Easily Used Kits to Evolve Reagents that Covalently Tag and Inactivate Proteins Foundation for Applied Molecular Evolution Steven Benner ABSTRACT Under PAR-19-253, the NIGMS seeks new technologies that create a positive feedback loop that drives science forward by allowing new questions to be asked and new discoveries to be made, which in turn drives the development of new technologies. The Benner group has, for 30 years, contributed to this NIGMS vision, developing new technologies for NextGen DNA sequencing and NextGen DNA synthesis, bioinformatic and evolutionary analyses, dynamic combinatorial chemistry for drug discovery, protein engineering, and new platforms that make multiplexed diagnostics easy, platforms used today to manage the COVID pandemic. Here, we offer the NIGMS another transformative tool, a platform to allow researchers to choose a protein target and create a reagent (an AEGISZyme) that chemically transforms bound proteins. Such reagents have been sought for 40 years with only limited success. We will focus on one transformation: AEGISZymes that add an acyl group to an amino group on a lysine of the bound target, where the acylation reagent is an ester. This acylation may inactivate the targeted protein, allow- ing researchers to test hypotheses about the role of that protein in biology. It may carry a payload which, when internalized with the target protein, carry drugs or stabilized AEGISZymes into a cell. It may fluorescently tag the protein to help clinicians cut away fluorescing cancer cells selectively as they resect a tumor. To achieve this transformative and innovative outcome, we will apply laboratory in vitro evolution (LIVE) to artificially expanded genetic information systems (AEGIS). The platform will be delivered by meeting 3 Aims: Aim 1. We will use AEGIS-LIVE to deliver AEGISZymes that acylate lysines in target proteins with pass/fail reaction times of <10 sec-1. To test this, we will create these AEGISZymes that use a co-substrate carrying an ester group for three targets. Rates of the selected AEGISZymes will be quantitated, specificity will be metricked against similar targets with slightly different amino acid sequences, and modification sites will be found, Aim 2. We will use AEGIS-LIVE to deliver AEGISZymes that acylate lysines on researcher-chosen targets with turnover, with pass/fail turnovers of >1000 and kcat/KM of >105 M-1 sec-1. Turnover rates will be metricked under physiological and laboratory conditions, and correlated to duplex stability from thermodynamic data. Aim 3. We will use AEGIS-LIVE to deliver mirror AEGISZymes that are stable in biological systems, including transport into cells. This will allow AEGISZymes to be used in biological media, to support nanotrain toxin delivery, and to set the stage to use these molecules in vivo. Aim 4. We will test the scope of the platform to address design parameters, such as how long random regions should be, how good loops are as full protein sur...