Antisense Oligonucleotide Pulldown and Silencing of Circular RNA <i>Nfix</i> In Vivo in Neonatal Mouse Lungs

RNA-based therapeutics are emerging as a powerful platform for disease treatment, and one of the novel RNA molecules with therapeutic potential is circular RNA (circRNA). The ubiquitously expressed circRNAs are covalently closed, single-stranded RNA molecules formed by backsplicing and are known to regulate gene expression by either sponging microRNAs (miRNAs) or sequestering RNA-binding proteins. Several assays, including computational prediction, luciferase reporter, circRNA silencing, and pulldown assays, have been developed for functional characterization of these circRNAs. In this study, we performed a pulldown assay using a biotin-labeled antisense oligonucleotide (ASO) against circNfix to confirm the interaction of circNfix with miR204-5p in neonatal mouse lungs. Furthermore, we designed a specific GapmeR targeting circNfix and delivered it intranasally to newborn mouse pups to evaluate the effect of this specific RNA silencing on the downstream pathway in the lungs of pups exposed to hyperoxia. This is the first time a circNfix GapmeR has been targeted in vivo in an experimental neonatal disease model by intranasal delivery. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Designing divergent primers Basic Protocol 2: Designing biotin-labeled ASO to pulldown circNfix and check its association with miR204-5p Basic Protocol 3: Designing circNfix GapmeR and intranasal administration in neonatal mouse pups.