Abstract
Immunoassays using affinity binders such as antibodies and aptamers are crucial for molecular biology. However, the advancement of analytical methods based on these affinity probes is often hampered by complex operational steps that can introduce errors, particularly in intricate environments such as intracellular settings and microfluidic systems. There is growing interest in developing molecular probes for wash-free assays that activate signals upon target detection. Here we report a systematic functional screening platform for switchable aptamer beacon probes that can achieve target-responsive detection. A stem–loop, hairpin-shaped beacon library was constructed on microbeads and screened using target-responsive fluorescence-activated sorting. The selected aptamer beacons exhibit strong affinities, triggering fluorescence only upon binding, thus enabling wash-free immunoassays for the detection of intracellular and membrane proteins. Computational modelling offers insights into aptamer binding and structural switching mechanisms, revealing how specific protein–aptamer interactions drive stem–loop unwinding and postbinding conformational changes critical for functional activation. This approach establishes a standardized platform for generating switchable aptameric tools, supporting their potential in advanced diagnostics and research.
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Data availability
The crystal structure of the HER2 ECD (PDB ID: 6BGT) obtained from the Protein Data Bank47 was supplemented with missing residues spanning positions 22–132, 603–609 and 629–652 using the Homology Model module integrated into the MOE2019 software (https://www.chemcomp.com/en/Products.htm). The main data supporting the results in this study are available within the article and its Supplementary Information. Source data are provided with this paper. The sequencing data files are too large to be publicly shared, but they are available from the corresponding author upon reasonable request. All other data generated in this study are available via figshare at https://doi.org/10.6084/m9.figshare.28934987 (ref. 54).
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Acknowledgements
We thank J. Sun from the National Center for Nanoscience and Technology, China, and X. Fang from the Hangzhou Institute of Medicine, Chinese Academy of Sciences, for their long-term collaboration and insightful discussions. We also extend our thanks to the staff at the Peking University Medical and Health Analysis Center and the State Key Laboratory of Natural and Biomimetic Drugs for their invaluable assistance with instrumental analysis. L.Z. discloses support for the research described in this study from the National Key R&D Program of China (grant nos. 2022YFA1304501 and 2023YFF1205902), the National Natural Science Foundation of China (grant nos. 22227805 and 22374004) and Excellent Young Scientists Fund Program (Overseas).
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X.C., P.Y. and L.Z. conceptualized and designed the research. X.C., P.Y., C.J., X.Z., T.W., Q.L., Y.C., H.S., H.X. and S.B. performed the experiments. X.C., P.Y. and L.Z. analysed the experimental data. W.L. and Z.Z. conducted computations. P.Y., X.C., W.L., Z.Z. and L.Z. prepared the figures and wrote the paper. All authors discussed the results and commented on the paper. L.Z. guided and supervised the research.
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Nature Biomedical Engineering thanks Hyun Gyu Park, Hao Yan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Schematic illustration and flow cytometry validation of the library construction.
The monoclonal microbeads library of aptamer beacons screening was constructed by emulsion PCR amplification, restriction enzyme-enabled site-specific modification, and exonuclease-digested single-strand generation, which were tested on primer-immobilized microbeads using. The library comprises a randomized region (pink), forward primers (blue and yellow), and reverse primer binding sites (yellow and green). Intramolecular complementary regions (yellow) and restriction enzyme cutting sites (letters) are embedded within. Fluorophores and quenchers are both attached onto dU bases.
Extended Data Fig. 2 Predicted structures of selected aptamer beacons against IL-2.
Each aptamer beacon for HER2 are illustrated by the predicted 2D structure (http://www.unafold.org/mfold/applications/dna-folding-form.php) and predicted 3D structure (http://biophy.hust.edu.cn/new/3dRNA/create).
Extended Data Fig. 3 Concentration-dependent fluorescence restoration of selected aptamer beacons (Cy3 and BHQ1) in response to gradient concentration of IL-2.
a, The target protein bound to the aptamer beacon, and the fluorescence of the aptamer beacon was restored. b, The fluorescence of AB-I2 was related to the change in the concentration of the target protein. c, The fluorescence of AB-I5 was related to the change in the concentration of the target protein. d, The fluorescence of AB-I5 was related to the change in the concentration of the target protein.
Extended Data Fig. 4 Specificity of AB-I2 Across Different Cell Types.
a, AB-I2 does not respond to cytokines produced by Raji cells. Experiments were performed in triplicate. Data are expressed as mean ± s.d of biological replicates. (t test statistic, two tailed, n = 3) b, AB-I2 does not respond to cytokines produced by THP-1 cells. Experiments were performed in triplicate. Data are expressed as mean ± s.d of biological replicate. (t test statistic, two tailed, n = 3).
