Nonstandard base pairs in RNA often play important roles in RNA structure and function. These non- Watson/Crick base pairs are abundant in the thousands of guide RNA:mRNA complexes in the unique and essential RNA editing system of trypanosomes. The role of these nonstandard base pairs is unclear but is essential to understand for the design of drugs that target these RNAs, stop RNA editing, and kill trypanosomes. Our central hypothesis is that the effect of nonstandard base pairs on RNA editing, stability, and structure in these RNA complexes is dependent on their nucleotide sequence context.
In specific aim 1, we test the effects of nonstandard base pairs on RNA editing efficiency with designed RNA editing substrates. Those substrates that lead to interesting effects on RNA editing will be selected for further study in specific aims 2 and 3.
In aim 2, we will determine the contribution of nonstandard base pairs to thermodynamic stability and relate this knowledge back to RNA editing efficiency.
In aim 3, we will determine the impacts of nonstandard base pairs on the overall shape ofthe RNA at low resolution (10-20 A) with small angle X-ray scattering and around the nonstandard base pair at high resolution (better than 2.5 A) using X-ray crystallography. The structural information will be interpreted in terms ofthe biochemical and thermodynamic data from aims 1 and 2. The pursuit of aim 3 will be greatly enhanced by the Oklahoma COBRE in Structural Biology through collaborative interactions with other COBRE and OSBN members, by mentoring from senior faculty members, and by access to COBRE Core Facilities. The results of this project will contribute to the development of better therapies to treat debilitating and often fatal infections with trypanosomal parasites, which infect 500,000 immigrants in the United States and threaten 600 million people worldwide.
This project uses biochemical, biophysical, and structural biology (S/ XS and X-ray crystallography) approaches to study the role of RNA structure in the unique RNA editing pathway found in trypanosomes. The results will define the common features of gRNA:mRNA duplexes that are targets for design of novel therapies that will kill trypanosomal parasites in infected patients by disabling RNA editing.
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