Abstract
Identification and characterization of circulating tumor cells (CTCs) from blood samples of patients with cancer can help monitor parameters such as disease stage, disease progression and therapeutic efficiency. However, the sensitivity and specificity of current multivalent approaches used for CTC capture is limited by the lack of control over the ligands’ position. In this Protocol Update, we describe DNA-tetrahedral frameworks anchored with aptamers that can be configured with user-defined spatial arrangements and stoichiometries. The modified tetrahedral DNA frameworks, termed ‘n-simplexes’, can be used as probes to specifically target receptor−ligand interactions on the cell membrane. Here, we describe the synthesis and use of n-simplexes that target the epithelial cell adhesion molecule expressed on the surface of CTCs. The characterization of the n-simplexes includes measuring the binding affinity to the membrane receptors as a result of the spatial arrangement and stoichiometry of the aptamers. We further detail the capture of CTCs from patient blood samples. The procedure for the preparation and characterization of n-simplexes requires 11.5 h, CTC capture from clinical samples and data processing requires ~5 h per six samples and the downstream analysis of captured cells typically requires 5.5 h. The protocol is suitable for users with basic expertise in molecular biology and handling of clinical samples.
Key points
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The procedure describes the design of targeted probes via orthogonal anchoring of aptamers, with varying stoichiometries, to the vertices of tetrahedral DNA frameworks. The frameworks, synthesized by using a single annealing step, are then characterized by using native polyacrylamide gel electrophoresis, atomic force microscopy, single-molecule fluorescence and transmission electron microscopy.
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The targeted tetrahedral DNA frameworks improve CTC capture efficiency when compared with alternative nucleic acid–based probes in whole-blood samples with the supernatant removed via centrifugation.
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Data availability
The main data discussed in this protocol are available in the supporting primary research paper29. The raw datasets are too large to be publicly shared but are available for research purposes from the corresponding authors upon reasonable request. Source data are provided with this paper.
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Acknowledgements
This work was financially supported by the National Key Research and Development Program of China (2021YFF1200300 and 2020YFA0909000), the National Natural Science Foundation of China (22025404, T2188102 and 22174132), the Natural Science Foundation of Shanghai (23ZR1438700), the Shanghai Municipal Health Commission (2022JC027) and the Innovative Research Group of High-Level Local Universities in Shanghai (SHSMU-ZLCX20212602).
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M.Li and X.Z. supervised the projects. Y.C., M.Lin and D.Y. designed and conducted the experiments. Y.C., M.Lin and S.W. analyzed the data. Y.C. and M.Li wrote the manuscript.
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Nature Protocols thanks Julian Alexander Tanner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Li, M. et al. J. Am. Chem. Soc. 141, 18910–18915 (2019): https://doi.org/10.1021/jacs.9b11015
This protocol is an update to: Nat. Protoc. 11, 1244–1263 (2016): https://doi.org/10.1038/nprot.2016.071
Supplementary information
Supplementary Information
Supplementary Figs. 1–7
Supplementary Data 5
Unprocessed gels for Supplementary Fig. 5
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Source Data Fig. 3
Unprocessed gels
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Chen, Y., Lin, M., Ye, D. et al. Functionalized tetrahedral DNA frameworks for the capture of circulating tumor cells. Nat Protoc 19, 985–1014 (2024). https://doi.org/10.1038/s41596-023-00943-3
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DOI: https://doi.org/10.1038/s41596-023-00943-3
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