Extended Data Fig. 5 Intracellular cytokine monitoring.
a, Selection of infectious agents. Using Lipofectamine 3000 to transfect AB-I2 into Jurkat cells, the distinction between activated and non-activated Jurkat cells can be achieved. b, Observation of the specific processes of activated and non-activated Jurkat cells through confocal microscopy. Lipofectamine 3000 was used to transfect AB-i2 into Jurkat cells that had been treated with ConA and Brefeldin A for 3 hours. After incubation for 1 hour, the cells were observed under a confocal microscope. c, Activated Jurkat cells can be observed under a confocal microscope with more obvious fluorescence than inactivated Jurkat cells. Three fields of view were randomly selected for each sample for shooting. Due to limited space, only one of the fields of view is shown here.
Extended Data Fig. 6 Predicted structures of selected aptamer beacons against HER2.
Each aptamer beacon for HER2 are illustrated by the predicted 2D structure (http://www.unafold.org/mfold/applications/dna-folding-form.php) and predicted 3D structure (http://biophy.hust.edu.cn/new/3dRNA/create).
Extended Data Fig. 7 Further characterization of AB-H10 interaction with HER2 protein.
a, Schematic diagram illustrating the principle of Pull-Down experiments to validate probe targets. Cells were lysed, and membrane proteins were extracted and incubated with biotinylated aptamer beacons, followed by capturing by streptavidin-coated microbeads. The complex was resolved by SDS-PAGE. b, Conducting Pull-Down experiments using various aptamer beacons on HER2( + ) AU565 cells, followed by Western Blot experiments with HER2 antibodies to confirm the protein bands originating from HER2.This experiment was repeated once with similar results. c, Schematic illustration of AB-H10 incubated with protein-immobilized microbeads. d, AB-H10 specifically binds to HER2-immobilized beads rather than EGFR-immobilized beads or empty beads.
Extended Data Fig. 8 Estimation of the half-life of aptamer beacons in different solution systems.
a, the half-life of AB-I2 in PBS. b, the half-life of AB-I2 in Binding Buffer. c, the half-life of AB-I2 in DMEM. d, the half-life of AB-I2 in DMEM + 10%FBS. e, the half-life of AB-H10 in PBS. f, the half-life of AB-H10 in Binding Buffer. g, the half-life of AB-H10 in DMEM.h, the half-life of AB-H10 in DMEM + 10%FBS. The solutions were sampled at different time points with three biological replicates, and fluorescence was detected using BioTek equipment. Data are expressed as mean ± s.d of biological replicates. The concentrations of AB-I2 and AB-H10 were 500 nM, and the incubation was performed at 37 °C. The solutions were analyzed by running them on a 15% PAGE gel, which was then stained with GelRed dye.
Extended Data Fig. 9 Intracellular Stability Estimation for AB-I2.
a, Estimate of the intracellular half-life of AB-I2 delivered by Lipo3000. Changes in fluorescence intensity of Cy3 in cells over incubation time. b, Intracellular degradation curve of AB-I2. Experiments were performed in triplicate. Data are expressed as mean ± s.d of biological replicates. c, Prediction of the Tm value of AB-I2 under intracellular ion concentrations. (http://www.unafold.org/mfold/applications/dna-folding-form.php).
Extended Data Fig. 10 Prediction and estimation of the melting temperature of AB-I2 and AB-H10.
a, Prediction and estimate of Tm value of AB-I2 in PBS. b, Prediction and estimate of Tm value of AB-I2 in Binding Buffer. c, Prediction and estimate of Tm value of AB-I2 in DMEM. d, Prediction and estimate of Tm value of AB-H10 in PBS. e, Prediction and estimate of Tm value of AB-H10 in Binding Buffer. f, Prediction and estimate of Tm value of AB-H10 in DMEM. (http://www.unafold.org/mfold/applications/dna-folding-form.php) Experiments were performed in triplicate. Data are expressed as mean ± s.d of biological replicates.
Supplementary information
Supplementary Information
Supplementary Figs. 1–4 and Tables 1–12.
Supplementary Video 1
Simulation of the mechanism of the HER2 aptamer beacon. Left: a 1-μs metadynamics simulation revealed the molecular mechanism of AB-H10 base-pair separation at the HER2 ECD binding site. Right: an unwound AB-H10 structure was generated using a protein structure modelling program, RoseTTAFold, and refined with a 10-ns MD simulation.
Source data
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Statistical source data and unprocessed gels.
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Source Data Extended Data Fig. 4
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Cheng, X., Yao, P., Jin, C. et al. Systematic functional screening of switchable aptamer beacon probes. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01503-8
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DOI: https://doi.org/10.1038/s41551-025-01503-8
