We have characterized structural and dynamic aspects of the d(C1G2C3G4A5A6T6T5C4G3C2G1) 12-mer duplex based on an analysis of the proton and phosphorus nuclear magnetic resonance parameters in the premelting and melting transition region. The self-complementary dodecanucleotide sequence forms a 12 base-paired duplex with 2-fold symmetry in solution. The six Watson-Crick imino protons are well resolved between 12.5 and 14.0 ppm and have been assigned to individual positions in the sequence. Fraying of the ends of the duplex is most pronounced at terminal base pair 1. This fraying extends into the interior of the duplex up to and including base pair 3 with increasing temperature in the premelting transition region. A temperature-dependent transition is detected in the dA·dT-containing tetranucleotide core of the duplex in the premelting temperature range, which is not observed at the flanking dG·dC-containing tetranucleotide regions. The 11 phosphodiesters in the 12-mer duplex exhibit 31P chemical shifts spread over a 0.45-ppm chemical shift range, suggestive of a distribution of O-P torsion angles and/or O-P-O bond angles at individual phosphodiester linkages along the sequence. Well-resolved resonances of nonexchangeable base protons are observed in the 12-mer duplex spectrum. Partial assignment of these resonances to specific base pairs in the 12-mer was accomplished with spin decoupling, nuclear Overhauser effect, and chemical modification studies. These base protons shift as average resonances during the helix-coil transition of the 12-mer duplex in 0.1 M phosphate solution with a transition midpoint of 72 ± 2 °C at the 10 nonterminal base pairs. Upfield shifts are observed at the base protons of the dodecanucleotide sequence on duplex formation, which reflect the stacking interactions in the double-helical state. The thermally induced transitions of the right-handed 12-mer duplex in 0.01 and 0.1 M NaCl were investigated by differential scanning calorimetry. In 0.01 M NaCl an enthalpy change of 90 kcal (mol of double strand)−1 was measured with a melting temperature of 65.5 °C. In 0.1 M NaCl an enthalpy change of 102 kcal (mol of double strand)−1 was measured with a melting temperature of 71.3 °C. Analysis of the shapes of the calorimetric heat capacity curves yields van't Hoff enthalpies of 94 kcal in 0.01 M NaCl and 74 kcal in 0.1 M NaCl. Thus, we conclude that in 0.01 M NaCl the transition approaches two-state behavior (ΔHcal = ΔHv.H.) while in 0.1 M NaCl the transition from duplex to strands involves intermediate states (ΔHv.H. < ΔHcal). The ratio of the van't Hoff and calorimetric enthalpies allows specification of the size of the cooperative unit. Thus, in 0.1 M NaCl 9 ± 1 base pairs of the 12-mer melt in a cooperative manner.
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