Engineering modular and tunable single-molecule sensors by decoupling sensing from signal output | Nature Nanotechnology

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Biosensors play key roles in medical research and diagnostics. However, the development of biosensors for new biomolecular targets of interest often involves tedious optimization steps to ensure a high signal response at the analyte concentration of interest. Here we show a modular nanosensor platform that facilitates these steps by offering ways to decouple and independently tune the signal output as well as the response window. Our approach utilizes a dynamic DNA origami nanostructure to engineer a high optical signal response based on fluorescence resonance energy transfer. We demonstrate mechanisms to tune the sensor’s response window, specificity and cooperativity as well as highlight the modularity of the proposed platform by extending it to different biomolecular targets including more complex sensing schemes. This versatile nanosensor platform offers a promising starting point for the rapid development of biosensors with tailored properties.

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All experimental data supporting the findings of this work as well as DNA origami cadnano files are available from Zenodo at https://doi.org/10.5281/zenodo.12168537 (ref. 67). Source data are provided with this paper.

The custom code for the analysis of single-molecule confocal scans is available at https://gitlab.lrz.de/tinnefeldlab/cospota.

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We thank T. Liedl and J. Rädler for providing access to transmission electron microscopy facilities and the members of the Tinnefeld lab for discussions, especially F. Steiner. We also thank M. Scheckenbach for initial AFM measurements. This work was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie actions (grant agreement number 840741, GLUCORIGAMI), the German Research Foundation (DFG, grant number GL 1079/1-1, project number 503042693 to V.G. and project number 201269156, SFB 1032 Project A13 and INST 86/1904-1 FUGG to P.T.) and the Bavarian Ministry of Science and the Arts through the ONE MUNICH Project ‘Munich Multiscale Biofabrication’. V.G. is also grateful for the support by a Humboldt Research Fellowship from the Alexander von Humboldt Foundation. M.P. acknowledges support by Studienstiftung des deutschen Volkes.

These authors contributed equally: Lennart Grabenhorst, Martina Pfeiffer.

Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Munich, Germany

Lennart Grabenhorst, Martina Pfeiffer, Thea Schinkel, Mirjam Kümmerlin, Gereon A. Brüggenthies, Jasmin B. Maglic, Florian Selbach, Alexander T. Murr, Philip Tinnefeld & Viktorija Glembockyte

Max Planck Institute for Medical Research, Heidelberg, Germany

Viktorija Glembockyte

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V.G. and P.T. conceived the idea and directed the project. V.G., P.T., L.G. and M.P. further conceptualized the research. L.G. designed the DNA origami sensor and performed oxDNA simulations. L.G., V.G., M.K. and J.B.M. tested and optimized the design of the sensor and the signal transduction element. F.S. and G.A.B. performed TEM measurements. L.G. and V.G. implemented the sensor tuning strategies and carried out and analysed single-molecule titration experiments with the help of G.A.B. and A.T.M.; T.S. and V.G. designed and carried out the sensor specificity studies. M.P. carried out and analysed antibody, nuclease detection and multiplexed detection assays. L.G. wrote the code for single-molecule data analysis. L.G. and V.G. designed and implemented the PDGF as well as the Cas9-based RNA detection schemes with the help of G.A.B. L.G. and V.G. prepared figures. V.G., L.G. and P.T. wrote the manuscript with additional input from M.P.; all authors reviewed and accepted the manuscript.

Correspondence to Philip Tinnefeld or Viktorija Glembockyte.

The authors declare no competing interests.

Nature Nanotechnology thanks Leo Chou, Mahipal Ganji and Felix Rizzuto for their contribution to the peer review of this work.

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Supplementary Texts 1 and 2, Figs. 1–28 and Tables 1–11.

Movie of oxDNA simulation of the nanosensor (front view).

Movie of oxDNA simulation of the nanosensor (side view).

Statistical source data for Fig. 1.

Statistical source data for Fig. 2.

Statistical source data for Fig. 3.

Statistical source data for Fig. 4.

Statistical source data for Fig. 5.

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Grabenhorst, L., Pfeiffer, M., Schinkel, T. et al. Engineering modular and tunable single-molecule sensors by decoupling sensing from signal output. Nat. Nanotechnol. (2024). https://doi.org/10.1038/s41565-024-01804-0

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Received: 23 November 2023

Accepted: 12 September 2024

Published: 07 November 2024

DOI: https://doi.org/10.1038/s41565-024-01804-0

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