Helix-Coil Transition of the Self-Complementary dG-dG-dA-dA-dT-dT-dC-dC Duplex

Abstract

The helix-coil transition of the octanucleotide self-complementary duplex dG-dG-d A-dA-dT-dTdC-dC has been monitored at the Watson-Crick protons, the base and sugar nonexchangeable protons and the backbone phosphates by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The melting transition of the octanucleotide monitored by ultraviolet absorbance spectroscopy is characterized by the thermodynamic parameters AH" = -216.7 kJ/mol and AS" (25 "C) = -0.632 kJ mol-' K-' in 0.1 M NaCl, 10 mM phosphate solution. Correlation of the transition midpoint values monitored by the ultraviolet absorbance studies at strand concentrations below 0.2 mM and by NMR studies at 5.3 mM suggest that both methods are monitoring the octanucleotide duplex-to-strand transition. The NMR spectra of the Watson-Crick ring NH protons of the octanucleotide duplex have been followed as a function of temperature. The resonance from the terminal dG-dC base pairs broadens out at room temperature while the resonances from the other base pairs broaden simultaneously with the onset of the melting transition. The nonexchangeable base and sugar H-1' protons are resolved in the duplex and strand states and shift as average peaks through the melting transition. The experimental shifts on duplex formation have been compared with calculated values based on ring-current and atomic diamagnetic anisotropy contributions for a B-DNA base-pair-overlap geometry in solution. Several nonexchangeable proton resonances broaden in the fast-exchange region during the duplex-to-strand transition and the excess widths yield a duplex dissociation rate constant for the octanucleotide of 1.9 x lo3 s-' at 32 "C (fraction of duplex = 0.86) in 0.1 M NaCl, 10 mM phosphate buffer. The 31P resonances of the seven internucleotide phosphates are distributed over 0.6 ppm in the duplex state, shift downfield during the duplex-to-strand transition and undergo additional downfield shifts during the stacked-to-unstacked strand transition with increasing temperature.The application of nuclear magnetic resonance (NMR) spectroscopy to monitor the duplex-to-strand transition of oligonucleotide and polynucleotide sequences has been recently reviewed and demonstrates the power of this technique to differentiate between individual base pairs in a duplex and, in addition, to monitor each base pair at the Watson-Crick hydrogen bonds, the nonexchangeable base and sugar protons and the backbone phosphates in solution [1,2].These investigations include studies on the exchangeable and nonexchangeable protons of the deoxy pentanucleotide (dT-dT-dG-dT-dT) . (dA-dA-dC-dAdA) [3,4] and deoxy block polymer (dC,,-dA,,) . (dT,,-dG,,) [5], and the self-complementary sequences dA-dT-dG-dC-dA-dT [6] and poly(dA-dT) [7]. In addition, the exchangeable protons in double-stranded and triple-stranded oligomer duplexes containing Watson-Crick and Hoogsteen base pair have been

Immunostimulatory CpG Oligonucleotides Form Defined Three‐Dimensional Structures: Results from an NMR Study

“…This suggests that this core portion of 4 forms a duplex structure that melts cooperatively. As expected, [43] the maximum in broadening occurs at a temperature below the midpoint of the transition visible by following chemical shift changes (Figure 8), so a "softening" of the structure sets in before the strands fully dissociate. This and the sequential NOESY cross-peak pattern observed for all but the 3'-terminal residue of 4, strongly suggest that the core hexamer forms a duplex similar to that of 3.…”

“…of broadening for a given resonance depends on the chemical shift difference between the duplex and single-stranded states. [43] Thus, the absence of distinct broadening does not necessarily mean that no transition occurs, but simply that the chemical shifts for the given resonance are (incidentally) similar for the two states. For protons where the chemical shift differences are significant, the maximum line width may be observed at a temperature below the melting point determined by chemical shift or UV-melting analysis.…”

“…For protons where the chemical shift differences are significant, the maximum line width may be observed at a temperature below the melting point determined by chemical shift or UV-melting analysis. [43] We interrogated local transitions in the DNA structures studied by plotting the line width against temperature. Duplex dissociation was thus confirmed for (3) 2 from a series of one-dimensional 1 H NMR spectra acquired at temperature intervals of 10 8C (Figure 4).…”

Mono and bidimensional1H NMR and inverse detected13C NMR of the duplex d(CA)3·d(TG)3

“…However, progressive addition of NaC1 and use of a spin-echo sequence for water suppression [14] allows the observation of two (70 mM NaC1) or three (140 mM NaC1) broad signals (Fig. 3) in the imino protons region, the ones resonating at about 14 ppm assignable to TA pairs and those resonating at about 13 ppm assignable to CG pairs [15]. The lowering of the temperature leads to the decrease of the exchange rate with water, due to the slowing down of the kinetics of base-pairing opening, and the sharpening of the lines of the imino protons is observed.…”

Study by 23Na-NMR, 1H-NMR, and Ultraviolet Spectroscopy of the Thermal Stability of an 11-Basepair Oligonucleotide

“…For instance, the partial unpairing of the double helix and the presence of unfolding intermediates have been reported (Cantor and Schimmel, 1980;Hopkins et al, 1993;SantaLucia et al, 1996). The melting process is generally followed by ultraviolet (UV) spectroscopy but can also be studied by other techniques, such as 1 H-and 31 P-NMR (Patel and Canuel, 1979;Patel et al, 1982a;Petersheim and Turner, 1983b;Roongta et al, 1990). When individual resonances corresponding to specific nucleotides can be monitored, NMR has the advantage of offering the possibility to verify the validity of the two-state model (Patel et al, 1982a;Petersheim and Turner, 1983b).…”