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Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs

Nature Biotechnology volume 37, pages 1287–1293 (2019)Cite this article

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Abstract

Fluorescent RNAs (FRs), aptamers that bind and activate fluorescent dyes, have been used to image abundant cellular RNA species. However, limitations such as low brightness and limited availability of dye/aptamer combinations with different spectral characteristics have limited use of these tools in live mammalian cells and in vivo. Here, we develop Peppers, a series of monomeric, bright and stable FRs with a broad range of emission maxima spanning from cyan to red. Peppers allow simple and robust imaging of diverse RNA species in live cells with minimal perturbation of the target RNA’s transcription, localization and translation. Quantification of the levels of proteins and their messenger RNAs in single cells suggests that translation is governed by normal enzyme kinetics but with marked heterogeneity. We further show that Peppers can be used for imaging genomic loci with CRISPR display, for real-time tracking of protein–RNA tethering, and for super-resolution imaging. We believe these FRs will be useful tools for live imaging of cellular RNAs.

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Fig. 1: Characterization of Pepper530 RMFP.
Fig. 2: Expanding spectral range of FRs.
Fig. 3: Multi-color imaging of genomic loci using CRISPR display of Pepper.

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The data that support the findings in this study are available upon reasonable request from the corresponding authors.

References

  1. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Battich, N., Stoeger, T. & Pelkmans, L. Image-based transcriptomics in thousands of single human cells at single-molecule resolution. Nat. Methods 10, 1127–1133 (2013).

    CAS  PubMed  Google Scholar 

  3. Li, F. et al. A covalent approach for site-specific RNA labeling in mammalian cells. Angew. Chem Int. Ed. Engl. 54, 4597–4602 (2015).

    CAS  PubMed  Google Scholar 

  4. Alexander, S. C., Busby, K. N., Cole, C. M., Zhou, C. Y. & Devaraj, N. K. Site-specific covalent labeling of RNA by enzymatic transglycosylation. J. Am. Chem. Soc. 137, 12756–12759 (2015).

    CAS  PubMed  Google Scholar 

  5. Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).

    CAS  PubMed  Google Scholar 

  6. Nelles, D. A. et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165, 488–496 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Daigle, N. & Ellenberg, J. LambdaN-GFP: an RNA reporter system for live-cell imaging. Nat. Methods 4, 633–636 (2007).

    CAS  PubMed  Google Scholar 

  8. Batra, R. et al. Elimination of toxic microsatellite repeat expansion RNA by RNA-targeting Cas9.Cell 179, 899–912 (2017).

    Google Scholar 

  9. Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nat. Methods 6, 331–338 (2009).

    CAS  PubMed  Google Scholar 

  10. You, M. & Jaffrey, S. R. Structure and mechanism of RNA mimics of green fluorescent protein. Ann. Rev. Biophys. 44, 187–206 (2015).

    CAS  Google Scholar 

  11. Ouellet, J. RNA Fluorescence with Light-Up Aptamers. Front. Chem. 4, 29 (2016).

    PubMed  PubMed Central  Google Scholar 

  12. Tutucci, E. et al. An improved MS2 system for accurate reporting of the mRNA life cycle. Nat. Methods 15, 81–89 (2018).

    CAS  PubMed  Google Scholar 

  13. Constantin, T. P. et al. Synthesis of new fluorogenic cyanine dyes and incorporation into RNA fluoromodules. Org. Lett. 10, 1561–1564 (2008).

    CAS  PubMed  Google Scholar 

  14. Dolgosheina, E. V. et al. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking. ACS Chem. Biol. 9, 2412–2420 (2014).

    CAS  PubMed  Google Scholar 

  15. Tan, X. et al. Fluoromodules consisting of a promiscuous RNA aptamer and red or blue fluorogenic cyanine dyes: selection, characterization, and bioimaging. J. Am. Chem. Soc. 139, 9001–9009 (2017).

    CAS  PubMed  Google Scholar 

  16. Autour, A. et al. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nature Commun. 9, 656 (2018).

    Google Scholar 

  17. Sando, A., Narita, A., Hayami, M. & Aoyama, Y. Transcription monitoring using fused RNA with a dye-binding light-up aptamer as a tag: a blue fluorescent RNA.Chem. Commun. 33, 3858–3860 (2008).

    Google Scholar 

  18. Babendure, J. R., Adams, S. R. & Tsien, R. Y. Aptamers switch on fluorescence of triphenylmethane dyes. J. Am. Chem. Soc. 125, 14716–14717 (2003).

    CAS  PubMed  Google Scholar 

  19. Sunbul, M. & Jaschke, A. Contact-mediated quenching for RNA imaging in bacteria with a fluorophore-binding aptamer. Angew. Chem. 52, 13401–13404 (2013).

