplet repeat sequences, CNG, where N = A, C, G, or T. In particular, it has been suggested that the daughter strand of the leading strand partially dissociates from the parent strand and forms a hairpin. The equilibrium between the fully duplexed parent:daugter species and the parent:hairpin species is dependent upon their relative stabilities and the rates of reannealing of the daughter strand back to the parent. These stabilities and rates are ultimately influenced by the sequence context of the DNA and its length. Previous work has demonstrated that longer strands are more stable than shorter strands and that the identity of N also influences the thermal stability [Paiva, A. M.; Sheardy, R. D. Biochemistry 2004, 43, 14218-14227]. Here, we show that the rate of duplex formation from complementary hairpins is also sequence context and length dependent. In particular, longer duplexes have higher activation energies than shorter duplexes of the same sequence context. Further, [(CCG):(GGC)] duplexes have lower activation energies than corresponding [(CAG):(GTC)] duplexes of the same length. Hence, hairpins formed from long CNG sequences are more thermodynamically stable and have slower kinetics for reannealing to their complement than shorter analogues. Gene expansion can now be explained in terms of thermodynamics and kinetics.

Influence of sequence context and length on the structure and stability of triplet repeat DNA oligomers.

Genetic expansion diseases have been linked to the properties of triplet repeat DNA sequences during replication. The most common triplet repeats associated with such diseases are CAG, CCG, CGG, and CTG. It has been suggested that gene expansion occurs as a result of hairpin formation of long stretches of these sequences on the leading daughter strand synthesized during DNA replication [Gellibolian, R., Bacolla, A., and Wells, R. D. (1997) J. Biol. Chem. 272, 16793-7]. To test the biophysical basis for this model, oligonucleotides of general sequence (CNG)(n), where N = A, C, G, or T and n = 4, 5, 10, 15, or 25, were synthesized and characterized by circular dichroism (CD) spectropolarimetry, optical melting studies, and differential scanning calorimetry (DSC). The goal of these studies was to evaluate the influence of sequence context and oligomer length on their secondary structures and stabilities. The results indicate that all single oligomers, even those as short as 12 nucleotides, form stable hairpin structures at 25 degrees C. Such hairpins are characterized by the presence of N:N mismatched base pairs sandwiched between G:C base pairs in the stems and loops of three to four unpaired bases. Thermodynamic analysis of these structures reveals that their stabilities are influenced by both the sequence of the particular oligomer and its length. Specifically, the stability order of CGG > CTG > CAG > CCG was observed. In addition, longer oligomers were found to be more stable than shorter oligomers of the same sequence. However, a stability plateau above 45 nucleotides suggests that the length dependence reaches a maximum value where the stability of the G:C base pairs can no longer compensate the instability of the N:N mismatches in the stems of the hairpins. The results are discussed in terms of the above model proposed for gene expansion.