High-Throughput RNA Structure Analysis

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It once required months to sequence a gene; whole bacterial genomes are now analyzed in this same period. In contrast, RNA secondary and tertiary structure analysis technology is almost unchanged from that in use 20 years ago. It still typically requires months to analyze the secondary structure of an RNA spanning a few hundred nucleotides. This is a serious deficiency because RNA function can only be understood in the context of the pattern of base pairing and tertiary interactions unique to each RNA. When 2'-amine groups in RNA are substituted for the 2'-hydroxyl normally found in RNA, nucleophilic attack of this amine to form a 2'-amide is catalyzed by the adjacent 3'-phosphodiester. Exploratory work indicates that it is possible to exploit a closely related principal to create a structure-specific approach for modifying the normal 2'-hydroxyl constituent of RNA nucleotides. The RNA 2'-hydroxyl will react with select anhydrides in a reaction that requires that the nucleotide is not base paired or involved in higher order tertiary interactions. The overarching research goal of this project is to use this 2'-hydroxyl-based chemistry to create a high-throughput technology for RNA structure analysis. Objectives for this project include: structure-function studies of anhydride-based 2'-hydroxyl chemistry, workflow optimization to facilitate high-throughput RNA structure analysis, and application of this technology to understand structural consequences of mutations in mitochondrial tRNAs.Broader Impacts. First, an efficient high-throughput RNA structure analysis technology has the potential to transform many fields of RNA science, perhaps in ways similar to how DNA sequencing has transformed genomic analyses. Second, the large expansion of the research endeavor at major universities in recent years has exacerbated a hyper-specialization in which teaching and science outreach often become divorced from research. Steadfast efforts will be continued to strive towards excellence in teaching, to incorporate undergraduate students into creative laboratory research, and to develop substantial science outreach activities for elementary school children.