    CAS  Google Scholar 

  20. Arora, A., Sunbul, M. & Jaschke, A. Dual-colour imaging of RNAs using quencher- and fluorophore-binding aptamers. Nucleic Acids Res. 43, e144 (2015).

    PubMed  PubMed Central  Google Scholar 

  21. Ying, Z. M., Wu, Z., Tu, B., Tan, W. & Jiang, J. H. Genetically encoded fluorescent RNA sensor for ratiometric imaging of MicroRNA in living tumor cells. J. Am. Chem. Soc. 139, 9779–9782 (2017).

    CAS  PubMed  Google Scholar 

  22. Braselmann, E. et al. A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells.Nat. Chem. Biol. 14, 964–971 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Perez-Gonzalez, C., Lafontaine, D. A. & Penedo, J. C. Fluorescence-based strategies to Investigate the structure and dynamics of aptamer–ligand complexes. Front. Chem. 4, 33 (2016).

    PubMed  PubMed Central  Google Scholar 

  24. Paige, J. S., Wu, K. Y. & Jaffrey, S. R. RNA mimics of green fluorescent protein. Science 333, 642–646 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Filonov, G. S., Moon, J. D., Svensen, N. & Jaffrey, S. R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J. Am. Chem. Soc. 136, 16299–16308 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Strack, R. L., Disney, M. D. & Jaffrey, S. R. A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nat. Methods 10, 1219–1224 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Song, W. et al. Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex. Nat. Chem. Biol. 13, 1187–1194 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Paige, J. S., Nguyen-Duc, T., Song, W. & Jaffrey, S. R. Fluorescence imaging of cellular metabolites with RNA. Science 335, 1194 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Song, W., Strack, R. L. & Jaffrey, S. R. Imaging bacterial protein expression using genetically encoded RNA sensors. Nat. Methods 10, 873–875 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Svensen, N. & Jaffrey, S. R. Fluorescent RNA aptamers as a tool to study RNA-modifying enzymes. Cell Chem. Biol. 23, 415–425 (2016).

    PubMed  PubMed Central  Google Scholar 

  31. Song, W., Strack, R. L., Svensen, N. & Jaffrey, S. R. Plug-and-play fluorophores extend the spectral properties of Spinach. J. Am. Chem. Soc. 136, 1198–1201 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Wang, P. et al. Photochemical properties of Spinach and its use in selective imaging. Chem. Sci. 4, 2865–2873 (2013).

    CAS  Google Scholar 

  33. Han, K. Y., Leslie, B. J., Fei, J., Zhang, J. & Ha, T. Understanding the photophysics of the spinach-DFHBI RNA aptamer-fluorogen complex to improve live-cell RNA imaging. J. Am. Chem. Soc. 135, 19033–19038 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Warner, K. D. et al. A homodimer interface without base pairs in an RNA mimic of red fluorescent protein. Nat. Chem. Biol. 13, 1195–1201 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Schwanhausser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).

    PubMed  Google Scholar 

  36. Huang, H. et al. A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore. Nat. Chem. Biol. 10, 686–691 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Warner, K. D. et al. Structural basis for activity of highly efficient RNA mimics of green fluorescent protein. Nat. Struct. Mol. Biol. 21, 658–663 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Trachman, R. J. 3rd et al. Structural basis for high-affinity fluorophore binding and activation by RNA Mango. Nat. Chem. Biol. 13, 807–813 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Prasanth, K. V. et al. Nuclear organization and dynamics of 7SK RNA in regulating gene expression. Mol. Biol. Cell 21, 4184–4196 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Novotny, I. et al. SART3-dependent accumulation of incomplete spliceosomal snRNPs in Cajal bodies. Cell Rep. 10, 429–440 (2015).

    CAS  PubMed  Google Scholar 

  41. Reid, D. W. & Nicchitta, C. V. Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosome profiling. J. Biol. Chem. 287, 5518–5527 (2012).

    CAS  PubMed  Google Scholar 

  42. Wang, X., Chen, X. & Yang, Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nat. Methods 9, 266–269 (2012).

    CAS  PubMed  Google Scholar 

  43. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Shao, S. et al. Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system. Nucleic Acids Res. 44, e86 (2016).

    PubMed  PubMed Central  Google Scholar 

  45. Wang, Y., Wang, Z. & Tanaka Hall, T. M. Engineered proteins with Pumilio/fem-3 mRNA binding factor scaffold to manipulate RNA metabolism. FEBS 280, 3755–3767 (2013).

    CAS  Google Scholar 

  46. Park, H. Y., Trcek, T., Wells, A. L., Chao, J. A. & Singer, R. H. An unbiased analysis method to quantify mRNA localization reveals its correlation with cell motility. Cell Rep. 1, 179–184 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Kennedy, M. J. et al. Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Albiez, H. et al. Chromatin domains and the interchromatin compartment form structurally defined and functionally interacting nuclear networks. Chromosome Res. 14, 707–733 (2006).

