Regulation of, and by RNA is crucial to a diversity of disease states. The ability of an RNA to change its 3-dimensional structure can be intimately linked to specific functions and interactions with other biomolecules (1). An RNA stem-loop structure identified at the translation start site of Collagen 1𝛂1 (Col1A1 5’SL), represents one such control element. It is known that interaction of this region with La-related protein 6 (Larp6) controls collagen synthesis, and hypothesized this occurs through dynamic RNA structure remodeling. The equilibrium between the cis and trans structures of Col1𝛂1 mRNA is unknown and has contributed to the mechanisms of the production of collagen type 1 proteins to be incomplete. We are interested in examining the Coll𝛂1 mRNA 5’SL remodeling equilibrium by developing a switchable RNA fluorescent-light up aptamer (FLAP) capable of detecting RNA remodeling. Here we reengineer the Mango II FLAP (Mango II) to create variant Mango II FLAPs to serve as fluorescent sensors of specific RNA remodeling. Feasibility assays analyzing the excitation and emission spectra indicate specific wavelengths where maximum fluorescent intensities are observed, and compared to determine fluorescent capabilities. Modified Mango II (Mango II V2) mirrors the original Mango II fluorescence within the appropriate limits, indicating successful creation of a variant Mango II FLAP. The next step, we will Integrate the variant Mango II aptamer into the 5’ stem loop of Col1𝛂1 mRNA, and test the switching between translationally active and repressed forms of Col1𝛂1 by fluorescence readout.
Shell, Audrey; Manuwai, Mauka; Hazen, Preston; Baggs, Eric; and Warner, Lisa, "Developing an RNA Fluorescent Light-Up Aptamer (FLAP) to Detect RNA Remodeling" (2021). 2021 Undergraduate Research Showcase. 32.