Probing specificity of RNA : ribonucleoprotein interactions through in vitro selection

RNA binding proteins play a crucial role in normal cellular functions. However, little work has been successful in developing a code of recognition that may be exploited for the creation of novel RNA binding proteins. Research presented here has attempted to discern applicable specificity rules of the most commonly found RNA binding motif, the ribonucleoprotein (RNP) domain. I have employed in vitro selection to facilitate this, as it has been utilized in the past to discover the natural binding sequences of nucleic acid binding proteins. The process of in vitro selection results in the evolution of nucleic acid binding species, or aptamers. In order to perform these experiments within the span of my graduate career, I have developed an automated robotic workstation that is capable of performing in vitro selection. I have performed experiments that validate this automated system, and have further confirmed that it can successfully generate aptamers to protein targets possessing high specificity and affinity to their protein ligand. Moreover, this automated selection system has been able to recapitulate the natural sequence and structural specificity of the RNP-containing protein target used as a model system here, U1A. This evolutionarily conserved spliceosomal protein is chosen due to the extensive amount of biochemical and structural data available regarding binding to its cognate RNA. Twenty-one in vitro selection experiments have been executed in an automated manner against U1A point mutants. The results of these selections suggest that in vitro selection can be used as a tool by which determinants of specificity may be elucidated. Additionally, these selections have uncovered new information regarding U1A cognate specificity and affinity not currently known to the community.