Antisense Oligo Pulldown of Circular RNA for Downstream Analysis.

Circular RNAs (circRNAs) are a large family of noncoding RNA molecules that have emerged as novel regulators of gene expression by sequestering microRNAs (miRNAs) and RNA-binding proteins (RBPs). Several computational tools have been developed to predict circRNA interaction with target miRNAs and RBPs with a view to studying their potential effect on downstream target genes and cellular physiology. Biochemical assays, including reporter assays, AGO2 pulldown, ribonucleoprotein pulldown, and biotin-labeled RNA pulldown, are used to capture the association of miRNAs and RBPs with circRNAs. Only a few studies have used circRNA pulldown assays to capture the associated miRNAs and RBPs under physiological conditions. In this detailed protocol, the circRNA of interest (e.g., circHipk2) was captured using a biotin-labeled antisense oligo (ASO) targeting the circHipk2 backsplice junction sequence followed by pulldown with streptavidin-conjugated magnetic beads. The specific enrichment of circRNA was analyzed using reverse transcription quantitative PCR (RT-qPCR). Furthermore, the ASO pulldown assay can be coupled to miRNA RT-qPCR and western blotting analysis to confirm the association of miRNAs and RBPs predicted to interact with the target circRNA. In summary, the specific pulldown of circRNA using this quick and easy method makes it a useful tool for identifying and validating circRNA interaction with specific miRNAs and RBPs.