Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Spatially resolved in vivo imaging of inflammation-associated mRNA via enzymatic fluorescence amplification in a molecular beacon

Nature Biomedical Engineering volume 6, pages 1074–1084 (2022)Cite this article

Subjects

Abstract

The in vivo optical imaging of RNA biomarkers of inflammation is hindered by low signal-to-background ratios, owing to non-specific signal amplification in healthy tissues. Here we report the design and in vivo applicability, for the imaging of inflammation-associated messenger RNAs (mRNAs), of a molecular beacon bearing apurinic/apyrimidinic sites, whose amplification of fluorescence is triggered by human apurinic/apyrimidinic endonuclease 1 on translocation from the nucleus into the cytoplasm specifically in inflammatory cells. We assessed the sensitivity and tissue specificity of an engineered molecular beacon targeting interleukin-6 (IL-6) mRNA in live mice, by detecting acute inflammation in their paws and drug-induced inflammation in their livers. This enzymatic-amplification strategy may enable the specific and sensitive imaging of other disease-relevant RNAs in vivo.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Imaging of inflammation-associated mRNA via enzymatic fluorescence amplification.
Fig. 2: Evaluation of APE1-activated signal-amplification strategy in test tubes.
Fig. 3: APE1 translocation-mediated imaging of IL-6 mRNA in inflammatory cells.
Fig. 4: Evaluation of the signal-amplification strategy for imaging of acute inflammation in vivo.
Fig. 5: Evaluation of the signal-amplification strategy for diagnosis of drug-induced hepatotoxicity in vivo.
Fig. 6: Comparison with clinical assays.

Similar content being viewed by others

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are too large to be publicly shared, yet they are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

References

  1. Medzhitov, R. Origin and physiological roles of inflammation. Nature 454, 428–435 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Medzhitov, R. Inflammation 2010: new adventures of an old flame. Cell 140, 771–776 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Liu, C. H. et al. Biomarkers of chronic inflammation in disease development and prevention: challenges and opportunities. Nat. Immunol. 18, 1175–1180 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Signore, A., Mather, S. J., Piaggio, G., Malviya, G. & Dierckx, R. A. Molecular imaging of inflammation/infection: nuclear medicine and optical imaging agents and methods. Chem. Rev. 110, 3112–3145 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Dorward, D. A., Lucas, C. D., Rossi, A. G., Haslett, C. & Dhaliwal, K. Imaging inflammation: molecular strategies to visualize key components of the inflammatory cascade, from initiation to resolution. Pharmacol. Ther. 135, 182–199 (2012).

    Article  CAS  PubMed  Google Scholar 

  6. O’Neill, L. A., Sheedy, F. J. & McCoy, C. E. MicroRNAs: the fine-tuners of Toll-like receptor signalling. Nat. Rev. Immunol. 11, 163–175 (2011).

    Article  PubMed  CAS  Google Scholar 

  7. O’Connell, R. M., Rao, D. S. & Baltimore, D. microRNA regulation of inflammatory responses. Annu. Rev. Immunol. 30, 295–312 (2012).

    Article  PubMed  CAS  Google Scholar 

  8. Femino, A. M., Fay, F. S., Fogarty, K. & Singer, R. H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).

    Article  CAS  PubMed  Google Scholar 

  9. Chen, K. H., Boettiger, A. N., Moffitt, J. R., Wang, S. & Zhuang, X. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Eng, C. L. et al. Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH. Nature 568, 235–239 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lee, J. H. et al. Highly multiplexed subcellular RNA sequencing in situ. Science 343, 1360–1363 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wang, X. et al. Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 361, eaat5691 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Ke, R. et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nat. Methods 10, 857–860 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Kaewsapsak, P., Shechner, D. M., Mallard, W., Rinn, J. L. & Ting, A. Y. Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein–RNA crosslinking. eLife 6, e29224 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Benhalevy, D., Anastasakis, D. G. & Hafner, M. Proximity-CLIP provides a snapshot of protein-occupied RNA elements in subcellular compartments. Nat. Methods 15, 1074–1082 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lizardi, P. M. et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19, 225–232 (1998).

