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Updated: Apr 6, 2026
A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer
Published on: September 13, 2022
Facile Discovery of Cell-Surface Protein Targets of Cancer Cell Aptamers
Tao Bing1, Dihua Shangguan2, Yinsheng Wang3
1From the ‡Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China; §Department of Chemistry, University of California, Riverside, CA92521-0403.
Abstract:
Cancer biomarker discovery constitutes a frontier in cancer research. In recent years, cell-binding aptamers have become useful molecular probes for biomarker discovery. However, there are few successful examples, and the critical barrier resides in the identification of the cell-surface protein targets for the aptamers, where only a limited number of aptamer targets have been identified so far. Herein, we developed a universal SILAC-based quantitative proteomic method for target discovery of cell-binding aptamers. The method allowed for distinguishing specific aptamer-binding proteins from nonspecific proteins based on abundance ratios of proteins bound to aptamer-carrying bait and control bait. In addition, we employed fluorescently labeled aptamers for monitoring and optimizing the binding conditions. We were able to identify and validate selectin L and integrin α4 as the protein targets for two previously reported aptamers, Sgc-3b and Sgc-4e, respectively. This strategy should be generally applicable for the discovery of protein targets for other cell-binding aptamers, which will promote the applications of these aptamers.
Insights
Researchers developed a new proteomic method to identify cancer biomarkers using cell-binding aptamers. This technique successfully identified protein targets for aptamers, advancing biomarker discovery and aptamer applications.
Area of Science:
- Biochemistry
- Proteomics
- Cancer Research
Background:
- Cell-binding aptamers are valuable tools for cancer biomarker discovery.
- Identifying specific protein targets for aptamers remains a significant challenge.
- Limited success in aptamer target identification hinders their application.
Purpose of the Study:
- To develop a universal method for discovering cell-surface protein targets of cell-binding aptamers.
- To overcome the barrier in identifying aptamer targets for enhanced biomarker discovery.
- To promote the broader application of aptamers in cancer research.
Main Methods:
- Developed a universal SILAC-based quantitative proteomic method.
- Differentiated specific aptamer-binding proteins from non-specific proteins using abundance ratios.
- Utilized fluorescently labeled aptamers for monitoring and optimizing binding conditions.
Main Results:
- Successfully identified Selectin L and Integrin α4 as protein targets for aptamers Sgc-3b and Sgc-4e.
- The method effectively distinguished specific aptamer-binding proteins.
- Validated the identified protein targets.
Conclusions:
- The developed SILAC-based proteomic strategy is a universal approach for aptamer target discovery.
- This method facilitates the identification of novel cancer biomarkers.
- The strategy is expected to significantly advance the use of aptamers in clinical applications.
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