DNA nanodevices detect an acidic nanolayer on the lysosomal surface

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DNA nanodevices detect an acidic nanolayer on the lysosomal surface

Data availability

The data supporting the findings of this study are available within the Article and its Supplementary Information. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant nos. 92354306 to H.X., 92253304 to L.Q., T2188102 to W.T., 22174039 to L.Q., 32421001 to H.X., 22404051 to Z.W. and 32570805 to M.H.), National Key Research and Development Program of China (grant nos. 2021YFA0910101 to L.Q. and 2022YFE0210100 to H.X.), National Science and Technology Major Project of China (grant no. 2025ZD0215900 to M.H.), the Science and Technology Project of Hunan Province 2021RC4022 to L.Q., the Health Commission of Zhejiang Province Grant WKJ-ZJ-2103 to W.T. and the Investigator Program from New Cornerstone Science Foundation to H.X.

Author information

Authors and Affiliations

  1. Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo and Biosensing, College of Chemistry and Chemical Engineering, College of Biology, Aptamer Engineering Center of Hunan Province, Hunan University, Changsha, China

    Yutong Zhang, Xin Wang, Yuting Zhuo, Zhimin Wang, Qiang Zhang, Yao He, Liping Qiu & Weihong Tan

  2. New Cornerstone Science Laboratory and Liangzhu Laboratory, the Second Affiliated Hospital and School of Basic Medical Sciences, Zhejiang University, Hangzhou, China

    Yutong Zhang, Meiqin Hu, Yaping Meng, Fangqian Huang, Ping Li, Danzhen Chen & Haoxing Xu

  3. The Key Laboratory of Zhejiang Province for Aptamers and Theranostics, Zhejiang Cancer Hospital, Hangzhou Institute of Medicine, Chinese Academy of Sciences, Hangzhou, China

    Yutong Zhang, Hui Wu, Yulin Du, Liping Qiu & Weihong Tan

  4. Zhejiang Key Laboratory of Pain Perception and Neuromodulation, Hangzhou, China

    Meiqin Hu

  5. Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Haoxing Xu

  6. Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai, China

    Weihong Tan

Authors

  1. Yutong Zhang
  2. Meiqin Hu
  3. Yaping Meng
  4. Xin Wang
  5. Fangqian Huang
  6. Ping Li
  7. Yuting Zhuo
  8. Danzhen Chen
  9. Zhimin Wang
  10. Qiang Zhang
  11. Hui Wu
  12. Yao He
  13. Yulin Du
  14. Haoxing Xu
  15. Liping Qiu
  16. Weihong Tan

Contributions

Y. Zhang, L.Q. and H.X. conceived and designed the research. Y. Zhang, M.H., Y.M., F.H., P.L., D.C., Y. Zhuo, Z.W., H.W., Y.H. and Y.D. conducted the experiments. Q.Z. and X.W. performed image analysis. Y. Zhang performed the data analysis. Y. Zhang, M.H., L.Q., H.X. and W.T. drafted and revised the manuscript. All authors commented on the manuscript.

Corresponding authors

Correspondence to Haoxing Xu, Liping Qiu or Weihong Tan.

Ethics declarations

Competing interests

H.X. is the scientific cofounder and a partial owner of Lysoway Therapeutics Inc. (Boston) and Lyso-X Therapeutics Inc. (Hangzhou). The other authors declare no competing financial interests.

Peer review

Peer review information

Nature Cell Biology thanks Peter Kreuzaler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Efficient decoration of an N3 tag on the inner plasma membrane (IPM).

