THERMODYNAMICS OF MELTING OF THE CIRCULAR DUMBBELL D[PCGC-TT-GCG-TT]

The conformational behavior of DNA minihairpin loops is sensitive to t he directionality of the base pair that closes the loop. Especially ta ilored circular dumbbells, consisting of a stem of three Watson-Click base pairs capped on each side with a minihairpin loop, serve as excel lent model compounds by means of which deeper insight is gained into t he relative stability and melting properties of hairpin loops that dif fer only in directionality of the closing pair: C-G vs G-C. For this r eason the thermodynamic properties of the circular DNA decamers 5'-d[p CGC-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 spectro scopy. Molecules I and II adopt dumbbell structures closed on both sid es 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, co ntains a partially disrupted closing G-C base pair in the 5'-GTTC-3' l oop, with the cytosine base placed in a syn orientation. The opposite 5'-CTTG-3' loop remains stable. A study of the noncircular hairpin str ucture III shows similar conformational behavior for the 5'-GTTC-3' lo op 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 estima ted to lie above 365 K. The T-m value of the duplex stem and the 5'-CT TG-3' loop of the L2L4 form of I is 352 +/- 2 K. The Delta H-0 is calc ulated as -89 +/- 10 kJ/mol. The T-m value determined for the individu al residues of the 5'-GTTC-3' loop lies 4 degrees-11 degrees lower. Th e 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 g oing from a two-membered to a four-membered loop, in agreement with th e enthalpy difference of +28 kJ/mol between the two loops at the T-m m idpoint. 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 mut ual 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 junction s (model for immobilized Holliday junctions) can be designed that cons ist af a single DNA strand that features-CTTG-caps on three of the fou r arms of the junction. (C) 1995 John Wiley & Sons, Inc.