Ashton A. Shaffer
Mentor: Dr. Susan A. White
Ribonucleic acid (RNA) is an essential macromolecule in biological systems. The tertiary structure of the RNA is critical because it directs protein recognition and binding which in turn enables functions like regulation. When RNA is double stranded, it typically adopts a helical structure that consists of traditional Watson-Crick base pairing in which hydrogen bonds form between the nitrogenous bases guanine-cytosine and adenine-uracil. In some instances, the structure of the helix is interrupted by a sharp bend, known as a kink-turn. Kink-turned RNA consists of two asymmetrical strands with nucleotides that base pair without conforming to Watson-Crick rules and usually contains three nucleotides that are unpaired altogether. The proteins that interact with kink-turned RNA are specifically structured to chemically recognize its unique shape and interact with it to form protein-RNA complexes. We intend to study the characteristics of kink-turned RNA in comparison to base-paired RNA in order to better understand how RNA-protein complexes form and function.
Our purpose is to experimentally compare kink-turned (KT) and base-paired (BP) RNA using a variety of different methods. First, we will synthesize and purify both variants (KT and BP) and use a method called analytical ultracentrifugation to perform sedimentation experiments, which utilize fluid dynamic properties to distinguish between differently structured substances. Secondly, we will perform chromatography experiments to determine whether or not the kink-turn affects the mobility of the RNA in the columns. Next, we will perform thermodynamic denaturation experiments to differentiate the relative stabilities of the two types of RNA. Finally, we will employ atomic force microscopy to better visualize the actual structural differences between KT and BP RNA. For each of these individual experiments, we will also test whether or not the presence of magnesium salts affect the shape or motility of the RNAs. Our ultimate goal is to be able to collect information about the structure and behavior of KT RNA by comparing it to BP RNA using the aforementioned methods. By substantiating our knowledge of the characteristics of kink-turned RNA, we will be better able to understand RNA-protein interactions in general and how structural differences affect their behavior and biological functioning.