Abstract
Many experimental studies require the isolation of multi-subunit RNA-protein complexes for detailed analysis and characterization. It would be highly advantageous if an investigator could site-specifically label the RNA molecule(s) in such a native complex with a probe, such as a flourophore, to aid the characterization and biochemical as well as structural study of the complex. However, the commonly used RNA labeling techniques require chemical modifications of RNA that either cannot be done on a natively purified RNA or may be deleterious to the structure of an assembly. I propose an RNA labelling strategy that is gentle and simple and accomplished following purification of a native RNA-protein complex. The strategy uses: a small truncated RNA binding protein (U1A), fused with ten or six consecutive Histidine residues or a His-tag at the N-terminus; a short RNA stem-loop recognized by U1A; and a chemical reagent that recognizes the His-tag fused to U1A. The combination of the His-tagged U1A bound to a short RNA stem-loop and the His-tag bound to a functional chemical probe can serve as a ‘binding module’. Such a binding module can be made to recognize a specific RNA sequence in a target RNA molecule by including in the RNA stem-loop of the module a single-stranded sequence that is complementary to a single-stranded sequence in the RNA target. Importantly, the His-tag on the U1A protein serves two uses: (1) for purification of the U1A protein; and (2) as the binding target for a reagent composed of a tris-NTA moiety conjugated with a functional probe, such as a gold nanoparticle or flourophore. In this thesis, I describe the preparation of the U1A protein, the RNA short stem-loop RNA molecules to which the U1A protein binds, and the design and preparation of the target RNA molecules to establish the utility of the ‘binding module’ approach for RNA labeling.