Single-molecule light-scattering imaging for in situ counting circular RNA

Abstract

The detection of circular RNA (circRNA) at the single-molecule level is essential for the elucidation of circRNA-mediated signaling pathways and its association with disease. Although many fluorescence-based methods have been used for the in situ imaging of circRNA, the inherent defects of fluorescent dyes, including high background signal and photobleaching, preclude sensitivity to the single-molecule level. Herein, leveraging the microRNA sponge feature of circRNA and the strong light-scattering signals of gold nanoparticles (AuNPs), we develop a circRNA-induced assembling of plasmon-coupled spherical nucleic acids (SNAs) strategy for in situ light-scattering imaging and counting of circRNA at the single-molecule level. The SNAs were constructed by modifying sponge probes on AuNPs, which were small and initially invisible. The microRNA sponge nature of circRNA led to the assembly of SNAs to form AuNP aggregates with enhanced light-scattering signals, which could be visualized as diffraction-limited spots irrespective of the interference signals produced by the intracellular environment. This method enabled the accurate quantification of the circRNA by counting the number of spots from a zero background, facilitating the in situ imaging and counting of individual circRNAs at the single-molecule level.