Select-to-Screen: Proximity-Driven In-Gel Encoded Library Synthesis & Screening

Encoded library technologies typically involve affinity selection to identify hits, which identifies binding but not necessarily function. Transfer of encoded libraries to solid-phase enables activity-based hit generation using high-throughput screening assays. This has been accomplished for DNA-encoded libraries, but not other encoded library formats. This lecture will discuss the first steps toward enabling activity-based mRNA display library screening. DNA-functionalized magnetic microspheres are used to template via bulk emulsification the formation of uniform hydrogel particles. The DNA templates are transcribed and the product mRNA is captured locally in the gel via hybridization. The captured mRNA is then translated and puromycin capture results in polyvalent, monoclonal display of the translated peptide (100 nM peptide in gel by HiBiT quantitation). The beads are analyzed by FACS and enrichments of > 50-fold were observed in a model screen. The ultra-miniaturized gel particle library format scalably transfers output from selection to focused libraries for functional screening.

Brian M. Paegel

Professor

UC Irvine

Brian M. Paegel (rhymes with “bagel”) earned his undergraduate degree in chemistry from Duke University and his doctoral degree in chemistry from UC Berkeley working on miniaturized and integrated DNA sequencing technology development in collaboration with the Human Genome Project. He pursued postdoctoral studies in chemical biology and molecular evolution at Scripps Research. There, he studied the continuous evolution of catalytic RNAs, developing microfluidics for automation, reaction monitoring, and droplet compartmentalization. He was the recipient of both a NIH National Research Service Award (F32) and a Pathway to Independence Award (K99/R00). In 2008, Paegel was appointed to the Scripps chemistry faculty, starting his independent career at the new east coast campus of Scripps Research in Jupiter, Florida. He received the NIH Director’s New Innovator award and an NSF CAREER award in recognition of his contributions in reaction miniaturization for enzyme evolution, and Scripps granted him tenure in 2017 for his work in the field of DNA-encoded libraries and drug discovery technology development. In 2019, Paegel rejoined the University of California System where he is Professor in the Departments of Pharmaceutical Sciences, Chemistry, and Biomedical Engineering at Irvine. His laboratory aims to deliver advanced parallel synthesis and screening platforms to support cross-disciplinary translational research initiatives. Paegel is deploying these platforms to eliminate the canonical sense of what is “druggable” within the cellular milieu and to democratize the discovery of new medicines. To this end, he has co-founded four start-up companies in the biotechnology and drug discovery space.

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Select-to-Screen: Proximity-Driven In-Gel Encoded Library Synthesis & Screening
Open to view video.  |   Closed captions available
Open to view video.  |   Closed captions available Encoded library technologies typically involve affinity selection to identify hits, which identifies binding but not necessarily function. Transfer of encoded libraries to solid-phase enables activity-based hit generation using high-throughput screening assays. This has been accomplished for DNA-encoded libraries, but not other encoded library formats. This lecture will discuss the first steps toward enabling activity-based mRNA display library screening. DNA-functionalized magnetic microspheres are used to template via bulk emulsification the formation of uniform hydrogel particles. The DNA templates are transcribed and the product mRNA is captured locally in the gel via hybridization. The captured mRNA is then translated and puromycin capture results in polyvalent, monoclonal display of the translated peptide (100 nM peptide in gel by HiBiT quantitation). The beads are analyzed by FACS and enrichments of > 50-fold were observed in a model screen. The ultra-miniaturized gel particle library format scalably transfers output from selection to focused libraries for functional screening.
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