a, Chemical structures of mDc-N3 and IPM-N3. b, The membrane penetration kinetics of Silorhodamine (SiR)-DBCO. Left: Representative confocal images of HeLa cells incubated with 10 μM SiR-DBCO at 37 °C for different time spans (scale bar, 10 µm). Right: Summary of time-dependent SiR fluorescence intensity (n = 30, 30, and 31 cells for the left to right groups, respectively). c, Schematic illustration of the experimental workflow for construction of IPM-N3. d, Representative confocal images of SiR-DBCO-pretreated HeLa cells processed with or without 10 μM mDc-N3 at RT for 10 min, and then incubated in fresh culture medium for different time spans (scale bar, 10 µm). e, Schematic illustration of the experimental workflow for quantification of SiR-DBCO within cells. f, Left: Construction of the standard calibration curve of SiR-DBCO. Right: Fluorescence spectra of the lysate of HeLa cells after treatment with 10 μM SiR-DBCO at 37 °C for 60 min. g, Quantification of IPM-DNs. Construction of the standard calibration curve of DNCy5 in buffer solution (Standard curve 1), DNCy5 on the outer membrane of cells (Standard curve 2), and grey value of Cy5 in confocal microscope images (Standard curve 3). h, Summary of time-dependent changes of SiR fluorescence ratio between the plasma membrane and whole cell in SiR-DBCO-pretreated HeLa cells incubated with mDc-N3 (Left to right groups: n = 10, 10, 13, and 12 cells for mDc-N3 treated group; n = 12, 12, 13, and 13 cells for control group without mDc-N3 treatment). i, Representative confocal images of HeLa cells treated with mDc-N3 for 45 min (scale bar, 10 μm, green fluorescence = DIO for plasma membrane staining). j, Cellular distribution of the N3 tag. Representative confocal images of HeLa cells processed with SiR-DBCO/mDc-N3 and then stained with 100 nM Golgi Apparatus (GA) tracker, Endoplasmic Reticulum (ER) tracker, or Mitochondria (Mito) tracker (scale bar, 10 µm). k, Golgi apparatus-mediated metabolic process of mDc-N3. Representative confocal images of HeLa cells pretreated with 10 μM palmitoylation inhibitor 2-bromopalmitate (2-BP) (upper) or 20 μM Golgi disrupter Brefeldin A (BFA) (lower) at 37 °C for 60 min, and then processed with SiR-DBCO/mDc-N3 (scale bar, 10 µm). l, The IPM orientation of the N3 tag. Representative confocal images of mSA-EGFP-transfected HeLa cells processed with mDc-N3, and then incubated with DBCO-PEG4-Biotin at RT for 30 min (scale bar, 10 μm (upper) and 2.5 μm (lower)). Statistical data are presented as mean ± s.d. (b) or mean ± s.e.m. (h), with n as randomly selected cells from 3 biological repeats. For panel d and i-l, 3 independent experiments are repeated with similar results.

Source data

Extended Data Fig. 2 IPM-stabilized DNA nanodevices are sequentially delivered to early endosomes and lysosomes through endosome maturation without affecting lysosomal functions.

