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
Open Access articles citing this article.
Journal of Nanobiotechnology Open Access 21 June 2023
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$99.00 per year
only $8.25 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
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.
Medzhitov, R. Origin and physiological roles of inflammation. Nature 454, 428–435 (2008).
Medzhitov, R. Inflammation 2010: new adventures of an old flame. Cell 140, 771–776 (2010).
Liu, C. H. et al. Biomarkers of chronic inflammation in disease development and prevention: challenges and opportunities. Nat. Immunol. 18, 1175–1180 (2017).
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).
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).
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).
O’Connell, R. M., Rao, D. S. & Baltimore, D. microRNA regulation of inflammatory responses. Annu. Rev. Immunol. 30, 295–312 (2012).
Femino, A. M., Fay, F. S., Fogarty, K. & Singer, R. H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).
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).
Eng, C. L. et al. Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH. Nature 568, 235–239 (2019).
Lee, J. H. et al. Highly multiplexed subcellular RNA sequencing in situ. Science 343, 1360–1363 (2014).
Wang, X. et al. Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 361, eaat5691 (2018).
Ke, R. et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nat. Methods 10, 857–860 (2013).
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).
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).
Lizardi, P. M. et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19, 225–232 (1998).
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).
Dirks, R. M. & Pierce, N. A. Triggered amplification by hybridization chain reaction. Proc. Natl Acad. Sci. USA 101, 15275–15278 (2004).
Yin, P., Choi, H. T., Calvert, C. R. & Pierce, N. A. Programming biomolecular self-assembly pathways. Nature 451, 318–322 (2008).
Choi, H. M. T. et al. Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat. Biotechnol. 28, 1208–1212 (2010).
Qing, Z. et al. In situ amplification-based imaging of RNA in living cells. Angew. Chem. Int. Ed. 58, 11574–11585 (2019).
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).
Wang, K. et al. Molecular engineering of DNA: molecular beacons. Angew. Chem. Int. Ed. 48, 856–870 (2009).
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).
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).
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).
Liu, Y. et al. RNA abasic sites in yeast and human cells. Proc. Natl Acad. Sci. USA 117, 20689–20695 (2020).
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).
Tanaka, T., Narazaki, M. & Kishimoto, T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb. Perspect. Biol. 6, a016295 (2014).
Zhang, J. et al. Rationally designed molecular beacons for bioanalytical and biomedical applications. Chem. Soc. Rev. 44, 3036–3055 (2015).
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).
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).
Park, S. et al. Imaging inflammation using an activated macrophage probe with Slc18b1 as the activation-selective gating target. Nat. Commun. 10, 1111 (2019).
Gilleron, J. et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 31, 638–646 (2013).
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).
Kleiner, D. E. et al. Hepatic histological findings in suspected drug-induced liver injury: systematic evaluation and clinical associations. Hepatology 59, 661–670 (2014).
Andrade, R. J. et al. Drug-induced liver injury. Nat. Rev. Dis. Prim. 5, 58 (2019).
Tujios, S. & Fontana, R. J. Mechanisms of drug-induced liver injury: from bedside to bench. Nat. Rev. Gastroenterol. Hepatol. 8, 202–211 (2011).
Chen, X. et al. Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. Nat. Biotechnol. 37, 1287–1293 (2019).
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).
Larsson, C., Grundberg, I., Söderberg, O. & Nilsson, M. In situ detection and genotyping of individual mRNA molecules. Nat. Methods 7, 395–397 (2010).
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).
He, L. et al. mRNA-initiated, three-dimensional DNA amplifier able to function inside living cells. J. Am. Chem. Soc. 140, 258–263 (2018).
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).
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).
Abbotts, R. & Madhusudan, S. Human AP endonuclease 1 (APE1): from mechanistic insights to druggable target in cancer. Cancer Treat. Rev. 36, 425–435 (2010).
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).
Sang, K. L. et al. Apurinic/apyrimidinic endonuclease 1 inhibits protein kinase C-mediated p66shc phosphorylation and vasoconstriction. Cardiovasc. Res. 91, 502–509 (2011).
Huang, E. et al. The role of Cdk5-mediated apurinic/apyrimidinic endonuclease 1 phosphorylation in neuronal death. Nat. Cell Biol. 12, 563–571 (2010).
Lorenz, R. et al. ViennaRNA Package 2.0. Algorithm. Mol. Biol. 6, 26 (2011).
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.).
The authors declare no competing interests.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
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
This article is cited by
Journal of Nanobiotechnology (2023)