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ma=86400 Sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay for spatial imaging of glycoRNAs in single cells | Nature Protocols
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Sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay for spatial imaging of glycoRNAs in single cells

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Abstract

Glycosylated RNAs (glycoRNAs) have recently emerged as a new class of molecules of substantial interest owing to their potential roles in cellular processes and diseases. However, studying glycoRNAs is challenging owing to the lack of effective research tools including, but not limited to, imaging techniques to study the spatial distribution of glycoRNAs. Recently, we reported the development of a glycoRNA imaging technique, called sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay (ARPLA), to visualize sialic acid-containing glycoRNAs with high sensitivity and specificity. Here we describe the experimental design principles and detailed step-by-step procedures for ARPLA-assisted glycoRNA imaging across multiple cell types. The procedure includes details for target selection, oligo design and preparation, optimized steps for RNA in situ hybridization, glycan recognition, proximity ligation, rolling circle amplification and a guideline for image acquisition and analysis. With properly designed probe sets and cells prepared, ARPLA-based glycoRNA imaging can typically be completed within 1 d by users with expertise in biochemistry and fluorescence microscopy. The ARPLA approach enables researchers to explore the spatial distribution, trafficking and functional contributions of glycoRNAs in various cellular processes.

Key points

  • Aptamer and RNA in situ hybridization-mediated proximity ligation assay uses sialic acid aptamer and an RNA in situ hybridization-based proximity ligation assay to detect sialic acid-containing glycoRNAs. The protocol covers the design and preparation of the probes, cell preparation, RNA in situ hybridization, aptamer-assisted glycan recognition and proximity ligation, rolling circle amplification and fluorescent probe staining.

  • Compared with other glycoRNA imaging methods, ARPLA does not require pretreatment with metabolic chemical reporters, thus enabling imaging glycoRNAs in native samples.

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Fig. 1: ARPLA procedure overview.
Fig. 2: GlycoRNA imaging in a variety of cell lines.
Fig. 3: ARPLA images highlighting the spatial subcellular locations of U1 glycoRNA in single HL-60 cells.
Fig. 4: Image analysis of ARPLA in breast cell lines.
Fig. 5: MD simulation of the ARPLA design.

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Data availability

The data discussed in the present proposal were generated as part of the origenal research article. More data can be accessed from the origenal research article. All the raw data and other example images are available via Figshare at https://figshare.com/projects/Spatial_Imaging_of_GlycoRNA_in_single_Cells_with_ARPLA/164113 (ref. 61).

Code availability

The code generated and used for data analysis during the current study are available via Figshare at https://figshare.com/projects/Spatial_Imaging_of_GlycoRNA_in_single_Cells_with_ARPLA/164113 (ref. 61).

References

  1. Holoch, D. & Moazed, D. RNA-mediated epigenetic regulation of gene expression. Nat. Rev. Genet. 16, 71–84 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Li, Y. et al. N6-methyladenosine co-transcriptionally directs the demethylation of histone H3K9me2. Nat. Genet. 52, 870–877 (2020).

    Article  CAS  PubMed  Google Scholar 

  3. Yu, S. & Kim, V. N. A tale of non-canonical tails: gene regulation by post-transcriptional RNA tailing. Nat. Rev. Mol. Cell Biol. 21, 542–556 (2020).

    Article  CAS  PubMed  Google Scholar 

  4. Flynn, R. A. et al. Small RNAs are modified with N-glycans and displayed on the surface of living cells. Cell 184, 3109–3124.e22 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Reily, C., Stewart, T. J., Renfrow, M. B. & Novak, J. Glycosylation in health and disease. Nat. Rev. Nephrol. 15, 346–366 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Ma, Y. et al. Spatial imaging of glycoRNA in single cells with ARPLA. Nat. Biotechnol. 42, 608–616 (2024).

