Three-way junction-mediated three-letter coded SDA cascade CRISPR/ Cas12a system for circRNA detection

Circular RNAs (circRNAs), a special type of non-coding RNAs with covalently closed circular structure, have emerged as promising liquid biopsy biomarker. However, the precise detection of circRNAs is severely hampered by interference from homologous linear RNAs, posing a significant obstacle for their applications in molecular diagnostics. Herein, a strategy named three-way junction (TWJ)-mediated three-letter coded strand displacement amplification (SDA) cascade CRISPR/Cas12a system (TTSC) has been devised to achieve precise detection of circRNAs. Three-letter coded SDA (TC-SDA) has been originally designed in this study for eliminating undesired interference from linear RNA and performing dual functionality in both discrimination and amplification. With this design, the proposed strategy elegantly distinguishes between circRNA and homologous linear RNA through proximity effect of TWJ structure and non-specific amplification blocking ability of the three-letter code. Ultimately, this strategy successfully identified circRNA down to 0.1 % abundance. In addition, benefiting from the synergistically accelerated multiple signal amplification in one step, TTSC strategy exhibited good linear relationship of 0.5-1000 pM with a detection limit of 80.6 fM. Notably, the recovery test indicated that the proposed strategy has good prospects for clinical applications. Therefore, TTSC is expected to provide an ideal platform for circRNA detection in biotechnology research and molecular diagnostics.