Yan Song, Niu RongLi, Wang Zheng, Lin XiuKun
School of Environmental Science and Engineering, Dalian Jiaotong University, Dalian 116028, China.
Sci China C Life Sci. 2007 Oct;50(5):630-6. doi: 10.1007/s11427-007-0078-1.
Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>10(13) different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.
胸苷酸合成酶（TS）是催化胸苷酸生物合成的一种必需酶，是癌症治疗中的关键治疗靶点。最近的研究表明，TS通过与自身mRNA上的两个不同序列相互作用而作为一种RNA结合蛋白发挥作用，从而抑制翻译效率。在本研究中，使用mRNA展示技术从一个大的肽库（>10¹³个不同序列）中分离出与TS RNA具有高亲和力的肽。对随机库进行了多达十二轮的体外筛选和扩增。将所选肽（第12轮，R12）与初始随机库（第0轮，R0）的氨基酸组成进行比较，所选肽中的碱性和芳香族残基显著富集，表明这些肽区域可能在肽-TS mRNA相互作用中很重要。根据其理化性质对每个随机位置的氨基酸进行分类，并将分布与初始随机库的分布进行比较，发现在位置1、12、17和18处有明显的碱性电荷特征，表明碱性侧链参与RNA结合。二级结构预测表明，与R0库相比，R12库所选肽具有螺旋倾向，且这些区域富含碱性残基。电泳凝胶迁移率变动分析和体外翻译分析表明，使用mRNA展示技术选择的肽可以特异性结合TS RNA并抑制TS mRNA的翻译。我们的结果表明，所鉴定的肽可作为新的TS抑制剂，并开发成一类新型抗癌药物。