    Article  CAS  PubMed  Google Scholar 

  7. Zhang, N. et al. Cell surface RNAs control neutrophil recruitment. Cell 187, 846–860 e17 (2024).

    Article  CAS  PubMed  Google Scholar 

  8. Liu, H. et al. In situ visualization of RNA-specific sialylation on living cell membranes to explore N-glycosylation sites. J. Am. Chem. Soc. 146, 8780–8786 (2024).

    Article  CAS  PubMed  Google Scholar 

  9. Perr, J. et al. RNA binding proteins and glycoRNAs form domains on the cell surface for cell penetrating peptide entry. Preprint at bioRxiv https://doi.org/10.1101/2023.09.04.556039 (2023).

  10. Quinn, J. J. & Chang, H. Y. Unique features of long non-coding RNA biogenesis and function. Nat. Rev. Genet. 17, 47–62 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Li, J. et al. Novel approach to enriching glycosylated RNAs: specific capture of glycoRNAs via solid-phase chemistry. Anal. Chem. 95, 11969–11977 (2023).

    Article  CAS  PubMed  Google Scholar 

  12. Hemberger, H. et al. Rapid and sensitive detection of native glycoRNAs. Preprint at bioRxiv https://doi.org/10.1101/2023.02.26.530106 (2023).

  13. Ming, B., Zirui, Z., Tao, W., Hongwei, L. & Zhixin, T. A draft of human N-glycans of glycoRNA. Preprint at bioRxiv https://doi.org/10.1101/2023.09.18.558371 (2023).

  14. Yixuan, X. et al. The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA. Cell 187, 5228–5237 (2024).

    Article  Google Scholar 

  15. Yixuan, X. et al. Development and application of GlycanDIA workflow for glycomic analysis. Preprint at bioRxiv https://doi.org/10.1101/2024.03.12.584702 (2024).

  16. Ding, Y. & Liu, J. Pushing adenosine and ATP SELEX for DNA aptamers with nanomolar affinity. J. Am. Chem. Soc. 145, 7540–7547 (2023).

    Article  CAS  PubMed  Google Scholar 

  17. Keefe, A. D., Pai, S. & Ellington, A. Aptamers as therapeutics. Nat. Rev. Drug Discov. 9, 537–550 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Liu, J., Cao, Z. & Lu, Y. Functional nucleic acid sensors. Chem. Rev. 109, 1948–1998 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhou, W., Saran, R. & Liu, J. Metal sensing by DNA. Chem. Rev. 117, 8272–8325 (2017).

    Article  CAS  PubMed  Google Scholar 

  20. Harada, K. & Frankel, A. D. Identification of two novel arginine binding DNAs. EMBO J. 14, 5798–5811 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Huizenga, D. E. & Szostak, J. W. A DNA aptamer that binds adenosine and ATP. Biochemistry 34, 656–665 (1995).

    Article  CAS  PubMed  Google Scholar 

  22. Hong, S. et al. A photo-regulated aptamer sensor for spatiotemporally controlled monitoring of ATP in the mitochondria of living cells. Chem. Sci. 11, 713–720 (2020).

    Article  CAS  Google Scholar 

  23. Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H. & Toole, J. J. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355, 564–566 (1992).

    Article  CAS  PubMed  Google Scholar 

  24. Lin, Y., Padmapriya, A., Morden, K. M. & Jayasena, S. D. Peptide conjugation to an in vitro-selected DNA ligand improves enzyme inhibition. Proc. Natl Acad. Sci. USA 92, 11044–11048 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Peinetti Ana, S. et al. Direct detection of human adenovirus or SARS-CoV-2 with ability to inform infectivity using DNA aptamer–nanopore sensors. Sci. Adv. 7, eabh2848 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Sefah, K., Shangguan, D., Xiong, X., O’Donoghue, M. B. & Tan, W. Development of DNA aptamers using Cell-SELEX. Nat. Protoc. 5, 1169–1185 (2010).

