Abstract 2085 Optimizing the Procedure for Synthesis of SHAPE Probe 2-methylnicotinic acid imidazolide (NAI)

RNA research has become a staple in medical advancements. A greater understanding of RNA structure is crucial to revealing the currently unknown functions of the transcriptome. There are multiple methods for analyzing RNA secondary structures but because of the complexity of these sequences, new methods of analyzing are continuously being introduced. Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is a method in which the ribose sugar is modified using a chemical probe and analyzed by primer extension for RNA secondary structure mapping. These reagents are highly reactive electrophiles with very short half-lives which has limited their commercial availability, are often synthesized right before the probing experiment, and used with limited purification. This leads to several assumptions and the need for additional control experiments because the implications or the presence of byproducts has not been explored in literature. Hence, we are creating a new robust synthetic protocol for an existing SHAPE reagent, 2-methylnicotinic acid imidazolide (NAI) with the intention to isolate high-purity imidazolide probes for SHAPE analysis. This is being done by optimizing the synthetic steps to remove any byproducts by simple washes with solvent, and filtration. Although we are developing the synthesis for NAI, our methods are extendable to other imidazolide probes and we believe our work will make these SHAPE reagents widely available for investigators with limited access to synthetic expertise and enable them to explore RNA structure using SHAPE technology. This work has been funded through Metropolitan State University of Denver through the Undergraduate Research and Creative Scholarship Program grant awarded to Luis Delgado-Garcia for spring 2024.