Prospective identification of parasitic sequences in phage display screens

Abstract

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones.

Directed evolution of artificial enzymes (XNAzymes) from diverse repertoires of synthetic genetic polymers

“…Selection pools are especially prone to 'ladder-type' by-product formation 27 in later rounds, as sequences are designed with complementarity that may be expected to be even more extensive in active XNAzymes under enrichment, as secondary structures and XNAzyme:substrate hybridization regions evolve. Emulsion PCR has been suggested as a potential solution to amplification problems in aptamer SELEX 28 and phage display 29 , but it has yet to be explored for XNAzyme selection.  crItIcal step Again, it is recommended to remove aliquots for analysis beginning at cycle 10 and to perform additional cycles only when an amplicon is not yet observed.…”

Multivalent pIX phage display selects for distinct and improved antibody properties

“…This was even more pronounced for the other pIII clone, which had a high thermal stability, but was expressed at very low level. Preferential amplification bias may obscure retrieval of desirable clones during library screening and has been reported for pIII libraries648. Our data indicate that the pIX-derived clones were selected and enriched primarily based on favorable target interaction, and therefore appear to be less affected by such target-unrelated effects.…”

A Review on Recent Patents and Applications of Inorganic Material Binding Peptides

“…However, these libraries still possess drawbacks. These include their ability to bind to non-specific undesired targets [131,132], under representation of the desired sequences and their properties due to having bias towards peptide/ protein position, composition, and expression [133] or pH and interaction specific intrinsic bias [30], thus many target specific strong MBPs are not found during the phage display process. Additionally, another important factor to consider is the limited fundamental understanding of peptide-material interactions which is a major bottle neck for generating peptide based inorganic nanomaterials with improved biostability/ compatibility, solubility and functionality and increased physico-chemical properties.…”