Glutathione‐Responsive Acyl‐Modifications for Targeted RNA Decaging and Prolonged Protein Synthesis

A series of disulfide-based, self-immolative acyl conjugates have been designed and tested for reversibly protecting the ribose 2'-hydroxy position of RNA. These modifications were found to be responsive to endogenous glutathione levels for efficient RNA recovery both in solution and cultured cells. Through this modification strategy, we achieve shielding of the biomolecule from both nuclease enzymes and translational machinery, allowing for a tunable release of RNA with improved cellular stability and minimal innate immune activation. The self-immolative RNA deprotection includes a cyclization step which exhibits differing kinetic profiles based on ring size and electronic properties. Moreover, we demonstrate sustained target protein production for both green-fluorescent protein and nanoluciferase in cell models. Both acylated mRNA models resulted in significant increases in protein expression compared to the unmodified mRNA control, with increases in protein expression of up to 600% and sustained higher protein expression of modified mRNA samples observed over 120 h in comparison to unmodified RNA. These exciting results highlight the versatility of our approach in the design of next-generation RNA-based prodrugs.