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Published on: January 7, 2017
Nanoscale mapping of nucleolin-aptamer interactions on lung cancer cells reveals binding affinity hierarchy and
Longyun Chen1,2, Rongrong Feng1,3,4, Qianhui Xu1,3,4
1Jiangxi Provincial Key Laboratory of Tissue Engineering (2024SSY06291), Gannan Medical University, Ganzhou 341000, People's Republic of China. zhaowd@gmu.edu.cn.
Abstract:
Nucleolin, a protein overexpressed on the surface of cancer cells, has emerged as a promising therapeutic target due to its high affinity interactions with aptamers. This study localized nucleolin on lung cancer and normal cells at single-molecule resolution using the single molecule recognition imaging mode of Atomic Force Microscopy (AFM) with three aptamers: 9FU-AS1411, AS1411, and CRO. The results revealed abundant nucleolin expression on lung cancer cells, while minimal levels were detected on normal cells. The binding affinities and interaction dynamics of these aptamers were systematically evaluated. Flow cytometry and AFM-based force spectroscopy demonstrated that 9FU-AS1411 exhibited the strongest unbinding forces (piconewton level) and higher dissociation activation energy compared to AS1411, indicating enhanced complex stability. In contrast, CRO showed negligible binding, confirming its lack of specificity. Further analysis via Kelvin Probe Force Microscopy (KPFM) revealed distinct surface potential decrements after aptamer interactions: 24.4 mV (9FU-AS1411), 11.7 mV (AS1411), and 2.5 mV (CRO), correlating with their binding strengths. These findings quantitatively rank aptamer affinity as 9FU-AS1411 > AS1411 >> CRO, supported by molecular-level mechanistic insights into electrostatic and structural interactions. This work pioneers high-resolution spatial mapping of nucleolin-aptamer interactions, offering novel methodologies for studying protein-aptamer binding kinetics and electrical properties at unprecedented precision (0.1 mV resolution). The approaches established here not only advance nucleolin-targeted cancer therapy but also provide a framework for investigating other protein-aptamer systems in biomedical research.
Insights
This study used Atomic Force Microscopy to show that the aptamer 9FU-AS1411 strongly binds to nucleolin on lung cancer cells, offering a new tool for cancer therapy development.
Area of Science:
- Biophysics
- Nanotechnology
- Cancer Research
Background:
- Nucleolin is a protein overexpressed on cancer cells, making it a target for aptamer-based therapies.
- Aptamers show high affinity for nucleolin, but their binding characteristics need detailed investigation.
Purpose of the Study:
- To spatially map nucleolin-aptamer interactions at single-molecule resolution using Atomic Force Microscopy (AFM).
- To quantitatively evaluate the binding affinities and interaction dynamics of different aptamers with nucleolin.
- To explore novel methodologies for studying protein-aptamer binding kinetics and electrical properties.
Main Methods:
- Single molecule recognition imaging mode of AFM to localize nucleolin on lung cancer and normal cells.
- AFM-based force spectroscopy and flow cytometry to assess aptamer binding affinities and unbinding forces.
- Kelvin Probe Force Microscopy (KPFM) to measure surface potential changes after aptamer interaction.
Main Results:
- Abundant nucleolin expression was observed on lung cancer cells, with minimal levels on normal cells.
- The aptamer 9FU-AS1411 demonstrated superior binding affinity and complex stability compared to AS1411, evidenced by higher unbinding forces and dissociation activation energy.
- CRO aptamer showed negligible binding, indicating a lack of specificity.
- KPFM revealed distinct surface potential decrements correlating with aptamer binding strengths: 24.4 mV (9FU-AS1411), 11.7 mV (AS1411), and 2.5 mV (CRO).
Conclusions:
- The quantitative ranking of aptamer affinity is 9FU-AS1411 > AS1411 >> CRO, supported by mechanistic insights into electrostatic and structural interactions.
- This study pioneers high-resolution spatial mapping of nucleolin-aptamer interactions, providing a framework for studying protein-aptamer systems.
- The developed methodologies advance nucleolin-targeted cancer therapy and offer precise tools for biomedical research.
