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Spatial imaging of glycoRNA in single cells with ARPLA

Nature Biotechnology volume 42, pages 608–616 (2024)Cite this article

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Abstract

Little is known about the biological roles of glycosylated RNAs (glycoRNAs), a recently discovered class of glycosylated molecules, because of a lack of visualization methods. We report sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay (ARPLA) to visualize glycoRNAs in single cells with high sensitivity and selectivity. The signal output of ARPLA occurs only when dual recognition of a glycan and an RNA triggers in situ ligation, followed by rolling circle amplification of a complementary DNA, which generates a fluorescent signal by binding fluorophore-labeled oligonucleotides. Using ARPLA, we detect spatial distributions of glycoRNAs on the cell surface and their colocalization with lipid rafts as well as the intracellular trafficking of glycoRNAs through SNARE protein-mediated secretory exocytosis. Studies in breast cell lines suggest that surface glycoRNA is inversely associated with tumor malignancy and metastasis. Investigation of the relationship between glycoRNAs and monocyte–endothelial cell interactions suggests that glycoRNAs may mediate cell–cell interactions during the immune response.

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Fig. 1: Schematic illustration of glycoRNA in situ imaging using ARPLA.
Fig. 2: Evaluation of ARPLA for glycoRNA imaging in HeLa cells.
Fig. 3: Spatial distributions of glycoRNAs revealed by ARPLA in HL-60 cells.
Fig. 4: Visualization of glycoRNAs in malignant transformation using ARPLA.
Fig. 5: Visualization of glycoRNA levels during THP-1 differentiation and activation by LPS.

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Data availability

The data generated and analyzed during the current study are available at https://figshare.com/projects/Spatial_Imaging_of_GlycoRNA_in_single_Cells_with_ARPLA/164113. Source data are provided with this paper.

Code availability

The code generated and used for data analysis during the current study are available at https://figshare.com/projects/Spatial_Imaging_of_GlycoRNA_in_single_Cells_with_ARPLA/164113.

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Acknowledgements

This research was supported by the US National Institutes of Health (GM141931 to Y.L. and GM133658 to S.S.Y.) and the Susan G. Komen Foundation (CCR19609287 to S.S.Y.). Additionally, the Robert A. Welch Foundation (grant F-0020 to Y. L.) supported the Lu group research programs at The University of Texas at Austin. We thank L. M. Mirica and J. Chan at the Department of Chemistry at University of Illinois Urbana–Champaign for providing SH-SY5Y and THP-1 cell lines and A. B. Baker at the Department of Biomedical Engineering at The University of Texas at Austin for providing the MDA-MB-231 cell line. We especially thank B. Belardi at the Department of Chemical Engineering and B. Xhemalce at the Department of Molecular Biosciences at The University of Texas at Austin for manuscript suggestions. Confocal imaging was performed at the Center for Biomedical Research Support Microscopy and Imaging Facility at The University of Texas at Austin (RRID: SCR_021756). We thank A. Webb and P. Oliphint at The University of Texas at Austin for providing advice on confocal imaging. We would like to thank Y. Wu, M. Banik and R. Yang for providing suggestions on the manuscript and for proofreading.

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  1. These authors contributed equally: Yuan Ma, Weijie Guo.

Authors and Affiliations

  1. Department of Chemistry, The University of Texas at Austin, Austin, TX, USA

    Yuan Ma, Quanbing Mou, Xiangli Shao, Mingkuan Lyu, Whitney Lewis, Zhenglin Yang & Yi Lu

  2. Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA

    Weijie Guo, Valeria Garcia, Linggen Kong & Carson Ward

  3. Interdisciplinary Life Sciences Graduate Programs, The University of Texas at Austin, Austin, TX, USA

    Weijie Guo, Valeria Garcia, Linggen Kong, Whitney Lewis, S. Stephen Yi & Yi Lu

  4. Department of Oncology, Livestrong Cancer Institutes, Dell Medical School, The University of Texas at Austin, Austin, TX, USA

    Xingxin Pan & S. Stephen Yi

  5. Oden Institute for Computational Engineering and Sciences (ICES), The University of Texas at Austin, Austin, TX, USA

    S. Stephen Yi

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Contributions

Y.M., W.G. and Y.L. conceived and designed the study. Y.M. and W.G. performed the experiments and analyzed the data. Q.M. assisted in designing and validating the ARPLA method. Y.M., M.L., C.W. and V.G. performed RNA blotting experiments. Y.M., W.G., X.S., Z.Y. and W.L. performed cell experiments. L.K. performed the MD simulations. X.P. and S.S.Y. analyzed the data. The manuscript was written by Y.M., W.G. and Y.L. Y.L. supervised the project.

