Tridecanucleotide duplex-loop equilibrium in solution

The authors have studied the tridecanucleotide CGCGAATTACGCG (I), which contains an additional A at position 9, compared to the sequence CGCGAATTCGCG (II) of which the crystal structure has been determined. In low concentration in low salt, II is known to exhibit a biphasic melting curve, which has been interpreted to indicate a loop structure. By contrast, under comparable conditions I exhibits no distinct melting up to 900C, and also gives two major peaks on reverse phase chromatography. They explain this behavior by a slow equilibrium between loop (L) and duplex (D) forms. They have characterized this equilibrium by proton NMR spectroscopy, and shown that it is fully reversible by monitoring the two T(Me) resonances, each of which occur in two environments. Lower temperature and higher concentration favor the D form. The midpoint of the transition at 1.5 mM is 0.30 and at 4.5 mM is 6.50. The enthalpy of formation of the duplex is -30 and -22 kcal/M, respectively, and from temperature jump and saturation transfer measurements, they have determined the activation energy to be 56 kcal/M, indicating the breakage of 3-4 bps per duplex. CGCGCGAATTACGCGCG is much more stable in the L-form. They are using 2D-NMR methods to define the conformation of the L and D forms of I