The purpose of this project is to test a new method of tagging compounds in combinatorial mixtures that should simplify structural analysis of the mixture components and increase throughput of screening of potential ligands for biological molecules. As opposed to many known tagging techniques in combinatorial chemistry, this method not only discriminates library components from one another, but also distinguishes between different positions of functional groups within the same molecule. As a result of this, the exact structure of each component in a diverse complex mixture can be derived from mass spectrometry. Because mixture synthesis can be performed much faster than synthesis of libraries of individual compounds, this method, that simplifies identification and characterization of individual mixture components, can offer an effective tool for discovery of new ligands for biopolymers. The above tagging method will be combined with affinity selection techniques and applied to screening ligands for RNA. RNA is an emerging drug target, and because of current lack of knowledge about RNA tertiary structure, combinatorial techniques are particularly effective in the discovery of new ligands. Our method of regiochemical tagging should allow one to quickly explore large diversity space of potential ligands and identify new RNA binders and inhibitors of RNA-protein complex formation. The initial work will be focused on finding new binders for the trans-activation responsive (TAR) region of the HIV-1 RNA, which plays a key role in the virus life cycle. A number of labeled scaffolds will be synthesized, and the libraries, that can be readily formed from them, will be exposed to the immobilized TAR, followed by dissociation of the complex by TAR-selective peptides, such as Tat and TRBP. Subsequent mass spectrometry analysis of the selected binders should yield complete structure of the selected binders that will then be scaled-up and used for further binding studies. The long-term goals of the project include use of the validated method to find ligands for other medicinally important RNA motifs, as well as other biopolymers.