a, Left: Representative confocal images of SDNCy5-engineered HeLa cells pulsed with 1 mg/mL Rhodamine-labeled dextran (RB-dextran) for 30 min and then chased for different time spans (scale bar, 10 μm). Right: Summary of colocalization ratio between RB-dextran and SDNCy5 (n = 12, 12, and 11 cells for the left to right groups, respectively). b, Left: Representative confocal images of HeLa cells pulsed with 1 mg/mL RB-dextran for 30 min and then chased for different time spans (scale bar, 10 µm, green fluorescence = LysoTracker for lysosome staining). Right: Summary of colocalization ratio between RB-dextran and LysoTracker (n = 10 cells for each group). c, Organellar localization of SDN. Left: Representative confocal images of Rab5-mCherry-transfected HeLa cells engineered with IPM-SDNCy5, and then pulsed with dextran and chased for 5 min and 60 min (scale bar, 5 μm). Right: Summary of time-dependent colocalization ratios between Cy5 and mCherry (n = 14 and 12 cells for 5 min and 60 min, respectively). d, Organellar localization of SDN. Left: Representative confocal images of HeLa cells decorated with IPM-SDNCy5, pulsed with dextran and chased for 60 min, then immuno-stained with anti-LAMP1 (lysosome), anti-GM130 (Golgi apparatus), and anti-EEA1 (early endosome) antibodies (scale bar, 10 μm). Right: Summary of colocalization ratios between SDN and organelle markers (n = 5, 7, and 10 images for the left to right groups, respectively). e, Left: Representative confocal images of LAMP1-mCherry-transfected HeLa cells engineered with SDNCy5 and then incubated in fresh culture medium for 0 or 180 min (scale bar, 10 μm). Right: Summary of fluorescence ratios between Cy5 and mCherry (fCy5/fmCherry) at indicated time points (n = 159 and 144 lysosomes for 0 min and 180 min, respectively). f, Left: Representative confocal images of HeLa cells pretreated with CLMLY-SDN, and then incubated with Magic Red (2000× dilution) at RT for 30 min (scale bar, 20 µm). Right: Summary of the fluorescence intensity of Magic Red (n = 34 and 35 cells for the groups without and with CLMLY-SDN pretreatment, respectively). g, Left: Representative confocal images of HeLa cells pretreated with CLMLY-SDN, and then incubated with 1 mM Pepstatin A BODIPY at RT for 30 min (scale bar, 20 µm). Right: Summary of the fluorescence intensity of Pepstatin A BODIPY (n = 27 cells for each group). Untreated HeLa cells were used as a positive control. For all panels, statistical data are presented as mean ± s.d. (a, b, c and d) or as box-and-whiskers graphs (f and g), where the box represents the range of 25%-75%, the solid line inside means the mean level, and the whiskers represent the minimum and maximum, with n as randomly selected images/cells from ≥3 biological repeats. Two-tailed Student’s t test was used to assess statistical significance.

Source data

Extended Data Fig. 3 Construction of pH-reporting DNA nanodevices for measuring juxta-lysosomal pH.

a, Construction of DNFAM/Cy5. PAGE analyses of different DNA samples. From lane 1 to 4: S1; S2; DN (S1 + S2); 20-bp DNA ladder. S1 was labeled with a cholesterol tag. S2 was labeled with a FAM, a Cy5, and a DBCO tag. b, Fluorescence spectrum of the FAM and Cy5 dyes in a permeability buffer solution (DPBS containing 10 μM nigericin and 10 μM valinomycin) of pH ranging from 4.5 to 8.0. The excitation wavelength for FAM and Cy5 was set at 488 nm (λem = 500-550 nm) and 633 nm (λem = 650-700 nm), respectively. c, Summary of the FAM fluorescence versus the buffer pH. Red dotted line represents the pKa value of the FAM dye (n = 3 independent assays). d, The f1/f0 ratio of DNFAM/Cy5 incubated with Ca2+, Na+, K+, or Cs+ of 10 nM versus 10 mM in Tris-HCl buffer solutions. The fluorescence ratio fFAM/fCy5 (FAM: λex = 488 nm, λem = 520 nm; Cy5: λex = 633 nm, λem = 665 nm) of 10 nM was set as f0, and that of 10 mM was set as f1 (n = 3 independent assays). e, Summary of calibrated cytosolic (bulk cytosol) pH and juxta-lysosomal (Juxta-LY) pH values of primary mouse astrocytes, MCF-7 cells, and PC12 cells (Left to right groups: n = 14 and 5 images for astrocyte, 5 and 4 images for MCF-7, 4 and 7 images for PC12). f, Summary of calibrated juxta-lysosomal pH values in WT and Tmem175 KO MEF cells, as well as WT and TMEM175 KO U2OS cells, as measured with CLMLY-SDNFAM/Cy5 (Left to right groups: n = 9 and 10 images for MEF, 14 and 15 images for U2OS). For all panels, statistical data are presented as mean ± s.e.m. (d) or box-and-whiskers graphs (e and f), in which the box represents the range of 25%-75%, the solid line inside indicates the mean level, and the whiskers represent the minimum and maximum, with n as randomly selected images/cells from ≥3 biological repeats. Two-tailed Student’s t test was used to assess statistical significance.

