A grand challenge in biosensor design is to develop a single-molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Here, we created a family of adaptable, turn-on maturation (ATOM) biosensors consisting of a monobody (circularly permuted at one of two positions) or a nanobody (circularly permuted at one of three positions) inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells coexpressing cyan, yellow and red ATOM sensors detected biosensor targets that were specifically localized to various subcellular compartments. Fluorescence activation involved ligand-dependent chromophore maturation with turn-on ratios of up to 62-fold in cells and 100-fold in vitro. Endoplasmic reticulum- and mitochondria-localized ATOM sensors detected ligands that were targeted to those organelles. The ATOM design was validated with three monobodies and one nanobody inserted into distinct fluorescent proteins, suggesting that customized ATOM sensors can be generated quickly.
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All raw and processed data, statistical analyses and images (with associated metadata) generated in this work have been deposited to the Figshare repository (https://doi.org/10.58120/upstate.23959239.v2). This work did not generate any code. Plasmids expressing ATOM biosensors have been deposited to Addgene (Addgene codes 209702–209721). All other resources used in this study will be made available upon request.
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We thank X. Wang and X.-J. Chen for advice on mitochondrial imaging and for the gift of HEK 293T and HeLa cells, R. Oot and S. Wilkens for the gift of MDA-MB-231 cells, M. Mollapour for the gift of COS-7 cells, M. Cosgrove for the gift of the WDR5 expression plasmid and S. Koide for the gift of the MBSH2 and SH2 genes. This work was supported by National Institutes of Health grant nos. F30 GM146428 to H.S. and R01 GM148448 to S.N.L.
The authors declare no competing interests.
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Nature Methods thanks Moritoshi Sato, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.
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Extended Data Fig. 1 Ligand-dependent ATOM turn-on in cells is not due to increased biosensor levels.
HEK 293T cells were co-transfected with one plasmid encoding y-ATOMWDR5, y-ATOMSH2, y-ATOMRAS, or y-ATOMmCh and a second plasmid expressing WDR5, SH2, hRAS, or mCh. After 48 h, cells were fixed, stained with an anti-GFP antibody conjugated with Alexa594, and imaged in the red channel (antibody; data depicted as gray points) and yellow channel (biosensor; data depicted in indicated colors). Each data point represents a cell (50–100 per experiment). Scale bars are 200 µm. The results are representative of three BR. Number of cells, means, standard deviations, and P values obtained by a two-tailed t-test for all valid comparisons are included in Extended Data Table 1.
Extended Data Fig. 2 r-ATOMWDR5, y-ATOMSH2, and c-ATOMRAS, co-expressed in the same cells, exhibit high turn-on values in the presence of their target ligands but not in the presence of noncognate ligands.
To match Fig. 4, the cyan channel (hRAS) is pseudocolored red, the yellow channel (SH2) is pseudocolored green, and the red channel (WDR5) is pseudocolored blue. HEK 293T cells were co-transfected with an equimolar mixture of three biosensor plasmids and a fourth plasmid encoding WDR5 (first row), SH2 (second row), and hRAS (third row). The fourth row shows the same experiment, but the fourth plasmid expressed all three ligands as described in the text. Scale bars are 200 µm. Turn-on ratios in each color channel (bottom plots) were calculated by dividing the average intensity of cells expressing the biosensor and its cognate ligand by the average of the intensities of cells expressing the same biosensor and the two negative control ligands. Each data point represents a cell (40–60 per experiment). P < 10−4 for all comparisons shown. The results are representative of three BR. Number of cells, means, standard deviations, and P values obtained by a two-tailed t-test for all valid comparisons are included in Extended Data Table 1.
Extended Data Fig. 3 y-ATOMWDR5 detects endogenous WDR5 and identifies nuclear localization in HEK 293T cells.
Staining with anti-GFP antibody (red) indicated that WT and R80A mutant y-ATOMWDR5 were both expressed in cytoplasm and nucleus, with highest levels in the cytoplasm. Imaging in the yellow channel (pseudocolored green) showed the WT biosensor was activated in the nucleus but in the OFF state in the cytoplasm. R80A y-ATOMWDR5 was dark in both cytoplasm and nucleus. Scale bars are 20 µm. Each data point represents one cell (61 cells per experiment). ****, P < 10−4; ns, not significant. Results are representative of 3 BR. Number of cells, means, standard deviations, and P values obtained by a two-tailed t-test for all valid comparisons are included in Extended Data Table 1.
Extended Data Fig. 4 Performance of NIR-FbGFP localized to cytoplasm, mitochondria, and ER of HEK 293T cells.
Cells were co-transfected with plasmids encoding NIR-FbGFP and Clover (tagged with indicated localization sequences) and imaged in the far-red channel to quantify compartment-specific turn-on. (b) The same cells were imaged in the green channel to determine relative Clover expression. Scale bars are 200 µm. Results are representative of 3 BR.
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Sekhon, H., Ha, JH., Presti, M.F. et al. Adaptable, turn-on maturation (ATOM) fluorescent biosensors for multiplexed detection in cells. Nat Methods 20, 1920–1929 (2023). https://doi.org/10.1038/s41592-023-02065-w