Use of conformationally locking antisense oligonucleotides for the targeted inhibition of precursor microRNAs

Antisense oligonucleotides (ASOs) are widely used as therapeutics. One class of ASOs enhances protein expression by sequestering the mature microRNA (miRNA) in a double-stranded structure within the RNA-induced silencing complex (RISC). An alternative approach for the targeted control of gene expression is to use ASOs that bind to precursor miRNAs (pre-miRNAs) and modulate their enzymatic processing. Here, we demonstrate that ASOs that disrupt the junction of pre-miR-31, a region we previously identified as a critical regulatory element, are potent inhibitors of Dicer/TRBP processing in vitro but do not inhibit Dicer/TRBP binding. Furthermore, we extend and validate this strategy to pre-miR-144, which has a similar junction-dependent structure-function relationship. Through analysis of pre-miRNA secondary structures, we find that nearly 20% of human pre-miRNAs are predicted to contain junctions, and we validate our ASO approach on several members of this group. Importantly, we also verify the application of junction-targeting ASOs for the specific inhibition of pre-miRNA processing in cellulo. Our study reemphasizes the important role of RNA structure in regulating Dicer/TRBP processing of pre-miRNAs and provides the framework to develop structure-informed ASOs that serve to inhibit miRNA production.