Optimization of the Allosterically Regulated RNA Aptamer 5GR-II for in vivo Implementation Using Fluorescence Detection
Wilkey, Clayton J.
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Allosterically regulated RNA aptamers have been observed in nature and have recently been exploited to target specific molecules of interest based on in vitro selection. RNA aptazymes have been developed as a detection method for small molecules that could potentially replace the use of antibodies, but a roadblock that has yet to be overcome is the straightforward implementation of the aptazymes into cellular context. This study optimizes the DFHBI-1T binding RNA aptamer “Broccoli” paired with the 5-HTP scaffolded guanine riboswitch (GR) via a communication module (CM). Varying ion concentrations were investigated at both 25°C and 37°C (for pseudo-cellular context) to determine if the 5GR-II aptamer could be implemented into a cellular environment. The data suggest that the Broccoli aptamer is K+ dependent to bind to DFHBI-1T in the absence of sufficient 5-HTP. Optimal fold induction and KD occurred with the 5GR-II (U5) aptamer in standard ion conditions (Mg2+ [10 mM], Na+ [250 mM], and K+ [50 mM]) at 37°C.