    CAS  PubMed  Google Scholar 

  49. Wang, C., Han, B., Zhou, R. & Zhuang, X. Real-time imaging of translation on single mRNA transcripts in live cells. Cell 165, 990–1001 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Yan, X., Hoek, T. A., Vale, R. D. & Tanenbaum, M. E. Dynamics of translation of single mRNA molecules in vivo. Cell 165, 976–989 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Morisaki, T. et al. Real-time quantification of single RNA translation dynamics in living cells. Science 352, 1425–1429 (2016).

    CAS  PubMed  Google Scholar 

  52. Wu, B., Eliscovich, C., Yoon, Y. J. & Singer, R. H. Translation dynamics of single mRNAs in live cells and neurons. Science 352, 1430–1435 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Raj, A. & van Oudenaarden, A. Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135, 216–226 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Eldar, A. & Elowitz, M. B. Functional roles for noise in genetic circuits. Nature 467, 167–173 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Battich, N., Stoeger, T. & Pelkmans, L. Control of transcript variability in single mammalian cells. Cell 163, 1596–1610 (2015).

    CAS  PubMed  Google Scholar 

  56. Meacham, C. E. & Morrison, S. J. Tumour heterogeneity and cancer cell plasticity. Nature 501, 328–337 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Miller, J. D., Tajima, S., Lauffer, L. & Walter, P. The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane. J. Cell Biol. 128, 273–282 (1995).

    CAS  PubMed  Google Scholar 

  58. Davis, J. H. & Szostak, J. W. Isolation of high-affinity GTP aptamers from partially structured RNA libraries. Proc. Natl Acad. Sci. USA 99, 11616–11621 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Ishida, H., Tobita, S., Hasegawa, Y., Katoh, R. & Nozaki, K. Recent advances in instrumentation for absolute emission quantum yield measurements. Coord. Chem. Rev. 254, 2449–2458 (2010).

    CAS  Google Scholar 

  60. Magde, D., Rojas, G. E. & Seybold, P. G. Solvent dependence of the fluorescence lifetimes of xanthene dyes. Photochem. Photobiol. 70, 737–744 (1999).

    CAS  Google Scholar 

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Acknowledgements

We thank S.R. Jaffrey at Cornell University for sharing the pAVU6+27-F30-2xdBroccoli construct, Y. Sun at Peking University for the dCas9-GFP construct, C.L. Tucker at Duke University for CIBN/CRY2 constructs. We thank Y. Tang, Y. Liang, L. Chen, Y. Yu and R. Yao for technical assistance and S. Tribuna for secretarial assistance. This research was supported by the National Key Research and Development Program of China (grant no. 2017YFA050400 to Y.Y.), NSFC (grant nos. 21425311 and 21877037 to L. Zhu.; nos. 91857202, 21937004, 31225008 and 31470833 to Y.Y. and no. 31600688 to X.C.), the Shanghai Science and Technology Commission (grant nos. 18JC1411900 and 16430723100 to Y.Y.), the Research Program of State Key Laboratory of Bioreactor Engineering (to Y.Y.) and the China Postdoctoral Science Foundation (grant nos. 2016M59027 and 2017T00277 to X.C.). J.L. was supported by US NIH grant nos. HL61795, HG007690 and GM107618, and by an American Heart Association grant no. D007382.

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Author notes

  1. These authors contributed equally: Xianjun Chen, Dasheng Zhang, Ni Su.

Authors and Affiliations

  1. Optogenetics and Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, China

    Xianjun Chen, Dasheng Zhang, Ni Su, Bingkun Bao, Xin Xie, Fangting Zuo, Lipeng Yang, Hui Wang, Li Jiang, Qiuning Lin, Mengyue Fang, Ningfeng Li, Xin Hua, Zhengda Chen, Chunyan Bao, Lixin Zhang, Yuzheng Zhao, Linyong Zhu & Yi Yang

  2. School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, China

    Dasheng Zhang, Bingkun Bao, Lipeng Yang, Li Jiang, Qiuning Lin, Ningfeng Li, Xin Hua, Chunyan Bao & Linyong Zhu

  3. CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China

    Xianjun Chen & Yi Yang

  4. School of Pharmacy, East China University of Science and Technology, Shanghai, China

    Xianjun Chen, Ni Su, Xin Xie, Fangting Zuo, Hui Wang, Mengyue Fang, Zhengda Chen, Yuzheng Zhao & Yi Yang

  5. Key Laboratory of Advanced Control and Optimization for Chemical Processed of Ministry of Education, School of information Science and Engineering, East China University of Science and Technology, Shanghai, China

    Jinjin Xu & Wenli Du

  6. Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Joseph Loscalzo

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  1. Xianjun Chen
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Contributions

Ideas were conceived by Y.Y., L. Zhu and X.C. Y.Y., L. Zhu, X.C., D.Z and N.S. designed the experiments and analyzed the data. D.Z. and B.B. synthesized the dyes. X.C. performed the SELEX experiment. X.C., X.X., F.Z., H.W. and M.F. characterized the aptamer in vitro and constructed plasmids. N.S. and X.C. performed the live cell imaging experiments. L.Y., L.J., Q.L., N.L., X.H., Z.C, C.B., J.X., W.D., L. Zhang, Y.Z. and J.L. gave technical support and conceptual advice. Y.Y., L. Zhu, X.C., D.Z. and J.L. wrote the manuscript.

