PROJECT SUMMARY The overall goal of this proposal is to develop a fundamentally new class of oligonucleotide agents – covalent aptamers – that will have a major impact on nucleic acid-based reagents and therapeutics. Covalent aptamers will be an enabling methodology behind new aptamer applications and will provide new solutions to persistent limitations of this compound class. Aptamers are engineered oligonucleotides that bind protein targets and have found applications in a multitude of areas, including therapeutics, diagnostics, drug delivery, and imaging. They bind their targets with affinity and specificity that rivals those of antibodies, while displaying lower production cost, higher production consistency, and the ability to easily introduce chemical modifications. Capitalizing on the ability to amplify and sequence nucleic acids, aptamers are being generated through SELEX, a powerful in vitro selection process. Our central hypothesis is that the formation of a covalent bond between an aptamer – or a functional motif delivered by the aptamer – and its target protein will provide unprecedented residence time, increase nuclease stability (or obviate the need for it), and facilitate aptamer selection. Furthermore, covalent bond formation enables new applications based on the aptamer platform. The following aims will test this hy- pothesis. Aim 1: The chemistry of covalent aptamers. To gain a comprehensive understanding of the design principles behind covalent aptamers and to enable broad applicability, we will explore their chemistry based on our promising preliminary results. We will explore applications in antibody drug conjugate assembly, as well as catalytic covalent bond formation. Aim 2: Cell-based applications of covalent aptamers. We will develop several applications that are enabled by covalent aptamers and that showcase their utility as chemical biology probes. We are focusing on cell-surface proteins due to the rich collection of aptamers that have been reported. Aim 3: Selection of covalent aptamers. For the de novo generation of covalent aptamers capable of selectively cross- linking to any chosen protein target without prior sequence and/or structural information, we will develop a gen- eralized in vitro selection process that adds distinct advantages to traditional SELEX.