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Correspondence to Yi Lu.

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Nature Biotechnology thanks Matthew Disney, Ryan Flynn 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 MD simulation of the structure of the ARPLA system.

(a) A representation of the ARPLA system with different sites (site 1-6) chosen to analyze distances; (b) A representative structure from the simulation with oxDNA, including the glycan probe, the RNA binding probe, the connector 1, and the connector 2. The circle of the connector 1 and the connector 2 tends to form a triangle structure, with two sides formed by DNA helix and one side formed by the ssDNA region in the connector 1; (c) The distributions of the distance between interested sites. 3 sets of distances were calculated with oxDNA tool12, including the distances between the edges of the connector 1 ssDNA region (site 1 and site 2), between the ends of spacers of the Glycan probe and the RNA binding probe (site 3 and site 4), and between the predicted glycan binding site36 and the center of the RISH site (site 5 and site 6). The distance between site 1 and site 2 is in the range of 1-14 nm, with an average around 7 nm. The results agree with the B-form DNA length of the connector 1 ssDNA region (43 nt, around 14.3 nm), with the consideration of ssDNA bending and folding. The distance between site 3 and site 4 is in the range of 5-15 nm, with an average around 10 nm. The distance between site 5 and site 6 is in the range of 5-20 nm, with an average around 15 nm.

Extended Data Fig. 2 Verification of ARPLA method using HeLa as a model cell line.

(a) Thermogram for the ITC titration of 20 µM Neu5Ac aptamer titrated by 1 mM Neu5Ac in aptamer binding buffer; (b) Integrated heat of the ITC titration for Neu5Ac aptamer and Neu5Ac, the black line represents the binding curve fitted with the ‘one set of binding sites’ model; (c) Blotting of total RNA from HeLa cells after metabolic labeling with Ac4ManNAz, or HeLa cells without metabolic labeling; (d) ARPLA-mediated glycoRNA imaging on the surface of HeLa, HL-60, and THP-1 cells. Scale bar: 20 μm (HeLa), 10 μm (HL-60, THP-1); (e) Transmission-through dye microscopic image of HeLa cell. The membrane permeable dye (CellTracker Orange CMRA) and the membrane impermeant quencher acid blue 9 (AB9) were applied to the same cells. AB9 cannot enter the cell with intact membrane and thus cannot quench the membrane permeable dye, so the cells with intact membrane show bright fluorescence signals from CellTracker Orange CMRA. Leaky or damaged membrane after permeabilization treatment allows for the quencher to enter the cell, resulting in reduced or diminished fluorescence of the cell. Scale bar: 40 μm. All experiments were repeated independently three times with similar results.

Extended Data Fig. 3 Evaluation of the generality of ARPLA.

(a) CLSM images of glycoRNAs, utilizing ARPLA with Glycan probes with Neu5Ac aptamer, Tn antigen aptamer, and GalNAc aptamer; (b) Visualization of glycoRNAs with various RNA sequences, including U3, U8, U35a, and Y5; (c) CLSM images of U1 glycoRNA in different cell lines, such as SH-SY5Y, PANC-1, and HEK293T. Scale bars (b,c): 40 µm; (d) Blotting of total RNA from SH-SY5Y cells after metabolic labeling with Ac4ManNAz, or SH-SY5Y cells without metabolic labeling; (e) Blotting of total RNA from PANC-1 cells after metabolic labeling with Ac4ManNAz, or PANC-1 cells without metabolic labeling; (f) Blotting of total RNA from HEK293T cells after metabolic labeling with Ac4ManNAz, or HEK293T cells without metabolic labeling.

Extended Data Fig. 4 3D visualization of the spatial distributions of U1 glycoRNA and CT-B in HL-60 cells.

Z-stack images were collected with the staining of U1 glycoRNA by ARPLA (green) and lipid raft by CT-B (red). The images were shown in z-slices format (a), orthographic projection (b), and maximum intensity projection (c). Scale bar: 2 µm. The z-stack colocalization was repeated independently three times with similar results.

Extended Data Fig. 5 Visualization of glycoRNAs in malignant transformation using ARPLA, related with Fig. 4.