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Correspondence to Linyong Zhu or Yi Yang.

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Integrated supplementary information

Supplementary Figure 1 Screening, optimization and characterization of Pepper530 RMFP.

(a) Fluorescence intensity of HBC in different environments. The fluorescence of HBC in solution or in constraining PMMA glass was excited with ultra-violet light (365 nm). (b) Fluorescence activation of HBC by RNA pools from each round of SELEX. One μM RNA aptamer in each round was mixed with 5 μM HBC and the fluorescence was measured. (c)-(f) Validation of the HBC aptamers from SELEX. (c) HBC fluorescence in the presence of different aptamers. One μM HBC was incubated with 5 μM aptamers. (d) Excitation (dashed) and emission (solid) spectra of D11-HBC (green) and D12-HBC (blue). (e) Imaging of D11 and D12 aptamers in E. coli. BL21 Star™ (DE3) cells expressing F30-D11 or F30-D12 were induced by adding IPTG at 37 oC for 4 h. The cells were plated on glass slides and incubated with 2 µM HBC in HEPES buffer (containing 5 mM Mg2+). Fluorescence was visualized using a confocal laser scanning microscope with a 488 nm laser. BL21 Star™ (DE3) cells transformed with empty vector or no vector were used as the controls. Scale bar, 5 μm. (f) FACS analysis of D11-HBC and D12-HBC complexes in BL21 Star™ (DE3) cells. The fluorescence of the cells was analyzed using a Beckman Cytoflex S with a 488/8 nm excitation filter and a 525/40 nm emission filter. (g)-(h) Identification of the D11 core motif responsible for fluorescence activation by truncation analysis. (g) The Mfold-predicted secondary structures of D11 and its truncation mutants. (h) Quantification of HBC fluorescence induced by D11 and its truncation mutants. The markedly reduced fluorescence of the T3 aptamer suggests that T2 is the minimal aptamer sequence required for activation of HBC fluorescence. Data represent the mean ± s.d. from three technical replicates. (i)-(k), Mutation analysis of T2 truncation. (i) Mutation sites (red bases) in T2 truncation. (j) Quantification of fluorescence of T2 mutants using 1 μM aptamer and 5 μM HBC. Data represent the mean ± s.d. from three technical replicates. (k) Magnesium dependence of Pepper and T2. One μM RNA aptamer was mixed with 5 μM HBC and the fluorescence signal of the complex was measured with different concentrations of MgCl2. Data represent the mean ± s.d. from three technical replicates. (l) Normalized UV-vis absorbance spectra of 5 μM Pepper530 (green) or free HBC (gray). (m) Polyacrylamide gel electrophoresis (PAGE) analysis of Pepper. F30-Pepper, F30-Broccoli and F30-dBroccoli were analyzed on an 8% native PAGE gel. After PAGE, the gel was first stained with HBC and DFHBI-1T and imaged at 488/30 nm excitation and 535/30 nm emission (left), then stained with SYBR Gold and imaged at 305 nm excitation and 590/30 nm emission (right). F30-Pepper is 103 nt in length; F30-Broccoli is 105 nt in length; F30-dBroccoli is 234 nt. The “ladder ss50” contains ssDNA of 50, 100, and 150 nt (multiples of 50 nt). (n) Pepper530 fluorescence in the presence of increasing concentrations of HBC. Data represent the mean ± s.d. from three technical replicates. (o) Thermostability of Pepper530 and Broccoli-DFHBI-1T. Data represent the mean ± s.d. from three technical replicates. (p) Effect of pH on the fluorescence of Pepper530 or Broccoli-DFHBI-1T. Data represent the mean ± s.d. from three technical replicates. (q) Measurement of the Kon and Koff for the association and disassociation of HBC to Pepper. (i) The ON kinetics of the association of HBC to Pepper. A sepharose bead coated by streptavidin protein (GE Healthcare) was labeled by Pepper RNA coupled with biotin at the 3’ end. Two-photon fluorescence images were taken immediately after the beads were incubated with 10 nM HBC. Scale bar, 10 μm. (ii) Quantitative analysis of the Pepper-HBC complex formation. The data were fitted to the formula of exponential rise to maximum (y = y0+a·(1-e-bx) (methods for details). Data represent the mean fluorescence of three sepharose beads. (iii) The OFF kinetics of the disassociation of HBC to Pepper. The beads in (i) were placed in buffer without HBC fluorophore. Scale bar, 10 μm. (iv) Quantitative analysis of the fluorescence of Pepper-HBC complex. The data were fitted to the formula of exponential decay (y = y0+a·e-bx) (methods for details). Data represent the mean fluorescence of three sepharose beads. (r) Potassium independence for Pepper-induced fluorescence. For RNA refolding, solutions containing 1 µM Pepper and 5 µM HBC in 40 mM HEPES, 5 mM MgCl2, with or without 100 mM KCl or LiCl were heated to 70 °C for 5 min and cooled to room temperature over 15 min. The Broccoli aptamer, which has essentially the same core structure as Spinach1 and thus contains the same G-quadruplex, was used as a positive control. Unlike Broccoli, whose fluorescence is highly dependent on potassium, Pepper’s fluorescence is independent of potassium, suggesting that it has no G-quadruplex structure and probably has a new structure distinct from previously reported FRs. Data represent the mean ± s.d. from three technical replicates. (s) Tolerance of 5’ flanking RNA by Pepper. Pepper was fused to the 3′ end of the 5S RNA, 7SK RNA, and U6 RNA, or by placing it between 50-nt-long flanking sequences derived from the human β-actin 3′ untranslated region. Pepper with F30, tRNA, or no RNA fusion scaffold were used as controls. Fluorescence was measured by mixing 1 µM Pepper RNA with the indicated flanking sequence and 5 µM HBC, and normalized to the fluorescence of Pepper without scaffold RNA. Data represent the mean ± s.d. from three technical replicates. (t) Mutation analysis of T2 truncation. Stem-loop indicated in Pepper structure was replaced by either one of the three randomly selected stem-loops of difference sequence. Quantification of fluorescence of Pepper with different stem-loops, using 1 μM aptamer and 5 μM HBC. Data represent the mean ± s.d. from three technical replicates. (u) Schematic representation of different tandem arrays of Pepper. (v) Quantification of fluorescence of different tandem arrays of Pepper using 1 μM aptamer and 20 μM HBC. Data represent the mean ± s.d. from three technical replicates. (w) FACS analysis of Pepper530 fluorescence in mammalian cells. 293T/17 cells expressing mCherry and Pepper or Broccoli were incubated with 0.2 μM HBC or 20 μM DFHBI-1T, respectively, before analysis. mCherry expressed from another plasmid was used for assessing transfection efficiency. Untransfected cells or cells expressing only F30 scaffold RNA were used as the control. The cells were analyzed in two channels: green (ex = 488/8 nm, em = 525/40 nm) and red (ex = 561/10 nm, em = 610/20 nm). (x) The same fluorescence images in Fig. 1f with increased contrast (5x for confocal and 10x for two-photon). Scale bar, 10 μm. (y) Live-cell imaging of different tandem arrays of Pepper using single-photon (‘confocal’) excitation and two-photon excitation in COS-7 cells. Scale bar, 20 μm. (z) Quantitation of the cellular fluorescence of different tandem arrays of Pepper. Data represent the mean ± s.d. (N = 100 cells). For a, d-f, l-m, q(i), q(iii), w-y, at least two independent experiments were carried out with similar results.

