PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

Brinton, Lindsey T.; Bauknight, Dustin K.; Dasa, Siva Sai Krishna; Kelly, Kimberly A.

doi:10.1371/journal.pone.0155244

Cited by 36 publications

(50 citation statements)

References 45 publications

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“…122 Nowadays, phage display has been further developed in combination with other techniques, such as microfluidics or better analysis software, to improve its selection efficiency. 74,123–125 The most widely used phages for phage display are shown in Figure 2.…”

Section: Viral Librariesmentioning

confidence: 99%

Virus-Derived Peptides for Clinical Applications

2017

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Novel affinity agents with high specificity are needed to make progress in disease diagnosis and therapy. Over the last several years, peptides have been considered to have fundamental benefits over other affinity agents, such as antibodies, due to their fast blood clearance, low immunogenicity, rapid tissue penetration, and reproducible chemical synthesis. These features make peptides ideal affinity agents for applications in disease diagnostics and therapeutics for a wide variety of afflictions. Virus-derived peptide techniques provide a rapid, robust, and high-throughput way to identify organism-targeting peptides with high affinity and selectivity. Here, we will review viral peptide display techniques, how these techniques have been utilized to select new organism-targeting peptides, and their numerous biomedical applications with an emphasis on targeted imaging, diagnosis, and therapeutic techniques. In the future, these virus-derived peptides may be used as common diagnosis and therapeutics tools in local clinics.

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Section: Viral Librariesmentioning

confidence: 99%

Virus-Derived Peptides for Clinical Applications

2017

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“…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 ).…”

Section: Resultsmentioning

confidence: 99%

Phage display peptide libraries: deviations from randomness and correctives

Ryvkin

Ashkenazy

Weiss-Ottolenghi

et al. 2018

Nucleic Acids Research

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Peptide-expressing phage display libraries are widely used for the interrogation of antibodies. Affinity selected peptides are then analyzed to discover epitope mimetics, or are subjected to computational algorithms for epitope prediction. A critical assumption for these applications is the random representation of amino acids in the initial naïve peptide library. In a previous study, we implemented next generation sequencing to evaluate a naïve library and discovered severe deviations from randomness in UAG codon over-representation as well as in high G phosphoramidite abundance causing amino acid distribution biases. In this study, we demonstrate that the UAG over-representation can be attributed to the burden imposed on the phage upon the assembly of the recombinant Protein 8 subunits. This was corrected by constructing the libraries using supE44-containing bacteria which suppress the UAG driven abortive termination. We also demonstrate that the overabundance of G stems from variant synthesis-efficiency and can be corrected using compensating oligonucleotide-mixtures calibrated by mass spectroscopy. Construction of libraries implementing these correctives results in markedly improved libraries that display random distribution of amino acids, thus ensuring that enriched peptides obtained in biopanning represent a genuine selection event, a fundamental assumption for phage display applications.

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“…Deep sequencing was performed with an Ion Torrent PGM (ThermoFisher Scientific) and a 318 v2 sequencing chip. For analysis of the phage sequences, TorrentSuite 5.2.2 software (ThermoFisher Scientific) was used and downstream bioinformatics sequence analyses were performed using PHASTpep 26 . This software uses a portion the sequence data that corresponds to displayed peptides and removes sequences that do not have the codons present in NEB libraries 26 .…”

Section: Methodsmentioning

confidence: 99%

“…For analysis of the phage sequences, TorrentSuite 5.2.2 software (ThermoFisher Scientific) was used and downstream bioinformatics sequence analyses were performed using PHASTpep 26 . This software uses a portion the sequence data that corresponds to displayed peptides and removes sequences that do not have the codons present in NEB libraries 26 . DNA sequences are translated into amino acid sequences and frequencies are calculated and normalized to the reference library frequencies for each peptide.…”

Section: Methodsmentioning

confidence: 99%

MRI-based molecular imaging of epicardium-derived stromal cells (EpiSC) by peptide-mediated active targeting

Straub¹,

Nave²,

Bouvain³

et al. 2020

Sci Rep

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After myocardial infarction (MI), epicardial cells reactivate their embryonic program, proliferate and migrate into the damaged tissue to differentiate into fibroblasts, endothelial cells and, if adequately stimulated, to cardiomyocytes. Targeting epicardium-derived stromal cells (EpiSC) by specific ligands might enable the direct imaging of EpiSCs after MI to better understand their biology, but also may permit the cell-specific delivery of small molecules to improve the post-MI healing process. Therefore, the aim of this study was to identify specific peptides by phage display screening to enable EpiSC specific cargo delivery by active targeting. To this end, we utilized a sequential panning of a phage library on cultured rat EpiSCs and then subtracted phage that nonspecifically bound blood immune cells. EpiSC specific phage were analyzed by deep sequencing and bioinformatics analysis to identify a total of 78 300 ± 31 900 different, EpiSC-specific, peptide insertion sequences. Flow cytometry of the five most highly abundant peptides (EP1, -2, –3, -7 or EP9) showed strong binding to EpiSCs but not to blood immune cells. The best binding properties were found for EP9 which was further studied by surface plasmon resonance (SPR). SPR revealed rapid and stable association of EpiSCs with EP9. As a negative control, THP-1 monocytes did not associate with EP9. Coupling of EP9 to perfluorocarbon nanoemulsions (PFCs) resulted in the efficient delivery of 19F cargo to EpiSCs and enabled their visualization by 19F MRI. Moreover, active targeting of EpiSCs by EP9-labelled PFCs was able to outcompete the strong phagocytic uptake of PFCs by circulating monocytes. In summary, we have identified a 7-mer peptide, (EP9) that binds to EpiSCs with high affinity and specificity. This peptide can be used to deliver small molecule cargos such as contrast agents to permit future in vivo tracking of EpiSCs by molecular imaging and to transfer small pharmaceutical molecules to modulate the biological activity of EpiSCs.

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PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

Cited by 36 publications

References 45 publications

Virus-Derived Peptides for Clinical Applications

Virus-Derived Peptides for Clinical Applications

Phage display peptide libraries: deviations from randomness and correctives

MRI-based molecular imaging of epicardium-derived stromal cells (EpiSC) by peptide-mediated active targeting

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