Site-Selective RNA Modification by Programmable DNA-Small Molecule Conjugates

Efficient strategies for site-selective modification of native RNA are highly important for advancing fundamental research of RNA biology and translational RNA therapeutics. Previous approaches generally rely on the catalytic properties of enzymes or nucleic acids, such as repurposed transferases or in vitro selected ribozymes. However, these methods suffer from sequence bias and complicated production processes and often require proper folding of the biomolecules, limiting their programmability, rational design, and robustness. Here, we present a site-selective RNA acylation strategy with a DNA-DMAP (4-dimethylaminopyridine) conjugate, considerably smaller in size than enzymes or ribozymes. This conjugate hybridizes to the target RNA region and catalyzes an acyl transfer reaction from pentafluorophenyl (PFP) esters to a proximal 2'-OH group. By programming the DNA-DMAP sequence, the predetermined sites and often their adjacent sites in RNA can be selectively acylated with high conversion and fast reaction rates, making this strategy applicable to RNAs of varying lengths, including short synthetic RNAs, native 5S ribosomal RNA (rRNA), and eGFP mRNA. The azide-bearing acyl donors enable further chemical functionalization at the acylated sites via click reaction, establishing this approach as a versatile and general platform for in vitro site-selective RNA labeling and functionalization.