Supplementary Figure 2 Fluorescence imaging of diverse RNAs tagged with Pepper in live cells.

(a) Quantification of the fluorescence of Pepper and its fusions with scaffold RNAs in 293T/17 cells. Data represent the mean ± s.d. (N = 60 cells). (b) Effects of scaffold RNAs on the stability of Pepper in living cells. 293T/17 cells transfected with plasmids expressing Pepper or its fusions with scaffolds were treated with 5 µg/ml Actinomycin D (an inhibitor of RNA polymerases) 24 hrs after transfection. Fluorescence of Pepper was recorded during the time course of the inhibitor treatment. Data represent the mean ± s.d. (N = 10 cells). (c) Effects of scaffold RNAs on the long-term stability of in vitro transcribed Pepper exposed to air at 37 oC. Fluorescence of Pepper and its fusions was measured in the presence of 5 μM HBC fluorophore. Data represent the mean ± s.d. from three technical replicates. (d) Detection of Pepper tagged 7SK, U6 and 5S RNAs by FISH imaging (red) counterstained with DAPI (blue). Cells were co-transfected with SC35-BFP, or SART3-BFP expressing plasmids. SC35 and SART3 were used to localize nuclear speckles and Cajal body in live cells, as they interact with 7SK and U6, respectively. BFP fluorescence (blue) in live cells was imaged before FISH procedures. Scale bar, 5 μm. (e) Fluorescence imaging of Broccoli-, Corn- and Pepper-tagged BFP or ACTB mRNAs. COS-7 cells were labeled with 20 μM DFHBI-1T, 10 μM DFHO and 1 μM HBC, respectively. BFP or ACTB mRNAs without aptamer tag were used as the controls. Scale bars, 20 μm. (f) Detection of BFP, GAPDH and ACTB mRNA by FISH (red) with DAPI counterstaining (blue). Scale bar, 10 μm. (g) Detection of TMED2, BCAP31 and RCN2 RNA by FISH (red) with DAPI counterstaining (blue). Scale bar, 10 μm. (h) and (j) Detection of 4Pepper tagged mRNAs of cytosolic and nuclear proteins (h) or ER and integral membrane localized proteins (j) by sequential live cell Pepper imaging (green) and fixed cell FISH imaging (red). For the study of ER and integral membrane proteins’ mRNA localization in (j), ER (blue) were visualized by co-transfecting the cell with Srprb-BFP (a subunit of the signal recognition particle receptor localizes to ER membrane2) expression plasmid. Scale bars, 10 μm. (i) and (k) Detection of mRNAs of cytosolic and nuclear proteins (i) or ER and integral membrane localized proteins (k) by FISH (red) with DAPI counterstaining (blue). Scale bar, 10 μm. For d-k, two independent experiments were carried out with similar results.

