What techniques can be used to identify and characterize circRNA-protein interaction?

One technique is to use an RNA pull-down assay. Biotinylated DNA oligo probes are used to pull down circRNAs and associated proteins in cell lysates. This can be followed by Western blotting or mass spectrometry to analyze circRNA binding proteins. Overexpression of specific circRNAs and appropriate controls can be used to confirm RNA-protein interactions quantitatively. Another method is the RNA binding protein immunoprecipitation Assay (RIP) followed by circRNA Sequencing. This involves the immunoprecipitation of a protein of interest, followed by analysis of associated RNA using circRNA sequencing. Comparison with appropriate controls can help correlate pulled down proteins and circRNAs, providing evidence of specific protein-circRNA interactions. A third method is analysis of circRNAs and protein co-localization by microscopy: Fluorescence in situ Hybridization (FISH/ISH) techniques can be used to detect circRNA transcripts, while immunofluorescence staining can detect the position and abundance of proteins with specific antibodies. Co-localized signals for both RNA and protein indicate complex formation, providing visual evidence of circRNA-protein interactions. Lastly, one can use an RNase protection assay (RPA). RNase H is used to cleave a target RNA molecule at a specific site hybridized with a DNA probe. Protein binding to the RNA prevents cleavage, indicating a site of interaction between protein and RNA. Multiple small oligonucleotide probes can be used to map the entire RNA sequence for sites of interaction.