Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection.

Takuya Terai,Tomoyuki Koike,Naoto Nemoto

doi:10.3390/molecules25061472

Abstract

Binding peptides for given target molecules are often selected in vitro during drug discovery and chemical biology research. Among several display technologies for this purpose, complementary DNA (cDNA) display (a covalent complex of a peptide and its encoding cDNA linked via a specially designed puromycin-conjugated DNA) is unique in terms of library size, chemical stability, and flexibility of modification. However, selection of cDNA display libraries often suffers from false positives derived from non-specific binding. Although rigorous washing is a straightforward solution, this also leads to the loss of specific binders with moderate affinity because the interaction is non-covalent. To address this issue, herein, we propose a method to covalently link cDNA display molecules with their target proteins using light irradiation. We designed a new puromycin DNA linker that contains a photocrosslinking nucleic acid and prepared cDNA display molecules using the linker. Target proteins were also labeled with a short single-stranded DNA that should transiently hybridize with the linker. Upon ultraviolet (UV) light irradiation, cDNA display molecules encoding correct peptide aptamers made stable crosslinked products with the target proteins in solution, while display molecules encoding control peptides did not. Although further optimization and improvement is necessary, the results pave the way for efficient selection of peptide aptamers in multimolecular crowding biosystems.

Highlights

Directed molecular evolution is a powerful technology to create polypeptides with desired properties
The formed complementary DNA (cDNA) display molecules are incubated with target-immobilized magnetic beads, and, after some washing steps, bound molecules are collected and their cDNA is amplified by polymerase chain reaction (PCR) (Figure S2, Supplementary Materials)
Whereas reverse transcription is performed before selection in most modern messenger RNA (mRNA) display procedures [10], cDNA display is different in that the reverse transcription primer site is incorporated into the puromycin linker and, the cDNA moiety is covalently linked to the peptide via the linker

Summary

Introduction

Directed molecular evolution is a powerful technology to create polypeptides with desired properties. Complementary DNA (cDNA) display (Figure 1a) [12] is a promising technology to identify peptide aptamers or single-domain antibodies from a large library with high throughput. In cDNA display, a special DNA linker (Figure S1, Supplementary Materials) featuring puromycin (an antibiotic that is incorporated at the C-terminal of a nascent peptide in the A site of ribosomes) is ligated to an Molecules 2020, 25, 1472; doi:10.3390/molecules25061472 www.mdpi.com/journal/molecules mRNA library, yielding an mRNA–linker complex. Whereas reverse transcription is performed before selection in most modern mRNA display procedures [10], cDNA display is different in that the reverse transcription primer site is incorporated into the puromycin linker and, the cDNA moiety is covalently linked to the peptide via the linker. We and others used cDNA display to identify peptides associated with proteins [12,13,14], lipid membranes [15], and small molecules [16,17]

Methods

Results

Conclusion