研究报告: 基于分子对接和步进序列群的蓖麻毒素核酸适配体序列优化

目的 有效结合分子对接预测和表面等离子体共振实验评价技术,获得亲和力更强、序列最短的最优适配体。方法 针对前期筛选出的靶向蓖麻毒素的3条80 nt单链DNA适配体（L14、P3、L7）,在明确各自二维随机区茎环序列与靶蛋白结合能力的基础上,以H-DOCK分子对接为指导,分别确定蓖麻毒素适配体随机区的最短结合单元,从而构建两端延长步进序列群,以表面等离子体共振技术测定序列群序列的亲和力和动力学参数,明确适配体的结合关键结构,从而筛选得到最优适配体。结果 3条全长适配体的随机区适配体L14r、P3r、L7r均可形成一定的茎环结构,其中L14r较L14的亲和力增强9倍、L7r增强2倍、P3r基本不变。对随机区适配体和蓖麻毒素进行分子对接,结果显示,L14r、P3r、L7r的对接分数值皆优于阴性序列40T,结合关键氨基酸个数分别为11、8、9个,存在距离小于5 ?的预测结合位点分别为20、12、15个,具有良好的与蓖麻毒素的结合能力。进一步明确了蓖麻毒素活性口袋所容纳的适配体最短结合单元L14rm、P3rm、L7rm的序列构成,在此基础上构建出两端延长步进序列群。针对该步进群,基于结合关键氨基酸个数、结合位点个数、对接得分等参数的变化和表面等离子体共振测定结果筛选出最优适配体。所获得的最优适配体L14rm、L7rm-2亲和力继续增强了1~2倍。结论 随机区适配体能有效地与蓖麻毒素结合,较之全长适配体亲和力更强,分子对接结合步进序列群设计,仅使用17条序列,便有效获得了3条最优适配体并明确其结合作用。3条结合蓖麻毒素的最优适配体——L14rm、P3r、L7rm-2的K_D值分别为（64±30）、（167±19）、（120±1）nmol/L,亲和力提高到全长适配体的14、1、4倍。

Review: Molecular Docking and Stepping Sequence Cluster Design Prompted Sequence Optimization of Ricin Nucleic Acid Aptamers

Objective To obtain the finest aptamers with stronger affinity and shortest sequences from the combination of molecular docking simulation and surface plasmon resonance (SPR) evaluation experimentation.Methods Towards three previously screened single-stranded DNA aptamers against ricin with 80 nt, L14, P3, L7, on the confirmation basis of binding ability between their 2D stem-loop sequences of random region with target proteins, the H-DOCK molecular docking was performed to guide the determination of their minimum aptamer binding motifs (MABM), and an elongated stepping sequence cluster was sequentially constructed. The affinity and kinetic parameters of the designed clusters were measured by SPR, in which the binding key structures of the selected aptamers were depicted, and finally the finest aptamers could be selected.Results All three random region aptamers, L14r, P3r and L7r, can form some certain hairpin structures, and the affinity of L14r is increased by nine times than L14, L7r increased by 2 times, and P3r kept the same. The results of molecular docking between the random region aptamers and ricin showed that, the docking scores of L14r, P3r and L7r were all lower than negative sequence of 40T, the number of key binding amino acids were 11, 8 and 9, and the predicted binding sites with distances less than 5 ? were 20, 12 and 15, respectively, indicating good binding ability with ricin. Further, the sequence composition of MABM, L14rm, P3rm and L7rm, were deduced from the binding structures confined in the ricin active pockets, and the elongated stepping sequence cluster was built. On the parameters including the number of key binding amino acids, binding sites, the docking scores, as well as the results of SPR evaluation, the finest aptamers were evolved, in which the affinity of L14rm and L7rm-2 continued to be increased by 1-2 times.Conclusion The random region aptamer can effectively bind ricin with stronger affinity than the full-length aptamer. Molecular docking and stepping sequence clusters design can aid the fast evolution of three finest aptamers from only 17 sequences as well as the investigation of binding interaction. The K_D values of the three finest aptamers against ricin, L14rm, P3r and L7rm-2, were (64±30), (167±19) and (120±1) nmol/L, respectively, and the affinity was increased to 14, 1 and 4 times of the full-length aptamers.