Source data

Extended Data Fig. 4 Juxta-lysosomal pH of WT and TMEM175 KO cells determined using a lysosome-targeted, genetically-encoded pH indicator.

a, Schematic illustration of the design of a genetically-encoded, lysosome-targeted, juxta-lysosomal pH probe, in which both pH-sensitive pHluorin and pH-insensitive mCherry are fused with lysosomal membrane protein TMEM192 at the cytosolic side (mCherry-TMEM192-pHluorin). b, Representative confocal images of WT and TMEM175 KO HeLa cells transfected with mCherry-TMEM192-pHluorin (scale bar, 10 μm). Right panel shows the summary of calibrated juxta-lysosomal pH values (n = 12 and 21 images for WT and TMEM175 KO cells, respectively). For panel b, statistical data are presented as box-and-whiskers graphs, where the box represents the range of 25%-75%, the solid line inside means the mean level, and the whiskers represent the minimum and maximum with n as randomly selected images/cells from 3 biological repeats. Two-tailed Student’ s t test was used to assess statistical significance.

Source data

Extended Data Fig. 5 TMEM175-mediated H+ flux regulates juxta-lysosomal pH.

a, Representative confocal images of TMEM175 KO HeLa cells transfected with D41A (a selective H+-conductance-deficient mutant with normal K+ permeability) or S45A (normal H+ conductance but with reduced K+-permeability), using CLMLY-SDNFAM/Cy5 (scale bar, 10 μm). Green cycles indicate transfected cells. Right panel shows the summary of juxta-lysosomal pH (n = 9 and 10 images for TMEM175 KO cells transfected with S45A and D41A, respectively). The red dashed line represents the juxta-lysosomal pH of TMEM175 KO HeLa cells. b, Arachidonic acid (ArA) induced juxta-lysosomal pH reduction in WT, but not TMEM175 KO HeLa cells. Representative images of CLMLY-SDNFAM/Cy5-engineered WT and TMEM175 KO HeLa cells treated with or without ArA (100 μM, scale bar, 10 μm). Right panel shows the summary of juxta-lysosomal pH (n = 8, 8, 9, and 7 images for the left to right groups, respectively). For all panels, statistical data are presented as box-and-whiskers graphs, where the box represents the range of 25%-75%, the solid line inside means the mean level, and the whiskers represent the minimum and maximum, with n as randomly selected images from 3 biological repeats. Two-tailed Student’s t test was used to assess statistical significance.

Source data

Extended Data Fig. 6 Surface conjugation with DNA duplexes for measuring the thickness of an acidic layer.

a, Schematic illustration of liposomes anchored with 15-bp, 29-bp, or 52-bp DNA duplex; Right panels show the Dynamic Light Scattering (DLS) profiles of a naked liposome and liposomes conjugated with DNA duplexes of given base pair numbers. b, Correlative analysis of the liposomal diameter versus the number of DNA base pairs (n = 3 independent experiments). c, Summary of DNA duplex thickness on the liposomal surface (n = 3 independent experiments). For all panels, statistical data are presented as mean ± s.d. Two-tailed Student’s t test was used to assess statistical significance.

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Extended Data Fig. 7 Simultaneous monitoring of both intra-lysosomal and juxta-lysosomal acidities.

a, Scheme diagram of the tandem tagBFP-mNectarine-LAMP1-sfGFP probe to monitor intra-lysosomal and juxta-lysosomal acidities simultaneously. The pH-sensitive mNectarine and sfGFP are fused with LAMP1 at cytosolic side and luminal side, respectively, with the tandem pH-insensitive tagBFP (BFP) fused at cytosolic side serves as internal reference. The fluorescence intensity of both mNectarine and sfGFP becomes stronger upon de-acidification (that is, pH elevation). b, Representative fluorescence images of WT and TMEM175 KO HeLa cells expressing tagBFP-mNectarine-LAMP1-sfGFP probes in response to DCPIB (100 μM; scale bar, 10 μm). c, Summary of ratio intensity of sfGFP to tagBFP (to indicate intra-lysosomal acidity) and mNectarine to tagBFP (to indicate juxta-lysosomal acidity) (n = 48, 47, 48, 47, 56, 60, 56 and 60 cells for the left to right groups, respectively). For all panels, statistical Data are presented as mean ± s.e.m. with n as randomly selected cells from ≥ 3 biological repeats. Two-tailed Student’s t test was used to assess statistical significance.

