Identification Of The Peptides That Bind Thymidylate Synthase RNA With High Affinity Using MRNA Display

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Previous study indicated that the level of gene amplification, transcription, translation, and posttranslation were all involved in regulating the expression of TS. In addition to its role in enzyme catalysis, there is evidence that TS also functions as an RNA-binding protein. TS is able to bind to its own mRNA to form protein-TS mRNA complex, and this interaction results in translational suppression of TS. When TS is bound by 5-fluorouracil (5-FU) or other antimetabolite, the RNA binding activity of TS is dramatically decreased. The effect of reduced RNA binding activity is relief of translational repression, a process that leads to increased synthesis of new TS protein. Thus, this apparent autoregulatory model provides a rational mechanism for the development of drug resistance in tumor cells. Using display technologies to isolate nucleic acids, peptides and proteins has been studied intensively in recent years. The display technologies now applied include phage display, ribosome display, and in vitro mRNA display. In vitro mRNA display is a new and effective technique for peptides selection, and the rationale of this technique is that a synthetic mRNA with puromycin could covalently link with the protein that it encodes. Thus peptide or protein selected involve with its encoding nucleic acid sequence, which allows for the identification of isolated peptides by DNA sequencing. This approach has been used in identification of many functional peptides. In this study, peptides binding to TS RNA with high affinity were isolated using mRNA display technique from a large peptide library (>1013 different sequences). The selection scheme was constructed, and the experimental conditions, including library synthesis, formation of RNA-peptide fusion and RNA immobilization were optimized. The randomized library was subjected up to twelve rounds of in vitro selection and amplification. The sequencing analysis of eighth and twelfth round pool, and secondary structure prediction were processed. Compared the amino acid composition of the selected peptides with those from the initial random library (round zero, R0), the basic amino acid residues in selected peptides were enriched significantly, and more than one half of the sequences had at least one pair of basic amino acid. The results also showed that the content of phenylalanine residues was also significantly increased. The increase of basic and acromatic residues in the selected peptides may play a key role in the protein-TS RNA interactions. Fifty three percent of the sequences determined from R12 (round 12) pool shared a consensus arginine at position 12. Categorizing the amino acids at each random position based on their physicochemical properties and compared the distributions to those of the initial random pool, obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represent a helical propensity compared with R0 pool, almost one third of the selected peptides from R12 pool had an obvious helical propensity (>50% of the residues were folded into helix), and the regions were rich in basic residues. It is likely that the combination of secondary structure and sequence characteristics affects the peptides binding to TS RNA. 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.