Enhanced loop DNA folding induced by thymine-CH3 group contact and perpendicular guanine-thymine interaction.

A remarkable stabilizing effect induced by T-CH3 group and perpendicular guanine-thymine interactions in the DNA loop conformation has been demonstrated for the d(TTTG) loop structure using UV melting, high resolution NMR, distance geometry, and molecular dynamics studies. Contrary to the previously published d(TTCG) sequence that exhibits no specific inter-nucleotide interaction, we have found that d(TTTG), which differs only by one nucleotide with the d(TTCG) sequence (C7 --> T7), forms a rather stable and well-defined loop structure. Two characteristic structural features account for the stabilization of an otherwise flexible loop structure; the second loop T (T6) residue folds into the minor groove and engages in perpendicular interaction with the G8-NH2, while the third loop T (T7) residue stacks well upon the closing T5 x G8 wobble base pair and exhibits good contacts with many of the loop T5 and T6 sugar protons, which may form a hydrophobic core in the loop region. The importance of the bulky T7-CH3 was also proved by the UV melting study; while d(TTCG) hairpin exhibits a lower melting point (74.5 degrees C) than d(TTTG) hairpin (80.5 degrees C), d(TT(5-methyl)CG) hairpin resumes the same higher melting point (80 degrees C). Similarly, the fact that the melting temperature (74 degrees C) of d(TTTI) is lower than that of d(TTTG) indicates the critical role played by the G8-NH2 group. Our structural studies of the d(TTTG) loop indicate that DNA and RNA use a different strategy to establish stable tertiary folds. Comparison with several other pyrimidine-rich loop hairpins suggests that different minor-groove folding modes exist for the folding thymidine residue.