(a,b) CLSM images of MCF-10A, MCF-7, MDA-MB-231 cells in control groups using DNA with scrambled sequences. (c) Agarose gel electrophoresis image of total RNA from MCF-10A, MCF-7, MDA-MB-231 cells. These cells were treated with Ac4ManNAz for 48 h before RNA extraction. All experiments were repeated independently 3 times with similar results.

Extended Data Fig. 6 Fluorescence imaging of bulk sialic acid on the cell surface of MCF-10A, MCF-7, and MDA-MB-231 cells.

(a) Representative cell fluorescent images of bulk sialic acid. These cells were metabolically labeled by Ac4ManNAz for 24 h, followed by incubation with DBCO-PEG4-biotin and Cy5-streptavidin for fluorescence imaging. Scale bars for cell image: 50 μm. (b) Bar plot of the mean fluorescent intensities, the data were calculated from 3 biological replicates. The plot is shown in mean ± SD. Unpaired two-tailed Student’s t-test determines the statistical significance as (*) p = 0.0352, (ns) p = 0.0793, n = 3 independent replicates.

Extended Data Fig. 7 Visualization of glycoRNA level during THP-1 differentiation and activation by LPS, related with Fig. 5.

(a, b) CLSM images of THP-1 monocyte, resting M0 macrophage, and activated M0 macrophage by LPS, which are treated with DNA probe with scrambled sequence to replace aptamer in ARPLA. (c) Blotting of total RNA from THP-1 cells after metabolic labeling with Ac4ManNAz or THP-1 cells without metabolic labeling. (d) Agarose gel electrophoresis image of total RNA from THP-1 monocyte, resting M0 macrophage, and activated M0 macrophage by LPS. These cells were treated with Ac4ManNAz for 48 h before RNA extraction. All experiments were repeated independently three times with similar results.

Extended Data Fig. 8 Investigation of glycoRNA levels during HL-60 differentiation.

(a) CLSM images of U1, U3, and U8 glycoRNA levels evaluated by ARPLA in HL-60 and dHL-60 cells; (b) CLSM images of HL-60, dHL-60 cells in control groups using DNA with scrambled sequences; (c) Quantitative analysis for relative fluorescence intensity of ARPLA in (a) and (b). Data in (c) are representative of three independent experiments, n = 5 frames. Data are mean ±S.D. The statistical significance is determined by unpaired two-tailed Student’s t-test as (ns) not significant, (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001. P (U1 HL60 vs. dHL60) = 0.0159, P (U3 HL60 vs. dHL60) = 0.0079, P (U8 HL60 vs. dHL60) = 0.0002.

Extended Data Fig. 9 Fluorescence imaging of bulk sialic acid on the cell surface of THP-1 monocyte, resting M0 macrophage (M0), LPS activated M0 macrophage (M0 + LPS).

(a) Representative images of total sialic acid. These cells were metabolically labeled by Ac4ManNAz for 24 h, followed by incubation with DBCO-PEG4-biotin and Cy5-streptavidin for fluorescence imaging. Scale bars for cell image: 50 μm. (b) Quantification of the mean fluorescent intensity of the images, n = 3 biological replicates. Data are mean ±S.D. Unpaired two-tailed Student’s t-test determines the statistical significance. (**) p = 0.0013, (*) p = 0.0469.

Extended Data Fig. 10 Cell attachment assay.

The average cell attachment levels in resting M0 macrophage, activated M0 macrophage by LPS, activated M0 macrophage after RNase treatment. Data are representative of three independent experiments, n = 6 technical repeats. Data are mean ±S.D. The statistical significance is determined by unpaired two-tailed Student’s t-test, (***) P < 0.0001, (**) P = 0.0051. Cell attachment assay was performed three times independently with similar results.

Supplementary information

Supplementary Information

Supplementary Table 1. DNA sequences used in the experiments (5′ to 3′). Supplementary Fig. 1. Quantification of ARPLA signal dots for individual cells using U1 glycoRNA in HeLa cells as an example. Supplementary Fig. 2. GlycoRNA gel blot in HeLa cell subfractions. Supplementary Fig. 3. Single-cell analysis of ARPLA dots in breast cell lines. Supplementary Fig. 4. Investigation of total glycoRNA levels during HL-60 differentiation by RNA blotting. Supplementary Fig. 5. Estimation of the limit of resolution.

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Unprocessed gels and blots for all the gels and blots shown in the manuscript.

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Ma, Y., Guo, W., Mou, Q. et al. Spatial imaging of glycoRNA in single cells with ARPLA. Nat Biotechnol 42, 608–616 (2024). https://doi.org/10.1038/s41587-023-01801-z

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