Abstract
We present a robust approach for cellular detection, imaging, localization, and quantification of human and viral encoded circular RNAs (circRNA) using amplified fluorescence in situ hybridization (ampFISH). In this procedure, a pair of hairpin probes bind next to each other at contiguous stretches of sequence and then undergo a conformational reorganization which initiates a target-dependent hybridization chain reaction (HCR) resulting in deposition of an amplified fluorescent signal at the site. By harnessing the capabilities of both ampFISH and single-molecule FISH (smFISH), we selectively identified and imaged circular RNAs and their linear counterparts derived from the human genome, SARS-CoV-2 (an RNA virus), and human cytomegalovirus (HCMV, a DNA virus). Computational image processing facilitated accurate quantification of circular RNA molecules in individual cells. The specificity of ampFISH for circular RNA detection was confirmed through an in situ RNase R treatment that selectively degrades linear RNAs without impacting circular RNAs. The effectiveness of circular RNA detection was further validated by using ampFISH probes with mismatches and probe pairs that do not bind to the continuous sequence in their target RNAs but instead bind at segregated sites. An additional specificity test involved probes against the negative strands of the circular RNA sequence, absent in the cell. Importantly, our technique allows simultaneous detection of circular RNAs and their linear counterparts within the same cell with single molecule sensitivity, enabling explorations of circular RNA biogenesis, subcellular localization, and functions.