2′-O-methylation and N6-methyladenosine enhance the oral delivery of small RNAs in mice

Studies have demonstrated that small RNAs originating from plants can be taken up by the human digestive system and play a role in regulating gene expression. However, the impact of naturally occurring RNA modifications in these small RNAs on their absorption has been underexplored. Here, we show that 2'-O-methylation (2'-O-Me), N6-methyladenosine (m⁶A), and 5-methylcytidine (m⁵C) exhibit high abundance in small RNAs (18-30 nt) derived from herbal decoction. 2'-O-Me and m⁶A promote the oral delivery of small RNAs <i>in vitro</i> and <i>in vivo</i>, whereas m⁵C did not show the same enhancement. To study the underlying mechanism of this enhancement, we used liposomes to simulate the negatively charged cell membrane and investigated the influence of these modifications on RNA-biomembrane interaction, independent of RNA stability. Our findings provide a novel perspective on the role of modifications in small RNAs, offering insights into their potential applications in oral delivery for oligonucleotides.