Abstract. Innovative approaches are needed to create therapeutics that target HIV. Existing drugs can prolong patient lifespan by targeting multiple facets of the viral life cycle, but next-generation therapies are needed that act on new targets — especially those that resist mutation — to improve long-term therapeutic compliance and outcome. HIV-1 TAR RNA is a validated drug target that resists mutations to interact with the viral protein Tat, thus giving rise to an RNA-protein complex essential for proviral transcription and HIV-1 propagation. So far, TAR has evaded discovery of compounds with sufficient affinity and selectivity to warrant pharmaceutical development. To address this challenge, we undertook a ‘semi-design and protein evolution’ approach that yielded many novel, high-affinity (KDs ~ 1.3 to 0.5 nM) TAR Binding Proteins (TBPs) using yeast display maturation. We then determined the 1.80 Å resolution co-crystal structure of one variant, TBP6.7, in complex with TAR, revealing that the major binding interface consists of evolved loop β2-β3, which reads out the TAR RNA major groove. We hypothesize that cyclic peptides comprising the TBP6.7 β2-β3 loop, or other TBP loops evolved in our lab, will be entry points to create a novel class of TAR binders. Indeed, the TBP6.7 β2-β3 hairpin retains affinity and specificity for TAR when fused to the small protein SUMO, signifying that the β2-β3 loop is necessary and sufficient for TAR recognition. Structural identification of the β-hairpin motif, and our use of semi-design and evolution make our approach fundamentally different from prior efforts to block the Tat-TAR interaction, while providing a robust experimental premise to pursue our aims: (Aim 1) Validate the observed TBP6.7-TAR interface and determine additional novel co-crystal structures of other TBPs evolved in our lab;; (Aim 2) synthesize and optimize cyclic peptides derived from Aim 1 that bind TAR and inhibit its interaction with Tat;; (Aim 3) Test cyclic peptides from Aim 2 using viral infectivity assays to investigate mechanisms of action, therapeutic indices, and pharmacological properties in animals. To our knowledge, no other group has used protein evolution and structural biology to develop HIV-1 TAR-targeted reagents. We are a team of experts, comprising two P.I.s, with strong records in protein evolution, peptide-based drug discovery, HIV therapeutic discovery, measuring cell penetration and toxicity of biologics (McNaughton), and structural biology of therapeutically-relevant RNAs, protein-RNA complexes, and biophysical analysis of protein-RNA interactions (Wedekind), as well as two collaborators: Harold Smith (University of Rochester), a leader in drug discovery and development, and CEO of OyaGen ...