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A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Published on: August 17, 2019
Automated DNA-Encoded Library Synthesis and Activity-Based Screening at the Attomole Scale
John P Burdick1, Erika M Cerna Arroyo1,2, Samantha R Levine1,2
1Department of Pharmaceutical Sciences, University of California, Irvine, California92697, United States.
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One-bead-one-compound DNA-encoded library (OBOC-DEL) technology enables synthesis and activity-based screening of diverse compound collections but requires complex microfluidic instrumentation and laborious manual bead handling. Here, we execute DEL synthesis on 2.8-μm-dia magnetic beads and encapsulate them in a functionalized polyacrylamide hydrogel to support in-gel activity assays. Magnetic-bead loading capacity was ∼10 amol of small molecule and ∼104 DNA encoding tags per bead. We developed a fully automated magnetic solid-phase synthesis using a KingFisher instrument. Bulk emulsification of DEL beads with assay reagents eliminated the need for microfluidics. Activity-based screening against Factor Xa identified enzyme inhibitors, including the positive control (rivaroxaban, incorporated into the library), which was recovered as the most abundant hit (replicate k class = 14). This ultraminiaturized platform uses commercially available automation and flow cytometry, making OBOC-DEL technology broadly accessible and distributable.
