Uracil‐Selective Cross‐Linking in RNA and Inhibition of miRNA Function by 2‐Amino‐6‐vinyl‐7‐deazapurine Deoxynucleosides

Abstract

MicroRNAs (miRNAs) regulate gene expression through RNA interference. Consequently, miRNA inhibitors, such as anti‐miRNA oligonucleotides (AMOs), have attracted attention for treating miRNA overexpression. To achieve efficient inhibition, we developed 2‐amino‐6‐vinylpurine (AVP) nucleosides that form covalent bonds with uridine counterparts in RNA. We demonstrated that mRNA cross‐linked with AVP‐conjugated antisense oligonucleotides with AVP were protected from gene silencing by exogenous miRNA. However, endogenous miRNA function could not be inhibited in cells, probably because of slow cross‐linking kinetics. We recently developed ADpVP, an AVP derivative bearing a 7‐propynyl group—which boasts faster reaction rate than the original AVP. Here, we synthesized dADpVP—a deoxy analog of ADpVP—through a simplified synthesis protocol. Evaluation of the cross‐linking reaction revealed that the reaction kinetics of dADpVP were comparable to those of ADpVP. In addition, structural analysis of the cross‐linked adduct discovered N3 linkage against uridine. Incorporating dADpVP into two types of miRNA inhibitors revealed a marginal impact on AMO efficacy yet improved the performance of target site blockers. These results indicate the potential of cross‐linking nucleosides for indirect miRNA function inhibition.