Supplementary Figure 3 Large-scale single-cell analysis of mammalian mRNA translation control.

(a) Schematic representation of light-induced Pepper expression based on the LightOn system. Upon blue light exposure, GAVPO binds to the UASG sequence and actives the transcription of BFP-4Pepper. (b) and (c) Consecutive imaging (b) and quantitative analysis (c) of light-induced BFP-4Pepper mRNA expression. Data were normalized to the 13 hr time point. Data represent the mean ± s.d. (N = 20 cells). Scale bars, 10 μm. (d) Pepper has little effects on mRNA translation, transcription, or degradation. Left, The effect of Pepper on mRNA translation. 293T/17 cells expressing BFP-4Pepper or BFP mRNA under the control of the LightOn system were cultured in light illumination for 24 hrs before BFP fluorescence was analyzed. Untransfected cells were used as the control. Right, the effect of Pepper on mRNA transcription and degradation. 293T/17 cells expressing BFP-4Pepper or BFP mRNA under the control of the LightOn system were illuminated for 6 hrs and then transferred to dark conditions. mRNA levels at the indicated time points were analyzed using RT-qPCR. Data were normalized to the BFP sample at time 0. Left, two independent experiments were carried out with similar results. Right, data represent the mean ± s.d. from three technical replicates. (e) FACS analysis of COS-7 cells expressing BFP, 4Pepper, BFP and 4Pepper from two separate vectors, or BFP-2A-sfGFP. (f) Flow cytometry analysis of BFP and its mRNA tagged with 4Pepper in single live cells at different time after transfection. (g) flow cytometry analysis of BFP, mCherry and SNAP-tag protein and their mRNA tagged with Pepper in single live cells. The data were fit to a Michaelis-Menten equation (green dashed line with R2 and Km as indicated) or a linear equation (black dashed line), respectively. (h) Fluorescence imaging and quantitative analysis of BFP, mCherry and SNAP-tag protein and their mRNA tagged with Pepper in single live cells. The data were fit to a Michaelis-Menten equation (R2 as indicated). Scale bars, 50 μm. For e-h, at least two independent experiments were carried out with similar results. (i) and (j) The effect of translation regulating agents on the coefficient of determination between BFP protein and its mRNA levels. Data in (j) represent the mean ± s.d. from three cell cultures. (k) and (l) The coefficient of determination between protein and their mRNA levels of different protein species. The mRNAs encoding cytosol and nuclear (orange bars), ER and integral membrane proteins (blue bars) were fused to BFP-4Pepper RNA. Data in (l) represent the mean ± s.d. from four to five cell cultures. (m) and (n) The coefficient of determination between BFP protein and its mRNA levels in different cell lines. Statistical comparison was performed by two-tailed t test. Data in (n) represent the mean ± s.d. from four to five cell cultures.

Supplementary Figure 4 Strategy to tune the spectra of fluorophores.

