In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation  

作　　者：YAN Song1,2, NIU RongLi2, WANG Zheng2 & LIN XiuKun2 1 School of Environmental Science and Engineering, Dalian Jiaotong University, Dalian 116028, China 2 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China 

出　　处：《Science China(Life Sciences)》2007年第5期630-636,共7页中国科学（生命科学英文版）

基　　金：Supported by the National Natural Science Foundation of China (Grant No. 30472043);the Department of Science and Technology of Shandong Province (Grant No. 2004C07)

摘　　要：Thymidylate synthase (TS),an essential enzyme for catalyzing the biosynthesisof thymidylate,is a critical therapeutic target in cancer therapy.Recent studies have shown that TSfunctions as an RNA-binding protein by interacting with two different sequences on its ownmRNA,thus,repressing translational efficiency.In this study,peptides binding TS RNA with highaffinity were isolated using mRNA display from a large peptide library (】10^(13) differentsequences).The randomized library was subjected up to twelve rounds of in vitro selection andamplification.Comparing the amino acid composition of the selected peptides (12th round,R12) withthose from the initial random library (round zero,R0),the basic and aromatic residues in theselected peptides were enriched significantly,suggesting that these peptide regions might beimportant in the peptide-TS mRNA interaction.Categorizing the amino acids at each random positionbased on their physicochemical properties and comparing the distributions with those of the initialrandom pool,an obvious basic charge characteristic was found at positions 1,12,17 and 18,suggestingthat basic side chains participate in RNA binding.Secondary structure prediction showed that theselected peptides of R12 pool represented a helical propensity compared with R0 pool,and the regionswere rich in basic residues.The electrophoretic gel mobility shift and in vitro translation assaysshowed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit thetranslation of TS mRNA.Our results suggested that the identified peptides could be used as new TSinhibitors and developed to a novel class of anticancer agents.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 (>1013 different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid com-position 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, sug-gesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categoriz-ing 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. Sec-ondary structure prediction showed that the selected peptides of R12 pool represented a helical pro-pensity 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.

关 键 词：thymidylate synthase  MRNA display  selection  AFFINITY PEPTIDES 

分 类 号：Q75[生物学—分子生物学]