    Article  CAS  PubMed  Google Scholar 

  17. Walker, G. T., Little, M. C., Nadeau, J. G. & Shank, D. D. Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. Proc. Natl Acad. Sci. USA 89, 392–396 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Dirks, R. M. & Pierce, N. A. Triggered amplification by hybridization chain reaction. Proc. Natl Acad. Sci. USA 101, 15275–15278 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Yin, P., Choi, H. T., Calvert, C. R. & Pierce, N. A. Programming biomolecular self-assembly pathways. Nature 451, 318–322 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Choi, H. M. T. et al. Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat. Biotechnol. 28, 1208–1212 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Qing, Z. et al. In situ amplification-based imaging of RNA in living cells. Angew. Chem. Int. Ed. 58, 11574–11585 (2019).

    Article  CAS  Google Scholar 

  22. Sokol, L. D., Zhang, X., Lu, P. & Gewirtz, A. M. Real time detection of DNA•RNA hybridization in living cells. Proc. Natl Acad. Sci. USA 95, 11538–11543 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wang, K. et al. Molecular engineering of DNA: molecular beacons. Angew. Chem. Int. Ed. 48, 856–870 (2009).

    Article  CAS  Google Scholar 

  24. Luby, B. M. & Zheng, G. Specific and direct amplified detection of microRNA with microRNA: argonaute-2 cleavage (miRACle) beacons. Angew. Chem. Int. Ed. 56, 13704–13708 (2017).

    Article  CAS  Google Scholar 

  25. Zhao, J., Chu, H., Zhao, Y., Lu, Y. & Li, L. A NIR light gated DNA nanodevice for spatiotemporally controlled imaging of microRNA in cells and animals. J. Am. Chem. Soc. 141, 7056–7062 (2019).

    Article  CAS  PubMed  Google Scholar 

  26. Mol, C. D., Izumi, T., Mitra, S. & Tainer, J. A. DNA-bound structures and mutants reveal abasic DNA binding by APE1 DNA repair and coordination. Nature 403, 451–456 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Liu, Y. et al. RNA abasic sites in yeast and human cells. Proc. Natl Acad. Sci. USA 117, 20689–20695 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Nath, S. et al. The extracellular role of DNA damage repair protein APE1 in regulation of IL-6 expression. Cell. Signal. 39, 18–31 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Tanaka, T., Narazaki, M. & Kishimoto, T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb. Perspect. Biol. 6, a016295 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Zhang, J. et al. Rationally designed molecular beacons for bioanalytical and biomedical applications. Chem. Soc. Rev. 44, 3036–3055 (2015).

    Article  Google Scholar 

  31. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  PubMed  Google Scholar 

  32. Du, L. et al. The study of relationships between pKa value and siRNA delivery efficiency based on tri-block copolymers. Biomaterials 176, 84–93 (2018).

    Article  CAS  PubMed  Google Scholar 

  33. Park, S. et al. Imaging inflammation using an activated macrophage probe with Slc18b1 as the activation-selective gating target. Nat. Commun. 10, 1111 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Gilleron, J. et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 31, 638–646 (2013).

    Article  CAS  PubMed  Google Scholar 

  35. Fishel, M. L. & Kelley, M. R. The DNA base excision repair protein Ape1/Ref-1 as a therapeutic and chemopreventive target. Mol. Asp. Med. 28, 375–395 (2007).

    Article  CAS  Google Scholar 

  36. Kleiner, D. E. et al. Hepatic histological findings in suspected drug-induced liver injury: systematic evaluation and clinical associations. Hepatology 59, 661–670 (2014).

    Article  PubMed  Google Scholar 

  37. Andrade, R. J. et al. Drug-induced liver injury. Nat. Rev. Dis. Prim. 5, 58 (2019).

    Article  PubMed  Google Scholar 

  38. Tujios, S. & Fontana, R. J. Mechanisms of drug-induced liver injury: from bedside to bench. Nat. Rev. Gastroenterol. Hepatol. 8, 202–211 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Chen, X. et al. Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. Nat. Biotechnol. 37, 1287–1293 (2019).

