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FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Published on: August 20, 2012
Rational engineering of a large Stokes shift fluorogenic sensor for sensing Notum in living systems and inhibitor
Xiaodong Li1, Mengru Sun2, Jiaqian Miao1
1State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
Abstract:
Notum is a serine hydrolase that negatively regulates Wnt/β-catenin signaling and is implicated in diseases including colorectal cancer (CRC). Here we report an enzyme-activatable fluorogenic sensor for dynamic monitoring and multidimensional imaging of human Notum (hNotum) in complex biological systems. Through iterative optimization of the hemicyanine reporter and the enzyme-recognition motif, we identified Oc-HNBI as an optimal hNotum substrate. Oc-HNBI shows high selectivity, strong binding affinity, rapid response, a large Stokes shift (142 nm), and a high quantum yield, providing exceptional spatial resolution and high imaging performance. The fluorogenic sensor was successfully applied for in situ imaging of hNotum activity in various biological settings, including 3D cell spheroids, cancer tissue sections, organ tissues, and in vivo mouse models. Additionally, Oc-HNBI was adapted to develop a high-throughput screening assay for discovering hNotum inhibitors. Using this assay, we identified Sennoside A, a natural compound, as a potent hNotum inhibitor with potential anti-CRC properties. Overall, this study presents a reliable fluorescent tool for sensing and imaging hNotum activity in living systems. This tool has the potential to advance the understanding of hNotum's role in human diseases and contribute to anti-CRC drug discovery.
Insights
Researchers developed a novel fluorescent sensor, Oc-HNBI, to image human Notum (hNotum) activity in biological systems. This tool aids in understanding colorectal cancer and discovering new inhibitors like Sennoside A.
Area of Science:
- Biochemistry
- Molecular Biology
- Chemical Biology
Background:
- Notum is a serine hydrolase that negatively regulates Wnt/β-catenin signaling.
- Dysregulation of Notum is linked to diseases, notably colorectal cancer (CRC).
- Dynamic monitoring of hNotum activity in complex biological systems is challenging.
Purpose of the Study:
- To develop and validate an enzyme-activatable fluorogenic sensor for human Notum (hNotum).
- To enable dynamic monitoring and multidimensional imaging of hNotum activity.
- To facilitate the discovery of hNotum inhibitors for potential anti-CRC therapies.
Main Methods:
- Iterative optimization of hemicyanine reporter and enzyme-recognition motif led to Oc-HNBI.
- Characterization of Oc-HNBI for selectivity, binding affinity, response time, Stokes shift, and quantum yield.
- Application of the sensor for in situ imaging in 3D cell spheroids, tissues, and in vivo models; development of a high-throughput screening assay.
Main Results:
- Oc-HNBI demonstrated high selectivity, strong binding affinity, rapid response, large Stokes shift (142 nm), and high quantum yield.
- Successful in situ imaging of hNotum activity in diverse biological settings, including 3D spheroids, tissue sections, organs, and in vivo mouse models.
- Identification of Sennoside A as a potent hNotum inhibitor using the developed screening assay, suggesting potential anti-CRC properties.
Conclusions:
- A reliable fluorescent sensor (Oc-HNBI) was developed for sensing and imaging hNotum activity in living systems.
- This tool enhances understanding of hNotum's role in human diseases, particularly colorectal cancer.
- The developed sensor and screening assay contribute to advancing anti-CRC drug discovery.
