RNA functional control by hydrolysis reversible acylation

Abstract

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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/610419v2_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1cbce3org.highwire.dtl.DTLVardef@b51bc4org.highwire.dtl.DTLVardef@31e96dorg.highwire.dtl.DTLVardef@d42578_HPS_FORMAT_FIGEXP M_FIG 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. C_FIG