Specific and sensitive detection of CircRNA based on netlike hybridization chain reaction.

Circular RNA (circRNA), as a new class of biomarker, plays an important role in the occurrence and development of cancer. However, the limitations of detection methods in recent years have severely restricted the related research of circRNA. Here, we have developed an effective circRNA detection method based on the thermostatic netlike hybridization chain reaction (HCR). It combines reverse transcription-rolling circle amplification (RT-RCA) with well-designed netlike HCR to achieve dual selection and dual signal amplification, which can eliminate the interference of linear isomers. This two-dimensional netlike HCR is composed of an ingeniously designed trigger chain and two hairpin fuel probes, which can generate a stable network structure with RT-RCA products containing multiple sets of repeats at a constant temperature, thereby producing enhanced fluorescent signals. Systematic studies reveal that the optimized netlike HCR system has higher detection efficiency for DNA strands containing multiple sets of repetitive sequences, can detect circRNA as low as 0.1 pM, and has excellent selectivity. By using human tumor cell lines and tissues, it has been verified that the netlike HCR-based method can accurately detect specific circRNA in real biological samples without RNase R enrichment, which provides a simple and useful platform for detecting low-abundance circRNA. Furthermore, the proposed strategy is also a potential method for detecting some genes containing repetitive sequences, such as telomere DNA, centromere DNA and ribosomal DNA (rDNA).