cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions

Abstract

We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA–protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a ‘ligation site’ for T4 RNA ligase, a ‘biotin site’ for solid-phase handling, a ‘reverse transcription primer site’ for the efficient and rapid conversion from an unstable mRNA–protein fusion (mRNA display) to a stable mRNA/cDNA–protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a ‘restriction enzyme site’ for the release of a complex from the solid support. This enables not only stabilizing mRNA–protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.

In vitro selection of a peptide antagonist of growth hormone secretagogue receptor using cDNA display

“…S3 and S4. Puromycin-linker and mRNA/cDNA-peptide fusions were prepared as previously described (43,65). Detailed procedures are provided in SI Materials and Methods.…”

“…Recently, we have developed an in vitro display system, termed cDNA display, which is an improved system of mRNA display; some peptides were successfully screened via this method (42)(43)(44)(45)(46). Because a peptide is displayed on a cDNA in cDNA display (instead of being displayed on mRNA, as with mRNA display), peptide-cDNA fusion libraries resist degradation by ribonucleases and can be used under severe selection conditions, such as cell culture medium.…”

Photoassisted Recovery of DNA Molecules for On-chip Directed Evolution

“…Recently, we have developed an in vitro evolution method termed cDNA display [9][10], which can evolve functional protein/peptide molecules using puromycin technology [11]. Moreover, immobilization of proteins and peptides on a microchip using the cDNA display [12] and other puromycin technologies [13][14] has already been achieved.…”

Establishment of a novel cell-based assay for screening small molecule antagonists of human interleukin-6 receptor

“…With the innovation of the polymerase chain reaction (PCR) technique and optimization of the Escherichia coli expression system, a screening assay based on phage display technology for identifying IL-6R antagonists was established and applied in the recent past [16,17] . It is a burgeoning and efficient screening technique, particularly for identifying candidates with a high affinity.…”