Proteolysis-Responsive Rolling Circle Transcription Assay Enabling Femtomolar Sensitivity Detection of a Target Protease Biomarker

Proteases play crucial roles in the malignant progression of tumor and thus have been regarded as biomarkers for many cancers. Although protease assays such as immunoassays and fluorogenic substrate probes have been developed, it remains challenging for them to give consideration to both sensitivity and accuracy. Here, we describe a proteolysis-responsive rolling circle transcription assay (PRCTA) for the ultrasensitive and accurate detection of protease activities by the rational integration of a protease-responsive RNA polymerase and rolling circle transcription. Taking cancer biomarker matrix metalloproteinase-2 (MMP-2) as the model, the PRCTA, which can transduce and amplify each proteolysis event catalyzed by MMP-2 into the output of multiple tandem fluorescent RNAs by in vitro transcription, is constructed for the sensitive analysis of MMP-2 activities. Such a rational integration greatly enhances the signal gain in PRCTA, and it enables the limit of detection of MMP-2 as low as 3 fM. The feasibility of PRCTA has been validated by the sensitive analysis of cellular MMP-2 activities of different cell lines with good accuracy, and the readout can be readily visualized by a fluorescence imaging system. Therefore, PRCTA has achieved the detection of target protease biomarkers with femtomolar sensitivity, exhibiting promising potential in biomedicine research and cancer diagnosis.