The long-term goal of this research program is to understand the molecular mechanism of RNA-protein recognition. The proposed research will investigate the role of stacking interactions in stabilizing RNA-protein complexes. Stacking interactions have been proposed to be a general mechanism by which proteins specifically recognize RNA, yet little is known about their energetic contribution to RNA-protein complex formation. A quantitative description of the contribution of stacking interactions to RNA-protein binding is essential for applications that seek to modify or control processes involving RNA. Experiments will focus on the RNP domain, one of the most prevalent RNA-binding domains. Proteins containing RNP domains are important in developmental pathways and are implicated in autoimmune diseases. RNP domains have three, well conserved aromatic amino acids in the RNA-binding region that are observed to participate in stacking interactions with RNA bases in structural studies. In order to probe the electronic factors that contribute to stacking interactions in RNA-RNA complexes, the aromatic residues will be systematically varied through substitutions of natural and unnatural amino acids. Small molecule model studies will be used to evaluate the stacking ability of the amino acid side chains. A correlation between the stacking ability of the amino acid side chain and the equilibrium dissociation constants of the RNA-RNA complexes will demonstrate that stacking interactions are important for binding. To probe cooperation between hydrogen bonding and stacking interactions in RNA-RNA complexes, individual hydrogen bonds will be eliminated in modified bases that are incorporated in the RNA binding site. The ability of proteins to use stacking interactions as a means to distinguish between RNA and DNA binding sites will be probed through an investigation of stacking interactions in a DNA-peptide complex. Finally, combinatorial experiments will reveal whether stacking interactions are required for RNA recognition by the RNP domain.
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