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DEL technology has gained popularity as an adjunct to high-throughput screening techniques, offering expanded capabilities for investigating novel chemical entities. Typically, DELs comprise mixtures of DNA-small molecule conjugates, in which the DNA tag reflects the synthesis history of the combinatorial library member. These DELs, termed "on-DNA" DELs, undergo screening using affinity selection, and the DNA is sequenced to decode compounds which bind to specific protein targets. With modern DNA sequencing technologies, DEL can explore unique binding interactions in a single experiment, which has led to the discovery of ligands for various targets and the identification of numerous clinical candidates. More recently, the use of DELs in drug discovery has also spurred computational intervention, as the inherent properties of combinatorial chemistry yield highly structured and modular datasets ripe for informatics and machine learning-based analyses.We have previously pioneered solid-phase one-bead-one-compound (OBOC) DEL technology, introducing an innovative approach to DEL synthesis that unlocks new avenues for screening. In this method, a DEL library member is polyvalently coupled to a bead via a photolabile bond, alongside covalently bound DNA-encoding tags. Upon UV-exposure, the library member is released into the surrounding solution where it may engage with and modulate the activity of a target of interest. We have previously demonstrated the efficacy of activity-based OBOC-DEL "off-DNA" screening for novel enzyme inhibitors, in competition-based fluorescence polarization screening, and for the prediction of compound potency.In this dissertation, I build on this prior work and present advances in DEL library design and synthesis principles alongside the development of assays that enable the use of DEL in in vitro systems. Chapter 1 contains a detailed protocol summarizing improved synthesis and quality control procedures for OBOC-DELs, accessible by both acade

Over the last few decades, the development and application of advanced fluorescent probes have revolutionized our ability to assess complex biological processes visually and quantitatively with unprecedented specificity. Employing these probes alongside sophisticated microscopy techniques, this dissertation explores the development and application of diverse technologies within biological research to enhance our understanding of RNA biology, drug interactions, and cellular mechanisms.The use of fluorescent probes in real-time metabolic labeling of RNA, both in cellulo and in vivo, is explored. This approach provides critical insights into RNA dynamics, enabling the precise measurement of RNA expression patterns that are pivotal for understanding cellular function in health and disease.The development of a fluorescent reverse-transcription assay for detecting covalent interactions between small molecules and RNA is detailed. This method offers valuable perspectives on RNA modifications and their potential implications in therapeutic development, particularly through the identification of small molecule binders that could lead to innovative treatment strategies. Novel photocrosslinking fluorescent probes were developed for a new spatial transcriptomics technology employing spatially restricted cell tagging by confocal microscopy and cellular isolation with fluorescence-activated cell sorting. This technique allows for high- resolution mapping of gene expression within specific cellular contexts, thus deepening our understanding of the spatial and temporal dynamics of gene activity crucial for comprehending complex biological phenomena.The exploration of drug permeability through the development of a new fluorescence cell-based assay highlights how this technology can assess the bioavailability of pharmacologically relevant molecules, particularly those that challenge the conventional boundaries of drug design. By enabling the direct measurement of compound accumulatio

High-throughput screening is the foundation for modern small molecule discovery, but DNA-encoded library (DEL) technology has emerged as an alternative approach to synthesizing and screening large collections for novel bioactive chemical matter. Currently, DEL screening is limited to affinity selection binding assays and, more recently, biochemical activity-based assays. This thesis describes the engineering of a cellular DEL activity assay that can serve as the basis for DEL screening on live cells. DEL beads were modified to support growth of cells and report on cellular activity using standard cellular reporter assays. Design of this platform took into account cell phototoxicity, cell viability and growth, and design of a model cellular signal transduction assay. This thesis successfully demonstrates proof-of-concept compatibility of the developed cellular assay with solid-phase DEL chemistry, paving the way for cellular phenotypic DEL screening technology.

High-throughput screening is the foundation for modern small molecule discovery, but DNA-encoded library (DEL) technology has emerged as an alternative approach to synthesizing and screening large collections for novel bioactive chemical matter. Currently, DEL screening is limited to affinity selection binding assays and, more recently, biochemical activity-based assays. This thesis describes the engineering of a cellular DEL activity assay that can serve as the basis for DEL screening on live cells. DEL beads were modified to support growth of cells and report on cellular activity using standard cellular reporter assays. Design of this platform took into account cell phototoxicity, cell viability and growth, and design of a model cellular signal transduction assay. This thesis successfully demonstrates proof-of-concept compatibility of the developed cellular assay with solid-phase DEL chemistry, paving the way for cellular phenotypic DEL screening technology.

DNA-encoded library (DEL) technology is a powerful next-generation drug discovery engine. DELs permit the design and synthesis of large, customized molecular libraries, which can be mined for new ligands by affinity selection. More advanced screening technology has emerged that enables direct activity-based DEL screening, but to date, this is limited to biochemical activity assays. Cellular assays, which are vital for discovering physiologically relevant novel chemical matter, are still incompatible with DEL. Here, I describe a prospective workflow that renders DEL compatible with cellular activity assays. Cells are grown directly on DEL beads, which effectively serve as microcarriers. The cells are engineered to secrete a reporter upon stimulation with an agonist, which is liberated from the DEL bead via photocleavage. The microcarrier is functionalized with affinity tag ligands, which capture the affinity-tagged secreted reporter. Toward this goal, I have engineered cell lines that secrete HaloTag-labeled fluorescent protein reporters, shown that reporter cell lines can grow and signal on microcarriers, and explored photocleavage conditions that permit efficient photochemistry while preserving cell viability. Additional studies were conducted to identify appropriate positive control ligands of a model cellular target, STING (stimulator of interferon genes). This thesis lays the groundwork for a DEL-compatible cellular assay paradigm that can be adapted to an array of cell-based assay strategies.

DNA-encoded library (DEL) technology is a powerful next-generation drug discovery engine. DELs permit the design and synthesis of large, customized molecular libraries, which can be mined for new ligands by affinity selection. More advanced screening technology has emerged that enables direct activity-based DEL screening, but to date, this is limited to biochemical activity assays. Cellular assays, which are vital for discovering physiologically relevant novel chemical matter, are still incompatible with DEL. Here, I describe a prospective workflow that renders DEL compatible with cellular activity assays. Cells are grown directly on DEL beads, which effectively serve as microcarriers. The cells are engineered to secrete a reporter upon stimulation with an agonist, which is liberated from the DEL bead via photocleavage. The microcarrier is functionalized with affinity tag ligands, which capture the affinity-tagged secreted reporter. Toward this goal, I have engineered cell lines that secrete HaloTag-labeled fluorescent protein reporters, shown that reporter cell lines can grow and signal on microcarriers, and explored photocleavage conditions that permit efficient photochemistry while preserving cell viability. Additional studies were conducted to identify appropriate positive control ligands of a model cellular target, STING (stimulator of interferon genes). This thesis lays the groundwork for a DEL-compatible cellular assay paradigm that can be adapted to an array of cell-based assay strategies.