HBC has a typical chromophore structure consisting of an electron donor and an acceptor linked by π conjugation. In this architecture, emission wavelength of the dye is usually determined by their HOMO-LUMO gap, which is closely related to the π-structure and capabilities of electron donor and acceptor3,4. Increasing the electron withdrawing capability of the electron acceptor group or electron donating capability of the electron donor group usually lowers the HOMO-LUMO gap, enhances the bathochromic shift, and red-shifts the fluorescence. Extending the π-conjugation also lowers the HOMO-LUMO gap and red-shifts the spectra5,6. Decrease the electron density of the π-conjugation may lower the electron donating ability of the electron donor group7, and enhance the HOMO-LUMO gap, which resulted blue-shifting of the spectra. We therefore achieved spectroscopic shifting of Pepper using the following strategies, by tuning the aromatic π-structure or adjusting the electron donor and acceptor capability of HBC dyes: Strategy 1: Extend the conjugation length with condensed aromatic rings such as benzothiophene and bithiophene, to obtain red-shifted HBC599 and HBC620. Strategy 2: Decrease the electron withdrawing capability of the electron acceptor group using 2-benzoxazole to obtain slightly blue-shifted HBC525. Strategy 3: Introduce nitrogen atoms into the benzene π -conjugation to generate pyridine and pyrazine conjugation, to obtain blue-shifted HBC497 and HBC508. Strategy 4: Ammoniate the hydroxyethyl group of HBC497, to obtain HBC485 with further blue-shifted spectra. The aliphatic amine group of HBC485 may be protonated by the phosphoric acid of Pepper in the complex. The protonated amine group shall decrease the electron donating ability of the electron donor group, which leads to blue-shifting the spectra of the probes.

Supplementary Figure 5 HBC and its analogues showed no significant toxicity in live cells.

HeLa cell viability was measured in the presence of different concentrations of the following dyes: (a) HBC; (b) HBC485; (c) HBC497; (d) HBC508; (e) HBC514; (f) HBC525; (g) HBC599; (h) HBC620. Cells were grown in DMEM medium containing different concentrations of each fluorophore for 48 hrs, after which their viability was measured by the CCK-8 assay. Data represent the mean ± s.d. from three cell cultures.

Supplementary Figure 6

CRISPR display of Pepper arrays for imaging genomic loci. (a) Schematic presentation of CRISPR display of different pepper arrays for imaging genomic loci. Different Pepper arrays were inserted into the tetraloop, loop2, or both the tetraloop and loop2. (b) Co-labeling of human centromeric-satellites using the modified sgRNA scaffold (red) and dCas9-GFP (green). The nucleus was stained with Hoechst 33342 (blue). Scale bar, 5 μm. (c) Total number of centromeres detected per cell for the three modified sgRNAs. Data represent the mean ± s.d. (N = 10 cells). (d) Quantification of the fluorescence intensities of genomic loci labeled with different Pepper modified sgRNAs. Data represent the mean ± s.d. (n≈400 loci from 10 cells). (e) Dual-color imaging of telomeres and centromeres using Pepper- and Broccoli-modified sgRNA in HeLa cells. Broccoli-DFHBI-1T fluorescence image (original in the insert) was shown with 10×-increased contrast for visualization. Inset, the original image. Scale bar, 10 μm. For b and e, two independent experiments were carried out with similar results.

Supplementary Figure 7

Real-time imaging of protein-RNA tethering. (a) Schematic representation of the protein-RNA tethered reporting system based on the interaction of tdMCP and the MS2 binding hairpin RNA. (b) Live cell imaging of Pepper-MS2. HeLa cells were transfected with plasmid expressing Pepper-MS2 and cultured for 30 hrs. Images were taken in the presence of 1 μM HBC620. Scale bar, 10 μm. (c) Imaging of HeLa cells expressing Pepper-MS2 and tdMCP-BFP targeted to different subcellular compartments: i, Histone 2B (Nucleic); ii, LifeAct (Filamentous actin); iii, α-tubulin; iv, a palmitoylation sequence (Membrane); v, a Golgi apparatus targeting signal; vi, TOMM20 (Outer mitochondrial membrane); vii, Srprb (ER membrane); viii, a peroxisomal targeting sequence. Scale bar, 10 μm. (d) Schematic representation of optogenetic manipulation of RNA localization. Upon blue light exposure, the CRY2-mCherry-tdMCP fusion protein interacts with plasma membrane-targeted CIBN and recruits Pepper-MS2 to the plasma membrane. Imaging (e) and quantification (f) of light-induced translocation of Pepper485 and mCherry to the plasma membrane. Both Pepper485 and mCherry fluorescence localized to the cytosol under dark conditions; however, they began accumulating at the plasma membrane upon light illumination. Cells were illuminated with a 458 nm laser (30 μW) spaced 17 min apart during three consecutive light-dark cycles. Scale bars, 10 μm. Data represent the mean ± s.d. (N = 5 cells). (g) Time course of CRY2-mCherry-tdMCP and Pepper-MS2 recruitment to the plasma membrane after illumination with a 458 nm laser (30 μW). Scale bar, 5 μm. (h) Quantification of plasma membrane and cytosolic CRY2-mCherry-tdMCP protein and Pepper-MS2 RNA in (g). Data represent the mean ± s.d. (N = 5 cells). (i) Fluorescence imaging of mCherry and Pepper485 in HeLa cells expressing CIBN, CRY2-mCherry-tdMCP, and Pepper-MS2 during dark recovery after initial blue light activation. Cells were firstly illuminated using a 458 nm laser (30 μW), and then imaged using a 900 nm two-photon excitation for Pepper485 fluorescence and a 561 nm excitation for mCherry fluorescence. Scale bar, 10 μm. (j) Quantification of plasma membrane and cytosolic CRY2-mCherry-tdMCP protein and Pepper-MS2 RNA. Data represent the mean ± s.d. (N = 5 cells). For b, c and i, at least two independent experiments were carried out with similar results.

