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Updated: Aug 8, 2025
Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs
Published on: June 12, 2018
FOF1-ATP synthase molecular motor biosensor for miRNA detection of colon cancer
Ying Gao1, Jie Zhang2, Jiexia Pan3
1College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, China; Zhejiang Moda Biotech Co., Ltd, Hangzhou 310018, China.
Aims:
To establish a FOF1-ATP synthase molecular motor biosensor to accurately identify colon cancer miRNAs.
Main Methods:
The FOF1-ATP synthase molecular motor is extracted by fragmentation-centrifugation and connected to the colon cancer-specific miR-17 capture probe in the manner of the ε subunit-biotin-streptavidin-biotin system. Signal probes are designed for dual-signal characterization to increase detection accuracy. The FOF1-ATPase rotation rate decreases when the signaling and capture probes are combined with the target miRNA, resulting in a decrease in ATP synthesis. miR-17 concentrations are determined by changes in ATP-mediated chemiluminescence intensity and signal probe-mediated OD450nm.
Key Findings:
The chemiluminescence intensity and OD450nm show a good linear relationship with the miR-17 concentration in the range of 5 to 200 nmol L-1 (R2 = 0.9985, 0.9989). The colon cancer mouse model is established for the blood samples, and miR-17 in serum and RNA extracts is quantitatively determined using the constructed sensor.
Significance:
The results are consistent with colon cancer progression, and the low concentration of miR-17 detecting accuracy is comparable to the PCR assay. In conclusion, the developed method is a direct, rapid, and promising method for miRNA detection of colon cancer.
