Y Tanaka, T Hori, M Tagaya, M Katahira, F Nishikawa, T Sakamoto, Y Kurihara, S Nishikawa, S Uesugi
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引用次数: 1
摘要
合成了基于“伪结”模型设计的三种最小化丁型肝炎病毒(HDV) RNA核酶系统变体,并利用核磁共振光谱分析了它们的三级相互作用。Rz-1是一种顺式核酶系统(裂解形式,56-mer),其中茎IV从基因组HDV RNA的活性区域中删除。Rz-1用稳定同位素13C和15N均匀标记。Rz-2是一种反式作用核酶体系(底物:8-mer,裂解位点的胞苷残基被2'- o -甲基胞苷取代;酶:16聚合体加35聚合体)。Rz-2在35mer酶的鸟苷残基中用稳定同位素进行了部分标记。Rz-4是一种反式作用核酶体系(底物:8mer,裂解位点的胞苷残基被2′- o -甲基胞苷取代;酶53mer),由Perrotta和Been设计。Rz-4具有相同的序列和一个额外的闭环干IV。从2D-NOESY和2D-HSQC(除了Rz-4)光谱中可以看出,每种核酶在溶液中形成“假结”型结构。此外,Rz-1的G38、Rz-2和Rz-4的G28和G29形成碱基对。这些新的碱基对在修饰的基因组HDV RNA的晶体结构中被观察到。从Rz-2的温度变化实验来看,G28的亚质子信号在50℃时比其他相应信号消失得更早。当Rz-2用MgCl2滴定时,信号变化最大。
NMR analysis of tertiary interactions in HDV ribozymes.
Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems designed on the basis of the "pseudoknot" model were synthesized and their tertiary interactions were analyzed by NMR spectroscopy. Rz-1 is a cis-acting ribozyme system (the cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. Rz-2 is a trans-acting ribozyme system (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-2 was partially labeled with stable isotopes in guanosine residues of enzyme 35mer. Rz-4 is a trans-acting ribozyme system (substrate: 8mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme 53mer) which was designed by Perrotta and Been. Rz-4 has the same sequence and an extra loop closing stem IV. From 2D-NOESY and 2D-HSQC (except for Rz-4) spectra, it was suggested each ribozyme forms "pseudoknot" type structure in solution. Additionally, it was found that G38 of Rz-1, G28 and G29 of Rz-2 and Rz-4 form base-pairs. These novel base-pairs are observed in the crystal structure of a modified genomic HDV RNA. From temperature change experiment of Rz-2, the imino proton signal of G28 disappeared at 50 degrees C earlier than the other corresponding signals. Upon MgCl2 titration of Rz-2, this signal showed the largest shift.