CIRDES: an efficient genome-wide method for in vivo RNA–RNA interactome analysis†
Complex RNA–RNA interactions underlie fundamental biological processes. However, a large number of RNA–RNA interactions remain unknown. Most existing methods used to map RNA–RNA interactions are based on proximity ligation, but these strategies also capture a huge amount of intramolecular RNA secondary structures, making it almost impossible to detect most RNA–RNA interactions. To overcome this limitation, we developed an efficient, genome-wide method, Capture Interacting RNA and Deep Sequencing (CIRDES) for in vivo capturing of the RNA interactome. We designed multiple 20-nt CIRDES probes tiling the whole RNA sequence of interest. This strategy obtained high selectivity and low background noise proved by qRT-PCR data. CIRDES enriched target RNA and its interacting RNAs from cells crosslinked by formaldehyde in high efficiency. After hybridization and purification, the captured RNAs were converted to the cDNA library after a highly efficient ligation to a 3′ end infrared-dye-conjugated RNA adapter based on adapter ligation library construction. Using CIRDES, we detected highly abundant known interacting RNA, as well as a large number of novel targets of U6 snRNA. The enrichment of U4 snRNA, which interacts with U6, confirmed the robustness of the identification of the RNA–RNA interaction by CIRDES. These results suggest that the CIRDES is an efficient strategy for genome-wide RNA–RNA interactome analysis.
- This article is part of the themed collection: Analyst Emerging Investigators