Deep sequencing of phage-displayed peptide libraries reveals sequence motif that detects norovirus

Abstract

Norovirus infections are the leading cause of non-bacterial gastroenteritis and result in about 21 million new cases and $2 billion in costs per year in the United States. Existing diagnostics have limited feasibility for point-of-care applications, so there is a clear need for more reliable, rapid, and simple-to-use diagnostic tools in order to contain outbreaks and prevent inappropriate treatments. In this study, a combination of phage display technology, deep sequencing and computational analysis was used to identify 12-mer peptides with specific binding to norovirus genotype GI.1 virus-like particles (VLPs). After biopanning, phage populations were sequenced and analyzed to identify a consensus peptide motif-YRSWXP. Two 12-mer peptides containing this sequence, NV-O-R5-3 and NV-O-R5-6, were further characterized to evaluate the motif's functional ability to detect VLPs and virus. Results indicated that these peptides effectively detect GI.1 VLPs in solid-phase peptide arrays, ELISAs and dot blots. Further, their specificity for the S-domain of the major capsid protein enables them to detect a wide range of GI and GII norovirus genotypes. Both peptides were able to detect virus in norovirus-positive clinical stool samples. Overall, the work reported here demonstrates the application of phage display coupled with next generation sequencing and computational analysis to uncover peptides with specific binding ability to a target protein for diagnostic applications. Further, the reagents characterized here can be integrated into existing diagnostic formats to detect clinically relevant genotypes of norovirus in stool.

De NovoDiscovery of High Affinity Peptide Binders for the SARS-CoV-2 Spike Protein

“…The peptides reported here are potential starting points for the development of affinity-based diagnostic tools. [32][33][34] High-affinity reagents without direct competition activity for native receptors could be used for virus-directed delivery of antiviral payloads, or for the development of proteasome or lysosome targeting chimeras (PROTACs 35 and LYTACs 36 ). Adapting peptide 1 to a chemiluminescence enzyme immunoassay or a similar assay could improve its sensitivity for detection of SARS-CoV-2.…”

Phage display peptide libraries: deviations from randomness and correctives

“…Not only did the amino acids previously over represented, drop to a reasonable expected base line, but the correction for G overuse reduced the UAG concentration as well. To assess the corrections taken to improve the randomness of our libraries we analysed the profile produced for the commercially available New England BioLabs (NEB) Ph.D.-7 library (see, for example ( 2 , 29 , 47 , 49 , 53 , 57 , 63 , 65 , 66 , 72–75 )). For this, we have compared the nucleotide and amino-acid distributions of the NEB library with that observed in our fourth generation unbiased library (Figure 1D ).…”

Mapping Protein–Protein Interaction Interface Peptides with Jun-Fos Assisted Phage Display and Deep Sequencing

“…The availability of next-generation DNA sequencing has greatly enhanced the analysis of both combinatorial random peptide and genomic phage display libraries. , Previous deep sequencing studies of genomic and cDNA libraries that were enriched for in-frame fusions showed a complex naive library that contained a diverse array of genes, as well as specific peptides and domains that were enriched from affinity selections. , However, due to the size and complexity of the naive genomic or cDNA libraries, a detailed analysis of the selection process was not performed. Our high coverage of a limited size DNA plasmid using a Jun-Fos based library and deep sequencing allowed a detailed analysis of the affinity selection process.…”