Structural analysis of telomere G-quartet and molecular design of new nucleic acid aptamer

在PS-d（TC_4）的4个核苷酸间位（T_<PS1>C_<PS2>C_<PS3>C_<PS4>C） （PSn：磷酸化基团）上均引入了p手性立体定义的硫代磷酸基团，并在PS-d（TC_4）可能的16种非对映体中合成了10种对映体，研究了p手性的意义对i基序结构和稳定性的影响。圆二色光谱的温度依赖关系表明，[all R_p]PS-d(TC_4) i基序的熔化温度（Tm）与母体低聚物PO-d（TC_4）相同，为31℃，而[all S_p]PS-d(TC_4) i基序的熔化温度大幅降低了11℃。单次用S_p取代R_p可使PS1、ps2、ps3位置的T_m降低3 ~ 4℃，PS4位置的T_m降低1℃，显示出抑制T_m的加性。对比[all R_p]PS-、[all S_p]PS-和PO-d（TC_4）的NOESY谱，发现[all S_p]异构体的残余H6-H3′和H2”- h4′NOE交叉峰弱于[all R_p]异构体和PO-d(TC_4)，表明C3′-端构型和糖苷键角发生了变化。C2/C3/C4 H2”和H4”质子与[all S_p]PS-d（TC_4）和PO-d（TC_4）的化学位移差异也表明了[all S_p]PS-d（TC_4）形成的i基序的结构变化。这些结果表明，硫代磷酸键上的S_p构型改变了糖-硫代磷酸键的构象和窄槽内的分子间相互作用，导致i基序结构的不稳定。

The P-chiral stereodefined phosphorothioate groups have been introduced into all the four internucleotide positions of d(T_<PS1>C_<PS2>C_<PS3>C_<PS4>C) (PSn : phosphorothioate group), and among possible 16 diastereomers of PS-d(TC_4), 10 stereomers have been synthesized to investigate the effects of the sense of the P-chirality upon the structure and stability of the i-motif structure. The temperature dependence of circular dichroism spectra showed melting temperature (Tm) of the [all R_p]PS-d(TC_4) i-motif was 31 ℃ identical to that of the parent oligomer, PO-d(TC_4), while that of the [all S_p]PS-d(TC_4) i-motif was largely decreased by 11℃. Single substitution of R_p with S_p caused a decrease of T_m by 3〜4℃ at positions of PS1, 2, and 3, and by 1℃ at that of PS4, showing the additive property of the T_m suppression. The comparison of the NOESY spectra between [all R_p]PS-, [all S_p]PS-, and PO-d(TC_4) showed that intra-residual H6-H3' and H2"-H4' NOE cross-peaks of the [all S_p] isomer are weaker than those of the [all R_p] isomer and PO-d(TC_4), indicating the change in the C3'-endo conformation and glycosidic bond angle. The structural alternation for the i-motif formed by [all S_p]PS-d(TC_4) is also suggested by the chemical shift differences of C2/C3/C4 H2"and H4' protons from those of [all R_p]PS-d(TC_4) and PO-d(TC_4). These results suggest that the S_p configuration at phosphorus of the phosphorothioate linkage change the sugar-phosphorothioate conformation and intermolecular interaction in the narrow groove, leading to the destabilization of the i-motif structure.