title: An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening.
abstract: DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.
authors.affiliation: Department of Chemistry and ‡Doctoral Program in Chemical and Biological Sciences, The Scripps Research Institute , 130 Scripps Way, Jupiter, Florida 33458, United States.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: MacConnell
authors.affiliation: Department of Chemistry and ‡Doctoral Program in Chemical and Biological Sciences, The Scripps Research Institute , 130 Scripps Way, Jupiter, Florida 33458, United States.
authors.forename: Alexander K
authors.initials: AK
authors.lastname: Price
authors.affiliation: Department of Chemistry and ‡Doctoral Program in Chemical and Biological Sciences, The Scripps Research Institute , 130 Scripps Way, Jupiter, Florida 33458, United States.
authors.forename: Brian M
authors.identifier: 0000-0002-6531-6693
authors.initials: BM
authors.lastname: Paegel
mesh_terms: D001483:Base Sequence; D002402:Cathepsin D / Q000037:antagonists & inhibitors; D020650:Combinatorial Chemistry Techniques / Q000295:instrumentation*; D004247:DNA / Q000737:chemistry*; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004867:Equipment Design; D015723:Gene Library; D006801:Humans; D056656:Lab-On-A-Chip Devices; D008863:Microspheres; D010436:Pepstatins / Q000737:chemistry / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D010436:Pepstatins; D004247:DNA; D002402:Cathepsin D; C031375:pepstatin
keywords: DNA-encoded synthesis; combinatorial compound libraries; miniaturized automation