Thermodynamics of melting of the circular dumbbell d.

The conformational behavior of DNA minihairpin loops is sensitive to the directionality of the base pair that closes the loop. Especially tailored circular dumbbells, consisting of a stem of three Watson-Click base pairs capped on each side with a minihairpin loop, serve as excellent model compounds by means of which deeper insight is gained into the relative stability and melting properties of hairpin loops that differ only in directionality of the closing pair: C-G vs G-C. For this reason the thermodynamic properties of the circular DNA decamers 5'-d[pCGC-TT-GCG-TT]-3' (I) and reference compounds 5'-d[pGGC-TT-GCC-TT]-3' (II) and 5'-d(GCG-TC-CGC)-3' (III) are studied by means of nmr spectroscopy. Molecules I and II adopt dumbbell structures closed on both sides by a two-membered hairpin loop. At low temperature I consists of a mixture of two slowly exchanging forms, denoted L2L2 and L2L4. The low-temperature L2L2 form is the fully intact minihairpin structure with three Watson-Crick C-G base pairs. The high-temperature form, L2L4, contains a partially disrupted closing G-C base pair in the 5'-GTTC-3' loop, with the cytosine base placed in a syn orientation. The opposite 5'-CTTG-3' loop remains stable. A study of the noncircular hairpin structure III shows similar conformational behavior for the 5'-GTTC-3' loop as found in I; a syn orientation for C(6) and two slowly exchanging imino proton signals for G(3). The melting point T-m of II was estimated to lie above 365 K. The T-m value of the duplex stem and the 5'-CTTG-3' loop of the L2L4 form of I is 352 +/- 2 K. The Delta H-0 is calculated as -89 +/- 10 kJ/mol. The T-m value determined for the individual residues of the 5'-GTTC-3' loop lies 4 degrees-11 degrees lower. The enthalpy Delta H-0 of melting the thymine residues in the 5'-GTTC-3' loop is calculated to be -61 +/- 7 kJ/mol. Thermodynamic data of the equilibrium between the slowly exchanging two- and four-membered loop conformers of I reveal an upper limit for Delta H-0 of +30 kJ/mol in going from a two-membered to a four-membered loop, in agreement with the enthalpy difference of +28 kJ/mol between the two loops at the T-m midpoint. For hairpin III the upper limit for Delta H-0 in going from a two-membered to a four-membered loop amounts to +/-21 kJ/mol. The mutual exchange rate between the L2 and L4 form in III is estimated as 13.6 s(-1). Our results clearly suggest that small four-way DNA junctions (model for immobilized Holliday junctions) can be designed that consist af a single DNA strand that features-CTTG-caps on three of the four arms of the junction.