Searching for RNA-Protein Interactions: Methodology to Screen Library of Artificial Proteins for RNA-Binding

Abstract

The development of functional and stable artificial proteins has become a major goal in synthetic biology. As this field advances, screening these proteins for various activity are crucial in finding protein structures that are functional. The goal of this thesis was to develop a methodology to screen an artificial protein library specifically for RNA-binding ability by expressing the pyr attenuation mechanism in Bacillus subtilis (B. subtilis) in Escherichia coli (E. coli) as a biorthogonal approach. In developing this assay, the pyr attenuator, which regulates transcription in B. subtilis depending on its secondary structure, was placed upstream of an antibiotic resistance gene. Because the attenuator’s interaction with the PyrR protein dictates downstream gene expression, cell survival in the presence of an antibiotic could serve as a preliminary measure of protein-RNA interactions. In this project, various conditions were tested to define the necessary screening conditions for a protein library. It was found that the addition of a small molecule, uracil, allowed for clearer assay thresholds. However, further work must refine this methodology for it to serve as a potential method to screen artificial protein libraries for RNA-binding abilities based on gene expression and cell survival. If successful, this method could be a powerful way to identify de novo proteins with RNA-binding ability.