Loop contributions to the folding thermodynamics of DNA straight hairpin loops and pseudoknots

Pseudoknots have diverse and important roles in many biological functions. We used a combination of UV spectroscopy and differential scanning calorimetry to investigate the effect of the loop length on the unfolding thermodynamics of three sets of DNA stem-loop motifs with the following sequences: (a) d(GCGCT_nGCGC), where n = 3, 5, 7, 9; (b) d(CGCGCGT₄GAAATTCGCGCGT_nAATTTC), where n = 4, 6, and 8; and (c) d(TCTCTT_nAAAAAAAAGAGAT₅TTTTTTT), where n = 5, 7, 9, and 11. The increase in loop length of the first set of hairpins yielded decreasing T_Ms and constant unfolding enthalpies, resulting in an entropy driven decrease in the stability of the hairpin (δG° = -7.5 to -6.1 kcal/mol). In the second set, the increase in the length of the loops yielded similar T_Ms and slight increases in the unfolding enthalpies. This translated into more stable pseudoknots with an increasing δG° from -13.2 to -17.1 kcal/mol. This effect can be rationalized in terms of the increased flexibility of the pseudoknot with larger loops optimizing base-pair stacking interactions. In the last set of molecules, the increase in the length of one of the loops yielded an increase in the T_Ms and larger increases in the enthalpies, which stabilize the pseudoknot significantly increasing the δG° from -8.5 to -16.6 kcal/mol. In this set, the thymine loop is complementary to the stem of A·T base pairs and the longer loops are able to form TA·T base triplets due to the partial folding of the thymine loop into the ceiling of the major groove of the duplex, thus yielding a net formation of 1-3 TAT/TAT base-triplet stacks at the middle of its stem, depending on the loop length.