RNA functional control by hydrolysis reversible acylation

Reversible 2′-OH acylation is a powerful strategy for switching RNA function, but existing systems often rely on nonphysiological or cytotoxic triggers for deacylation. Here we present EST1A , a hydrolysis-responsive 2′-OH acylating reagent whose RNA adducts are efficiently removed by endogenous esterases in vitro and in cellulo . EST1A acylates model oligonucleotides, an EGFP-targeting antisense strand, and reporter mRNAs, thereby modulating their activity; notably, the acylated antisense strand shows enhanced EGFP knockdown in HepG2 cells. By tuning carboxylesterase and cholinesterase activity and comparing EST1A- acylated mCherry mRNA across noncancerous and cancer-derived cell lines, we reveal a positive correlation between intracellular esterase activity and functional recovery of acylated RNA. These results establish EST1A -mediated, hydrolysis-responsive 2′-OH acylation as a simple platform for enzyme-guided, cell-selective activation of RNA function and point toward esterase-activated RNA therapeutics. Entry for the Table of Contents Liu et al. introduce EST1A , a hydrolysis-responsive 2′-OH acylating reagent whose RNA adducts are removed by endogenous esterases or histidine, enabling reversible control of RNA function. By exploiting differences in esterase activity between noncancerous and cancer-derived cell lines, EST1A -treated mRNA exhibits enzyme-guided and cell-selective translational reactivation.