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 gu anine-thymine interactions in the DNA loop conformation has been demonstrat ed for the d(TTTG) loop structure using UV melting, high resolution NMR, di stance geometry, and molecular dynamics studies. Contrary to the previously published d(TTCG) sequence that exhibits no specific inter-nucleotide inte raction, we have found that d(TTTG), which differs only by one nucleotide w ith the d(TTCG) sequence (C7 --> T7), forms a rather stable and well-define d loop structure. Two characteristic structural features account for the st abilization of an otherwise flexible loop structure; the second loop T (T6) residue folds into the minor groove and engages in perpendicular interacti on with the G8-NH2, while the third loop T (T7) residue stacks well upon th e closing T5.G8 wobble base pair and exhibits good contacts with many of th e loop T5 and T6 sugar protons, which may form a hydrophobic core in the lo op region. The importance of the bulky T7-CH3 was also proved by the UV mel ting study; while d(TTCG) hairpin exhibits a lower melting point (74.5 degr eesC ) than d(TTTG) hairpin (80.5 degreesC ), d(TT5-methylCG) hairpin resum es the same higher melting point (80 degreesC ). Similarly, the fact that t he melting temperature (74 degreesC ) of d(TTTI) is lower than that of d(TT TG) 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 strat egy to establish stable tertiary folds. Comparison with several other pyrim idine-rich loop hairpins suggests that different minor-groove folding modes exist for the folding thymidine residue.