In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation.
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.
Yan
Song
S
School of Environmental Science and Engineering, Dalian Jiaotong University, Dalian 116028, China.
Niu
RongLi
R
Wang
Zheng
Z
Lin
XiuKun
X
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology; D002626:Chemistry, Pharmaceutical / Q000379:methods; D003001:Cloning, Molecular; D015723:Gene Library; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic
D000970:Antineoplastic Agents; D010455:Peptides; D012333:RNA, Messenger; D012313:RNA; D013940:Thymidylate Synthase