Construction of a fluorescence sensing platform based on RISC and CRISPR/Cas12a for the assay of Ago2 enzyme activity

Purpose: Argonaute 2 (Ago2) is a protein with nucleic acid endonuclease activity and is ubiquitous in eukaryotic cells. Ago2 is a core member of RNA interference and is involved in important gene regulation in the body and the development of multiple tumors by affecting oncogenic or oncogenic gene expression. Ago2 is a potential tumor biomarker. Therefore, the assay of Ago2 activity can help monitor the disease progression and prognosis and contribute to the study of the mechanism of disease development. Methods: Ago2-based nucleic acid endonuclease activity cleaves the target hairpin nucleic acid chain and progressively stimulates the hybridization chain reaction (HCR) in the sensing strategy, causing signal amplification to form specific dsDNA, which then triggers the powerful non-selective single nucleic acid chain cleavage activity of the CRISPR/cas12a system and releases the fluorescence signal in the reporter probe. Results: The sensitive assay of Ago2 enzyme activity was achieved using the biofunctional properties of RNAinduced silencing complex (RISC) and CRISPR/Cas12a. Under the optimized conditions, the fluorescence signal intensity of the assay was positively correlated with the activity concentration of Ago2, with an excellent linear response range of 0.02-20 nM and a limit of detection (LOD) of 10 pM. When applied to the assay of Ago2 in cell lysates, the experimental recovery was 93.33%-107.08%, and the sensing platform was able to detect the Ago2 content in the presence of interference specifically. Conclusion: Therefore, the fluorescence sensing platform successfully constructed in this paper is suitable for the assay of endogenous Ago2 activity in complex samples, and the assay method has the advantages of high specificity, sensitive detection, easy operation and low instrumentation requirements, which is expected to be applied to the actual sample assay in clinical settings. Meanwhile, it provides an assay direction for the biofunctional analysis of nucleases and a feasible direction for applying RISC and Crispr/Cas12a in sensing analysis.