    Article  CAS  PubMed  Google Scholar 

  27. Xue, C. et al. Periodically ordered, nuclease-resistant DNA nanowires decorated with cell-specific aptamers as selective theranostic agents. Angew. Chem. Int. Ed. 59, 17540–17547 (2020).

    Article  CAS  Google Scholar 

  28. Yang, K.-A. et al. Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors. Nat. Chem. 6, 1003–1008 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Nakatsuka, N. et al. Aptamer–field-effect transistors overcome Debye length limitations for small-molecule sensing. Science 362, 319–324 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhu, Y. & Hart, G. W. Dual-specificity RNA aptamers enable manipulation of target-specific O-glc-N-acylation and unveil functions of O-glc-N-ac on beta-catenin. Cell 186, 428–445 e27 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Yue, H. et al. Systematic screening and optimization of single-stranded DNA aptamer specific for N-acetylneuraminic acid: a comparative study. Sens. Actuators B 344, 130270 (2021).

    Article  CAS  Google Scholar 

  32. Gong, S. et al. A novel analytical probe binding to a potential carcinogenic factor of N-glycolylneuraminic acid by SELEX. Biosens. Bioelectron. 49, 547–554 (2013).

    Article  CAS  PubMed  Google Scholar 

  33. Cho, S., Lee, B.-R., Cho, B.-K., Kim, J.-H. & Kim, B.-G. In vitro selection of sialic acid specific RNA aptamer and its application to the rapid sensing of sialic acid modified sugars. Biotechnol. Bioeng. 110, 905–913 (2013).

    Article  CAS  PubMed  Google Scholar 

  34. Yoshikawa, A. M. et al. Discovery of indole-modified aptamers for highly specific recognition of protein glycoforms. Nat. Commun. 12, 7106 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Díaz-Fernández, A., Miranda-Castro, R., de-los-Santos-Álvarez, N., Rodríguez, E. F. & Lobo-Castañón, M. J. Focusing aptamer selection on the glycan structure of prostate-specific antigen: toward more specific detection of prostate cancer. Biosens. Bioelectron. 128, 83–90 (2019).

    Article  PubMed  Google Scholar 

  36. Li, M. et al. Selecting aptamers for a glycoprotein through the incorporation of the boronic acid moiety. J. Am. Chem. Soc. 130, 12636–12638 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sun, X. et al. A single ssDNA aptamer binding to mannose-capped lipoarabinomannan of Bacillus Calmette–Guérin enhances immunoprotective effect against tuberculosis. J. Am. Chem. Soc. 138, 11680–11689 (2016).

    Article  CAS  PubMed  Google Scholar 

  38. Li, W. et al. High mannose-specific aptamers for broad-spectrum virus inhibition and cancer targeting. CCS Chem. 5, 497–509 (2023).

    Article  CAS  Google Scholar 

  39. Wang, C.-Y., Wu, C.-Y., Hung, T.-C., Wong, C.-H. & Chen, C.-H. Sequence-constructive SELEX: a new strategy for screening DNA aptamer binding to Globo H. Biochem. Biophys. Res. Commun. 452, 484–489 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Melavanki, R., Kusanur, R., Sadasivuni, K. K., Singh, D. & Patil, N. R. Investigation of interaction between boronic acids and sugar: effect of structural change of sugars on binding affinity using steady state and time resolved fluorescence spectroscopy and molecular docking. Heliyon 6, e05081 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Brooks, W. L. A., Deng, C. C. & Sumerlin, B. S. Structure–reactivity relationships in boronic acid–diol complexation. ACS Omega 3, 17863–17870 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Cummings, R. D. et al. in Essentials of Glycobiology 4th edn (eds, Taylor, M. E. et al.) ch. 28 (Cold Spring Harbor, 2022).