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Extended Data Fig. 8 TMEM175 regulates lysosomal distribution in primary mouse neurons.

a, Summary of juxta-lysosomal acidity determined by using the genetically-encoded mCherry-TMEM192-pHluorin indicator (n = 16, 15, 18, and 13 images for the left to right groups, respectively). The increase of fluorescence ratio (pHluorin vs. mCherry) corresponds to the reduction of juxta-lysosomal acidity (that is, pHjx-LY elevation). b, Representative confocal images of WT and Tmem175 KO mouse neurons treated with or without 3 μg/mL α-synuclein preformed fibril (pff) for 7 days, and stained with anti-LAMP2 and anti-MAP2 antibodies (white dotted line represents soma region; scale bar, 10 μm). c, Summary of LAMP2 immunofluorescence in the soma relative to whole cell (n = 14, 12, 24, and 14 neurons for the left to right groups, respectively). For all panels, statistical data are presented as mean ± s.e.m. with n as randomly selected images/cells from 2 biological repeats. Two-tailed Student’s t test are used to assess statistical significance.

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Extended Data Fig. 9 RILP acts as a juxta-lysosomal pH sensor in regulating lysosome retrograde transport.

a, RILP mediates nutrient-sensitive regulation of lysosome perinuclear positioning. Representative confocal images of LAMP1-transfected WT and TMEM175 KO HeLa cells treated with Torin 1 (200 nM, scale bar, 10 μm). b, RILP clustering is decreased in Baf-A1-treated WT cells or TMEM175 KO HeLa cells. Left: Representative confocal images of WT (treated with or without 1 μM Baf-A1) and TMEM175 KO HeLa cells transfected with RILP-EGFP (scale bar, 10 μm). Right: Summary of LAMP1 intensity ratios on RILP puncta relative to whole-cell (n = 52, 50, and 44 cells for the left to right groups, respectively). Green line represents RILP positively-transfected cells. c, Sequence alignment analyses reveal conserved histidine residues in RILP. RILP and RILP-like protein 1 from different species, including human, mouse, danio rerio (DANRE), bos taurus (BOVIN), and xenopus tropicalis (XENLA), were used for analysis. d, Left: Representative confocal images of WT HeLa cells transfected with WT or mutant (H30A, H130A, H203A, H160A, H160D, H160K, H205A, H280A, H332A, H388A, or H390A) RILP-EGFP (scale bar, 10 μm). Right: Summary of the intensity ratios of LAMP1 versus RILP puncta in whole cell (n = 45, 30, 22, 46, 37, 32, 36, 28, 31, 38, 28, and 44 cells for the left to right groups, respectively). Green solid cycles indicate RILP-transfected cells. e, Representative confocal images of RILP KO HeLa cells transfected with various RILP-EGFP WT and mutant constructs (scale bar, 20 μm). Green solid lines indicate RILP-transfected cells. For all panels, statistical data are presented as mean ± s.e.m. with n as randomly selected cells from 3 biological repeats. Two-tailed Student’s t test are used to assess statistical significance.

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Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Figs. 1–16, Supplementary Tables 1 and 2, unprocessed western blot images, References, and light microscopy reporting table.

Reporting Summary

Supplementary Movie 1

Living imaging of intracellular localization of SDN. Time-lapse confocal imaging of LAMP1–mCherry- or EEA1–mCherry-transfected HeLa cells engineered with SDNCy5 for 120 min (scale bar, 5 μm).

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Zhang, Y., Hu, M., Meng, Y. et al. DNA nanodevices detect an acidic nanolayer on the lysosomal surface. Nat Cell Biol (2026). https://doi.org/10.1038/s41556-025-01855-y

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