凝血酶适体DNA四螺旋构象去折叠机制的NMR研究

凝血酶适体DNA[thrombin binding DNA aptamer d (G₁G₂T₃T₄G₅G₆T₇G₈T₉G₁₀G₁₁T₁₂T₁₃G₁₄G₁₅)，TBA]是对凝血酶有极高亲和性、并能有效抑制凝血酶凝血功能的单链DNA适体. 凝血酶适体DNA在K⁺等离子存在时呈现出椅式构象，其中2个堆积的四碱基体（G-quartet）构成椅子的主体部分，而1个TGT loop环和2个TT loop环分别构成椅子的靠背和椅脚. 我们测定了在K⁺存在时凝血酶适体DNA中亚氨基质子的交换速率， 发现位于TGT loop环和TT loop环内的亚氨基质子G6、G5和G14由于受TGT loop环和TT loop环的保护有比较小的交换速率，而位于环之外的亚氨基质子G2、G11和G15有较大的交换速率；TGT loop环稳定性同TT loop环相似；碱基T4、T13和T9在稳定凝血酶适体DNA结构中起着很大的作用. 这些证据进一步支持了凝血酶适体DNA的去折叠机制：位于TGT loop环和TT loop环之外的碱基对G1G15、G2G14和G5G11首先断裂其Hoogsteen氢键，而TGT loop环、TT loop环和其内的Hoogsteen氢键保持完好；当温度进一步升高时，TGT loop环和两个TT loop环打开环状结构，凝血酶适体DNA的椅式构象彻底解体，转变为自由单链. 

NMR Study of the Unfolding Mechanism for the Thrombin-Binding DNA Aptamer d (GGTTGGTGTGGTTGG)

15mer DNA d(GGTTGGTGTGGTTGG) is a thrombin-binding DNA aptamer(TBA),with inhibits clotting,of a high affinity with thrombin. In the presence of K~ ,TBA forms a right-handed-helix chair-like conformation with two stacked G-quartets and a regular phosphodiester backbone.The two G-quartets are linked by two TT loops at one end and a TGT loop at the other.The Hydrogen exchange rates of the imino protons of TBA in the presence of K~ were measured in this study.The results show that the G2,G11 and G15 iminos outside both the TGT loop and the two TT loops exchange faster,and in comparison the G6,G5 and G14 iminos have lower exchange rates because of the protection from the TGT loop and the TT loops.We also found that the stability of the TGT loop is similar to the two TT loops,and that the bases T4,T13 and T9 play more important roles in keeping the hydrogen bond stable in the K~ : TBA complex than in the Sr~(2 ): TBA complex.Evidence was obtained for the unfolding mechanism for the thrombin-binding aptamer.The data show that the hydrogen bonds between G2 and G14,G11 and G5,G15 and G1 outside the loops break first,and when the temperature increases further,the TGT loop and the two TT loops also become random coils.