oShark: A Modified Nuclease-Resistant Light-Up Aptamer

Owing to their three-dimensional folding, light-up RNA aptamers selectively bind and activate the fluorescence of their cognate fluorogen. These molecules have a wide scope of applications in vivo and in vitro. For instance, light-up RNA aptamers can be used as fluorescent reporters for the specific detection of target molecules. However, their use in complex media, especially in the extracellular environment, is restrained by the low chemical stability and rapid degradation of the RNA backbone, which impacts their half-life and, thus, their function. Although introducing chemical modifications, such as 2'-fluorinated nucleotides, can increase the aptamer chemical stability, it often interferes with their folding and/or affects fluorogen recognition and activation. In this work, we used a directed evolution strategy combining microfluidic-assisted screening and rational design to develop oShark, a fluorinated light-up aptamer that activates the fluorescence of the fluorogenic dye dimer Gemini-552 while preserving the aptamer integrity and function even in challenging extracellular biological media. The selected mutations likely act by promoting optimal folding of the modified aptamer, allowing it to reach the same level of fluorescence and brightness as the unmodified parental oCoral RNA. We further engineered oShark by fusing it with the light-up RNA Mango-II and optimizing the connecting sequence. This yielded Lemon-oShark, the first modified dual light-up aptamer.