    Article  CAS  PubMed  Google Scholar 

  40. Mustoe, A. M., Lama, N. N., Irving, P. S., Olson, S. W. & Weeks, K. M. RNA base-pairing complexity in living cells visualized by correlated chemical probing. Proc. Natl Acad. Sci. USA 116, 24574–24582 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Larsson, C., Grundberg, I., Söderberg, O. & Nilsson, M. In situ detection and genotyping of individual mRNA molecules. Nat. Methods 7, 395–397 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Xue, C. et al. Target-induced catalytic assembly of Y-shaped DNA and its application for in situ imaging of microRNAs. Angew. Chem. Int. Ed. 57, 9739–9743 (2018).

    Article  CAS  Google Scholar 

  43. He, L. et al. mRNA-initiated, three-dimensional DNA amplifier able to function inside living cells. J. Am. Chem. Soc. 140, 258–263 (2018).

    Article  CAS  PubMed  Google Scholar 

  44. Kishi, J. Y. et al. SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues. Nat. Methods 16, 533–544 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Shah, F. et al. Exploiting the Ref-1–APE1 node in cancer signaling and other diseases: from bench to clinic. NPJ Precis. Oncol. 1, 19 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Abbotts, R. & Madhusudan, S. Human AP endonuclease 1 (APE1): from mechanistic insights to druggable target in cancer. Cancer Treat. Rev. 36, 425–435 (2010).

    Article  CAS  PubMed  Google Scholar 

  47. Thakur, S. et al. APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions. Exp. Mol. Med. 46, e106 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Sang, K. L. et al. Apurinic/apyrimidinic endonuclease 1 inhibits protein kinase C-mediated p66shc phosphorylation and vasoconstriction. Cardiovasc. Res. 91, 502–509 (2011).

    Article  CAS  Google Scholar 

  49. Huang, E. et al. The role of Cdk5-mediated apurinic/apyrimidinic endonuclease 1 phosphorylation in neuronal death. Nat. Cell Biol. 12, 563–571 (2010).

    Article  CAS  PubMed  Google Scholar 

  50. Lorenz, R. et al. ViennaRNA Package 2.0. Algorithm. Mol. Biol. 6, 26 (2011).

    Google Scholar 

Download references

Acknowledgements

This work was supported financially by the National Natural Science Foundation of China (22125402 (to L.L.) and 22004023 (to J.Z.)), the Youth Innovation Promotion Association CAS (to J.Z.), the National Key R&D Program of China (2021YFA1200104, to L.L.) and the Strategic Priority Research Program of Chinese Academy of Sciences (XDB36000000, to L.L. and Y.Z.).

Author information

Author notes

  1. These authors contributed equally: Chuangui Sheng, Jian Zhao.

Authors and Affiliations

  1. CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, China

    Chuangui Sheng, Jian Zhao, Zhenghan Di, Yuliang Zhao & Lele Li

  2. College of Materials Science and Optoelectronic Technology, University of Chinese Academy of Sciences, Beijing, China

    Chuangui Sheng, Jian Zhao, Zhenghan Di, Yuliang Zhao & Lele Li

  3. Advanced Research Institute of Multidisciplinary Science, Beijing Institute of Technology, Beijing, China

    Yuanyu Huang

  4. GBA Research Innovation Institute for Nanotechnology, Guangdong, China

    Yuliang Zhao & Lele Li

Authors
  1. Chuangui Sheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jian Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhenghan Di
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuanyu Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yuliang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lele Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.S., J.Z. and L.L conceived the idea and designed all the experiments. C.S. and J.Z. performed the experiments. Y.H. provided the PEGylated tri-block co-polymer. C.S., J.Z., Z.D., Y.Z. and L.L. analysed and discussed the results. C.S., J.Z. and L.L. wrote the manuscript. All authors reviewed the manuscript and approved the final version.

Corresponding author

Correspondence to Lele Li.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Biomedical Engineering thanks Li-Qun Gu 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.

Supplementary information

Supplementary information

Supplementary methods, figures and tables.

Reporting Summary

Source data

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sheng, C., Zhao, J., Di, Z. et al. Spatially resolved in vivo imaging of inflammation-associated mRNA via enzymatic fluorescence amplification in a molecular beacon. Nat. Biomed. Eng 6, 1074–1084 (2022). https://doi.org/10.1038/s41551-022-00932-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-022-00932-z

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing