Bright and pH-sensitive Baby Spinach aptamer with RNA triplex fusion

Herein, we developed a fluorescent RNA aptamer as a pH-sensitive probe for monitoring the intercellular pH condition. We demonstrated that the designed RNA triplex structure can undergo pH-sensitive structural changes and function as a pH-nanoswitch. We then combined a previously reported fluorescent aptamer with an RNA pH-nanoswitch to facilitate it becoming pH-sensitive. Using the triplex-fused fluorescent aptamer, named Bright Baby Spinach aptamer, we successfully demonstrated that this pH probe can quickly and sensitively respond to intercellular changes in pH. Surprisingly, we found that Bright Baby Spinach aptamer showed a strong fluorescence up to 13-fold higher than that of the original aptamer in cells. A possible reason for this enhancement was that the RNA triplex structure may contribute to the appropriate folding of the aptamer to bind and stack with the fluorescent ligand 3,5-difluoro-4-hydroxybenzylidene imidazolinone. Thus, fluorescence-enhanced pH-sensitive Bright Baby Spinach aptamer has the potential for rapidly and sensitively responding to intracellular changes in pH.