A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells

Journal name:
Nature Nanotechnology
Year published:
Published online


The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5–130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.

At a glance


  1. Design and characterization of Clensor and ClensorTf.
    Figure 1: Design and characterization of Clensor and ClensorTf.

    a, Structure and working principle of Clensor. P, sensing module (pink line) containing a Cl-sensitive fluorophore, BAC (green filled circle); D2, normalizing module (brown line) containing a Cl-insensitive fluorophore, Alexa 647 (red filled circle); D1, targeting module (orange line). In the presence of Cl, BAC undergoes collisional quenching, whereas fluorescence of Alexa 647 is Cl-independent. b, Modified sensor design for targeting to the recycling pathway (ClensorTf). D1Tfapt, targeting module modified with an RNA aptamer (Tfapt) against the human transferrin receptor (cyan line). c, Fluorescence emission spectra of Clensor at the indicated [Cl] obtained using λExBAC = 435 nm (green) and λExAlexa 647 = 650 nm (red). d, In vitro Cl calibration profile of Clensor showing normalized Alexa 647 and BAC fluorescence intensity ratio (R/G) versus [Cl]. R/G values at different chloride concentrations were normalized to the value at 5 mM chloride. e, Plot of KSV for Clensor versus pH. Error bars indicate the mean of three independent experiments ± s.e.m.

  2. Programming Clensor delivery to specific endocytic organelles.
    Figure 2: Programming Clensor delivery to specific endocytic organelles.

    a, ClensorA647 internalization by haemocytes in the presence (+mBSA) and absence (−mBSA) of excess competitor ligand maleylated BSA (mBSA, 10 μM) and autofluorescence (AF) in Drosophila haemocytes. Error bars indicate the mean of three independent experiments ± s.e.m. (n = 20 cells). b, Trafficking of Clensor (ClensorA647) in haemocytes isolated from flies expressing YFP-Rab5 (upper row), YFP-Rab7 (middle row) and GFP-LAMP (lower row) at the indicated chase times. ClensorA647-positive vesicles are shown in red and YFP-Rab5/YFP-Rab7/GFP-LAMP vesicles are shown in green. Scale bars, 10 μm. c, Competition experiments with ClensorTfA647 and excess unlabelled transferrin (Tf, 25 μM). Normalized intensities of cells pulsed with ClensorTfA647 in the presence (+Tf) and absence (–Tf) of Tf and ClensorA647 are shown. Error bars indicate the mean of three independent experiments ± s.e.m. (n = 15 cells). d, Co-localization of ClensorTfA647 with transferrin (TfA568) in S2R+ cells. Scale bars, 10 μm.

  3. Quantitative performance of Clensor within subcellular organelles.
    Figure 3: Quantitative performance of Clensor within subcellular organelles.

    a, Alexa 647 channel and respective pseudocolour R/G map of Drosophila haemocytes pulsed with Clensor and clamped at 5, 40 and 80 mM Cl. Scale bars, 10 μm. b, Histograms showing typical spread of R/G ratios of vesicles clamped at 5, 40 and 80 mM Cl (n ≈ 10 cells, ≥50 endosomes). c, In vitro and intracellular fold change in R/G ratios of Clensor at 5 and 120 mM Cl. d, Normalized R/G intensity (Alexa 647/BAC) ratios inside the endosomes, plotted as a function of [Cl], yield the intracellular calibration profile (red), which is overlaid on the in vitro chloride calibration profile (black). Error bars indicate the mean of three independent experiments ± s.e.m. (n ≈ 10 cells, ≥50 endosomes).

  4. Spatiotemporal mapping of [Cl−] along the endolysosomal pathway using Clensor in living cells.
    Figure 4: Spatiotemporal mapping of [Cl] along the endolysosomal pathway using Clensor in living cells.

    a, Representative pseudocolour R/G map of live haemocytes isolated from wild-type Drosophila and labelled with Clensor. Scale bar, 10 μm. b, Histograms of R/G ratios of EE at 5 min (green), LE at 60 min (orange) and LY at 120 min (red) (n ≈ 20 cells, ∼100 endosomes/lysosomes). c,d, RT-PCR analysis of total RNA isolated from 3rd instar larvae of UAS-DmClC-b RNAi/Coll GAL4 larvae (DmClC-b RNAi) (c) and UAS-DmClC-c RNAi/Coll GAL4 larvae (DmClC-c RNAi) (d). Lanes marked as CS correspond to PCR-amplified cDNA of the indicated gene products (DmClC-b and DmClC-c) from wild-type Drosophila, and lanes marked as RNAi are for the corresponding gene products isolated from 3rd instar larvae of UASDmClC-b RNAi/Coll GAL4 (c) and UAS-DmClC-c RNAi/CollGAL4 (d) flies. RpL32 was used as a loading control. eg, Box plots showing [Cl¯] distributions for EE (e), LE (f) and LY (g) in haemocytes of the indicated genetic background (n ∼ 15 cells, ∼75 endosomes/lysosomes). h,i, Box plots showing pH distributions in EE (h) and LE (i) in haemocytes of the indicated genetic background using the FD10 dual excitation method. j, Box plots showing pH distribution in LY in haemocytes of the indicated genetic background using I4LY A488/A647 (n > 15 cells, >50 endosomes/lysosomes. In ej: CS, wild-type; b RNAi, RNAi against DmClC-b; b mutant, DmClC-b mutant; c RNAi, DmClC-c RNAi. Boxes represent 25–75% of the population. Horizontal lines within boxes represent the median. Filled circles represent mean of the data obtained for each indicated genotype. Error bars on filled circles represent s.e.m.

  5. ClensorTf maps [Cl−] within recycling endosomes (REs).
    Figure 5: ClensorTf maps [Cl] within recycling endosomes (REs).

    a,b, RNAi knockdown of DmClC-c and DmClC-b in Drosophila S2R+ cells. PCR-amplified cDNA of the indicated gene products (DmClC-b and DmClC-c) isolated from untreated and RNAi treated Drosophila S2R+ cells for RNAi against DmClC-c (a) and RNAi against DmClC-b (b). RpL32 was used as loading control.


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Author information


  1. National Centre for Biological Sciences, TIFR, GKVK, Bellary Road, Bangalore 560065, India

    • Sonali Saha,
    • Saheli Halder &
    • Yamuna Krishnan
  2. Department of Chemistry, University of Chicago, 929E, 57th Street, E305A, GCIS, Chicago, Illinois 60637, USA

    • Ved Prakash,
    • Kasturi Chakraborty &
    • Yamuna Krishnan


S.S., K.C., V.P. and Y.K. conceived and designed the experiments. S.S., V.P. and K.C. performed the experiments. S.H. designed the I4LYA488/A647 used herein. S.S., V.P., K.C. and Y.K. analysed the data. S.S. and Y.K. co-wrote the paper. All authors commented on the manuscript.

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