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Measuring cellular force using DNA-based tension probes: from ensemble to single-molecule studies

Nature Protocols (2025)Cite this article

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Abstract

Understanding how cells sense and respond to mechanical forces is crucial for many biological processes, including adhesion, migration, differentiation and immune activation. In this protocol, we describe two advanced DNA-based tension probes, the reversible shearing DNA-based tension probe (RSDTP) and ForceChrono probe, which provide powerful tools for studying mechanotransduction in living cells. RSDTPs enable dynamic quantification of forces ranging from 4 to 60 pN, offering the advantage of reversibility without ligand depletion, making them ideal for ensemble force measurements across populations of cells. ForceChrono probes not only measure the magnitude of force but also capture its duration and loading rate, providing essential insights into the temporal dynamics of single-molecule force transmission. We detail the fundamental principles, design strategies and step-by-step procedures for synthesizing, purifying and applying these probes, including surface preparation, cell experiments, image acquisition and data analysis. In addition, we describe the computational tools for image analysis. Together, these probes enable a detailed analysis of cellular mechanobiology, with applications in integrin mechanobiology and cell adhesion biology. This protocol is suitable for researchers with a background in cell biology, molecular biology, surface chemistry, optical imaging and data analysis and can be completed by a graduate student in 3–4 days.

Key points

  • This protocol describes the use of two advanced DNA-based tension probes, the reversible shearing DNA-based tension probe (RSDTP) and ForceChrono probe, which provide powerful tools for studying mechanotransduction in living cells.

  • Compare to other probes, RSDTP enables reversible and repeatable measurements to be made across a wide range of forces, while ForceChrono probes capture force duration and loading rate, providing essential insights into the temporal dynamics of single-molecule force transmission.

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Fig. 1: Overview of the RSDTP and ForceChrono probe protocol.
Fig. 2: Development and optimization of the RSDTPs.
Fig. 3: Development of ForceChrono probes for real-time imaging of individual integrin-mediated mechanical forces in living cells.
Fig. 4: The workflow of this protocol.
Fig. 5: The workflow of the synthesis of RSDTPs and ForceChrono probes.
Fig. 6: Purification results during synthesis of probes.
Fig. 7: The workflow of functionalized glass surface preparation.
Fig. 8: A preliminary experiment to determine the mixing ratio between the ForceChrono probe and the RGD-DNA-tether for single-molecule imaging.

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Data availability

The main images and analysis data in this protocol are available in the supporting primary research papers29,30. Additional source data have been uploaded to the online repository Figshare68 (https://doi.org/10.6084/m9.figshare.30016708.v1). Source data are provided with this paper.

Code availability

The data analysis tool (MATLAB code) used in this protocol can be accessed at https://doi.org/10.5281/zenodo.1489039365.

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Acknowledgements

We sincerely thank the Jie Yan laboratory for their guidance and support in the theoretical calculations of DNA mechanics.

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Author notes

  1. These authors contributed equally: Piyu Wu, Yuru Hu.

Authors and Affiliations

  1. State Key Laboratory of Metabolism and Regulation in Complex Organisms, TaiKang Center for Life and Medical Sciences, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan, China

    Piyu Wu, Yuru Hu, Hongyun Li, Wei Chen & Zheng Liu

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  1. Piyu Wu
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  2. Yuru Hu
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  3. Hongyun Li
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Contributions

Z.L. supervised the project. P.W. and Y.H. designed and carried out the experiments and performed data analysis. H.L. wrote the code for data-analysis tools for the ForceChrono probes. P.W., Y.H., W.C. and Z.L. wrote and edited the manuscript.

Corresponding author

Correspondence to Zheng Liu.

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Key references

Li, H. et al. Nat. Cell Biol. 23, 642–651 (2021): https://doi.org/10.1038/s41556-021-00691-0

Hu, Y. et al. Cell 187, 3445–3459.e15 (2024): https://doi.org/10.1016/j.cell.2024.05.008

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Supplementary Note 1, Supplementary Figures 1–5 and Supplementary Tables 1–6

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Unprocessed gels of Fig. 6d

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Wu, P., Hu, Y., Li, H. et al. Measuring cellular force using DNA-based tension probes: from ensemble to single-molecule studies. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01277-y

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