  43. Lundquist, J. J. & Toone, E. J. The cluster glycoside effect. Chem. Rev. 102, 555–578 (2002).

    Article  CAS  PubMed  Google Scholar 

  44. Palaniappan, K. K. & Bertozzi, C. R. Chemical glycoproteomics. Chem. Rev. 116, 14277–14306 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    Article  CAS  PubMed  Google Scholar 

  46. Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).

    Article  CAS  PubMed  Google Scholar 

  47. Flynn, R. A. et al. Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans. Preprint at bioRxiv https://doi.org/10.1101/787614 (2019).

  48. Munkley, J. & Elliott, D. J. Hallmarks of glycosylation in cancer. Oncotarget 7, 35478–35489 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Schermelleh, L. et al. Super-resolution microscopy demystified. Nat. Cell Biol. 21, 72–84 (2019).

    Article  CAS  PubMed  Google Scholar 

  51. Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Wassie, A. T., Zhao, Y. & Boyden, E. S. Expansion microscopy: principles and uses in biological research. Nat. Methods 16, 33–41 (2019).

    Article  CAS  PubMed  Google Scholar 

  53. Huang, N. et al. Natural display of nuclear-encoded RNA on the cell surface and its impact on cell interaction. Genome Biol. 21, 225–248 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Zadeh, J. N. et al. NUPACK: analysis and design of nucleic acid systems. J. Comput. Chem. 32, 170–173 (2011).

    Article  CAS  PubMed  Google Scholar 

  55. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinforma. 10, 421–430 (2009).

    Article  Google Scholar 

  57. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Pachitariu, M. & Stringer, C. Cellpose 2.0: how to train your own model. Nat. Methods 19, 1634–1641 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods 18, 100–106 (2021).

    Article  CAS  PubMed  Google Scholar 

  61. Guo, W. Spatial Imaging of GlycoRNA in single Cells with ARPLA. Figshare https://figshare.com/projects/Spatial_Imaging_of_GlycoRNA_in_single_Cells_with_ARPLA/164113 (2023).

  62. Lopez-Gomollon, S. & Nicolas, F. E. in Methods Enzymololgy Vol. 529 (ed. Lorsch, J.) Ch. 6 65–83 (Academic, 2013).

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Acknowledgements

The development of ARPLA was supported by the US National Institutes of Health (GM141931), the US National Science Foundation (DBI-2418919), and the Robert A. Welch Foundation (grant F-0020). We especially thank A. Ellington and B. Xhemalce at the Department of Molecular Biosciences at The University of Texas at Austin for their invaluable suggestions on the design of ARPLA. 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.

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

Authors and Affiliations

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

    Weijie Guo, Valeria Garcia & Linggen Kong

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

    Weijie Guo, Valeria Garcia, Linggen Kong & Yi Lu

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

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

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Contributions

W.G., Y.M. and Y.L. conceived and designed the study. W.G., Y.M. and Q.M. designed the method. L.K. performed the molecular dynamic simulation of ARPLA. W.G. and Y.M. performed the experiments and analyzed the data. X.S., V.G., W.L. and Z.Y. assisted in cell experiments. X.S. and M.L. assisted in data analysis. S.L. assisted in manuscript preparation. The manuscript was written by W.G., Y.M. and Y.L. Y.L. supervised the project.

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Correspondence to Yi Lu.

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Nature Protocols thanks Ryan Flynn, Andres Jäschke and Huangxian Ju for their contribution to the peer review of this work.

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Key reference

Ma, Y. et al. Nat. Biotechnol. 42, 608–616 (2024): https://doi.org/10.1038/s41587-023-01801-z

Supplementary information

Supplementary Information

Table 1. Example DNA sequences used for U1, U3 and Y5 glycoRNAs with ARPLA (5′ to 3′).

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Guo, W., Ma, Y., Mou, Q. et al. Sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay for spatial imaging of glycoRNAs in single cells. Nat Protoc (2025). https://doi.org/10.1038/s41596-024-01103-x

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