UC Irvine

DNA-encoded library (DEL) technology has revolutionized modern high-throughput drug discovery and delivered a powerful screening platform into the hands of ordinary academic laboratories, greatly expanding the community of researchers dedicated to discovering novel therapeutics. Traditionally, DELs are combinatorially synthesized small-molecule libraries accompanied by a unique DNA tag that upon sequencing reveals its synthetic history. These “on-DNA” DELs are screened for bioactive compounds through affinity selection, in which a resin-bound target of interest is incubated with the entire library, enabling high-affinity binders to be selected for identification and validation. DEL screens have been employed against a wide range of desirable therapeutic targets, including those previously considered undruggable, and generated numerous leads, some of which have progressed to clinical trials. As a potent force for early-stage drug discovery, DEL technology has continued to grow and mature, overcoming previous limitations, and solidifying its role as a mainstream screening platform. Consequently, the demand for more efficient, economical, and accessible DEL technologies has risen— particularly with the growing need for a more streamlined and automatable early lead identification process.

Our laboratory has further advanced DEL technology by introducing a bead scaffold to individual library members, pioneering one-bead-one-compound (OBOC) solid-phase DELs with new and previously unattainable capabilities. In this approach, both the library member and encoding tag are polyvalently displayed on a solid-phase DEL bead, wherein the small molecule can be cleaved from the bead via a UV-inducible photolabile linker. Solid-phase DELs are synthesized on polymer resin and screened using integrated microfluidic water-in-oil droplet architecture.Solid-phase DELs, however, are labor intensive to build, limited in screening throughput, and accompanying microfluidics present a high barrier to entry for researchers seeking to leverage the advantages of solid-phase DEL screening over traditional solution-phase DELs. These limitations necessitate the need for a new model of solid-phase DEL synthesis and screening that is automatable in nature, higher throughput, and, importantly, accessible to a wider range of research groups.

In this dissertation I address the current limitations by describing adoption and evolution of the OBOC-DEL model onto miniaturized magnetic particles, liberating solid-phase DEL screening from the constraints of microfluidics and streamlining the construction of DELs through automated synthesis. In Chapter 1 I present the application of off-DNA DEL screening in interrogation of a high-value target class considered unattainable for traditional on-DNA DELs, nucleic acid binding proteins. Using a fluorescence polarization assay, fragment-like ligands of HIV reverse transcriptase were identified as competitive binders to its nucleic acid-binding site. In Chapter 2 I describe the design and characterization of a next generation magnetic bead solid-phase DEL screening platform. A small 576-member 2-cycle DEL is constructed using automated synthesis and encoding and screened against model therapeutic target, factor Xa in emulsion droplets using magnetic hydrogel-encapsulated beads. Chapter 3 describes a statistical review into high throughput assay quality determination for replicate solid-phase DEL screening. In Chapter 4 I address current limitations of the magnetic OBOC-DEL platform and discuss it's potential future applications. Collectively, these studies establish the foundation for the next generation of solid-phase DEL screening as an automatable and distributable platform for early-stage drug discovery.