In Vitro Selection Assays: New Approaches and Applications in DNA-encoded Libraries and Activity-based Probes

Dr. Casey Krusemark, Assist. Professor, MCMP, Purdue University
RHPH 164
Date / Time: 
Thursday, April 18, 2019 - 4:00pm

: The in vitro selection of synthetic molecule libraries allows a collective querying of function for many molecules simultaneously. Inspired by natural selection-driven evolution, the signal for these assays is DNA allele frequency change within a population in response to selective pressure. As an assay, this approach has several advantages over assays employed in traditional small molecule screening campaigns, such as improved throughput and lower cost. We present a critical evaluation of in vitro selection assays with regard to their application to discovery from DNA-encoded libraries and also to selection-based sensing, a new assay approach we have developed that uses DNA-linked probes to detect enzyme activity by DNA sequencing or quantitative PCR. These selections include commonly employed affinity purifications, as well as enzyme substrate selections, and also selections enabled by covalent crosslinking of DNA-linked ligands to target proteins. We demonstrate substrate-based selection assays for protein kinase, protease, and transferase activities. With crosslinking selections, protein targets are covalently conjugated to proteins via affinity labeling or by active site-labeling electrophiles. Crosslinking has significant benefits for selection against target proteins in crude cell lysates, inside live cells, and the exterior of live cells. We present quantitative assay parameters of these specific approaches and their application with DNA-encoded libraries of peptidomimetics against protein targets, including protein kinases, chromodomains of the CBX family, and the delta opioid receptor. In addition, we explore the application of selection-based sensing for enzyme activity detection in complex proteomic samples and in the screening of conventional small molecule libraries for enzyme inhibition by DNA sequence analysis.

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