Next-generation phage display: integrating and comparing available molecular tools to enable cost-effective high-throughput analysis

dc.contributorVirginia Techen
dc.contributor.authorDias-Neto, Emmanueen
dc.contributor.authorNunes, Diana N.en
dc.contributor.authorGiordano, Ricardo J.en
dc.contributor.authorSun, Jessicaen
dc.contributor.authorBotz, Gregory H.en
dc.contributor.authorYang, Kuanen
dc.contributor.authorSetubal, João C.en
dc.contributor.authorPasqualini, Renataen
dc.contributor.authorArap, Wadihen
dc.description.abstractBackground: Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges. Methodology/Principal Findings: We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at .1,000-fold faster rate, and reduce costs ,250-fold for generating 106 ligand sequences. Conclusions/Significance: Our analyses demonstrates that whereas this approach correlates with the traditional colonycounting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger sequencing and is proposed as the method of choice over a broad range of phage display applications in vitro, in cells, and in vivo.en
dc.description.sponsorshipThis work was funded by grants from the National Institutes of Health and the U.S. Department of Defense, and by awards from AngelWorks, the Gillson-Longenbaugh Foundation, and the Marcus Foundation (all to RP and WA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.en
dc.identifier.citationDias-Neto E, Nunes DN, Giordano RJ, et al. Next-generation phage display: integrating and comparing available molecular tools to enable cost-effective high-throughput analysis. PLoS One. 2009;4:e8338.
dc.publisherPublic Library of Scienceen
dc.rightsIn Copyrighten
dc.subjectDNA sequencesen
dc.subjectPeptide librariesen
dc.subjectPolymerase chain reactionen
dc.titleNext-generation phage display: integrating and comparing available molecular tools to enable cost-effective high-throughput analysisen
dc.title.serialPLoS ONEen
dc.typeArticle - Refereeden


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