Project Summary/Abstract A broadly applicable cure for HIV infection remains elusive, in part because many of the viral and cellular proteins that could be targeted in cure strategies are neither enzymes nor receptors, poorly suited to conventional approaches to drug discovery. The proposed research will optimize a system for developing molecules that direct the degradation of these “undruggable” target proteins. Such an approach could be used to target proteins that are critical for immune evasion by HIV, such as the accessory proteins, or cellular proteins critical for the maintenance of viral latency, a critical barrier to viral eradication. This approach involves the innovative combination of PROteolysis-TArgeting Chimeras (PROTACs), bi- functional molecules that recruit target proteins to an E3 ubiquitin ligase to trigger their degradation, with RNA aptamers, short RNAs that can be selected to bind a protein target regardless of whether the protein is known to bind RNA in vivo. The developed RNA-based PROTACs will, with the RNA aptamer end, bind their target proteins, and, with the PROTAC end, recruit cellular machinery that ubiquitinates the target protein, thereby directing its destruction. Unlike typical drug targeting, the RNA-PROTAC need not fit into a particular site on the protein or directly inhibit its activity to function in this system. Methods to identify novel RNAs with specific protein- binding activity are well-described. High-affinity single-stranded short RNAs can be isolated in vitro from large libraries by a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX), yielding low or sub-nanomolar protein ligands. In this proof-of-concept study, two HIV proteins that bind specifically to known RNA sequences, Rev and Tat, will be employed as prototype targets. First, their known RNA targets will be conjugated to PROTACs using CLICK chemistry, delivered to cells and then tested for target degradation to optimize and validate the methods to link targeting RNAs to small molecules that recruit E3 ligases. We will also use SELEX to identify novel aptamers as the target-binding moiety, evolving modified RNAs for optimal ligand binding. The conjugation of the RNA aptamer to the PROTAC by CLICK chemistry requires the incorporation of modified ribonucleotides, which, when present in different ratios could affect target binding. The advantage of the proposed procedure is that the SELEX process will be performed with variable amounts of modified ribonucleotides to optimize targeting of the proteins of interest. In the case of our prototype targets, Rev and Tat, the sequences of the optimal modified RNA aptamers determined by SELEX may be different from their known RNA targets. The proposed experiments will develop a new strategy for drug design targeting HIV. Future studies will target other “undruggable” viral and cellular targets that could sensitize HIV-infected cells to immune surveillance and contribute to th...