Supplementary Figure 8 Comparison of Peppers’ and MS2 system’s Performance for RNA imaging in COS-7 Cells.

(a) Schematic representation of constructs expressing NLS-tdMCP-FPs, or mRNAs fused with 4P4M dual tag. mRNAs without 4P4M tag were used as controls. (b) and (c) Fluorescence imaging of Pepper620 (red) and tdMCP-EGFP (green) in cells expressing BFP mRNA (b) or ACTB mRNA (c), with or without 4P4M tag. (d) and (e) Fluorescence imaging of Pepper530 (green) and tdMCP-mCherry (red) in cells expressing BFP mRNA (d) or ACTB mRNA (e), with or without 4P4M tag. (f-i) Quantification of the fluorescence of MS2 system channel (tdMCP-EGFP or tdMCP-mCherry) and Pepper channel (Pepper530 or Pepper620) in the cytosol of cells expressing mRNAs with or without 4P4M tag. These data showed much higher signal/background contrast was detected in the Pepper channel comparing to the MS2 system channel. Statistical comparison was performed by two-tailed t test. Data represent the mean ± s.d. (N = 50 cells). (j) Schematic representation of constructs for the targeting of ACTB mRNA to outer membrane of mitochondria through TOMM20-tdPCP-BFP tethering. (k) and (m) Fluorescence imaging of tdMCP-EGFP and Pepper530 (k) or tdMCP-mCherry and Pepper620 (m) in cells expressing mitochondrial localized ACTB mRNA with 4P4M tag. Cells were co-transfected with ACTB-4P4M-4xPP7, TOMM20-tdPCP-BFP and NLS-tdMCP-EGFP (k) or NLS-tdMCP-mCherry (m) plasmids. The fluorescence signals of the MS2 system (EGFP or mCherry) were measured in cells before HBC ligands incubation. The fluorescence signals of Pepper were measured by the enhanced fluorescence in the same cells after incubation with HBC ligands, which reflected the formation of Pepper complex spectral matched to FPs of MS2 system (Pepper530 for EGFP, Pepper620 for mCherry). (l) and (n) Quantification of the MS2 system’s or Pepper’s fluorescence signal of ACTB mRNA tagged with 4P4M tag, and TOMM20-tdPCP-BFP fluorescence in mitochondria and cytosol of the cells. Statistical comparison was performed by two-tailed t test. N.S., no significant difference. Data represent the mean ± s.d. (N = 50 cells). Scale bar, 10 µm. For b-e, k and m, at least two independent experiments were carried out with similar results.

Supplementary Figure 9 Performance comparison of Peppers and Riboglow platform.

(a) Structures of Cbl-5xPEG-ATTO 590 and HBC620. Cbl-5xPEG-ATTO 590 is a macromolecule (MW>2000) that is hard to traverse plasma membrane. (b) Excitation and emission spectra of Pepper620 and Riboglow (the complex of RNA tag A and Cbl-5xPEG-ATTO 590). (c) Fluorescence activation of Cbl-5xPEG-ATTO 590 and HBC620 upon binding with their specific aptamers measured at 590 nm excitation 640 nm emission. Data represent the mean ± s.d. from three technical replicates. (d) and (e) Live cell imaging of G3BP1-BFP and ACTB-(A)4x (d) or ACTB-4Pepper (e) during stress granules (SGs) formation in cells treated with NaAsO2. (A)4x is the tandem tetramer of RNA tag A. G3BP1 protein is a granule-associated RNA-binding protein and a marker to localize SGs. Scale bar, 10 µm. (f) and (g) Line profiles of the fluorescence from G3BP1-BFP and ACTB-(A)4x (f) or ACTB-4Pepper (g) of the dashed lines in (d) and (e). (h) Schematic representation of constructs expressing 4Pepper or A(4x) alone from Pol II (U6 promoter), or mRNAs fused with 4Pepper or A(4x) tag from Pol III (CMV promoter). (i) Live cell imaging of Riboglow or Pepper620, and their fusions transcribed by Pol II or Pol III. Scale bar, 10 µm. (j) Quantitative analysis of the fluorescence of Riboglow or Pepper620, and their fusions in cells. Statistical comparison was performed by two-tailed t test; N.S., no significant difference. Data represent the mean ± s.d. (N = 50 cells). (k) Schematic representation of constructs for the targeting of 4Pepper or A(4x) fused ACTB mRNA to outer membrane of mitochondria through TOMM20-tdMCP-BFP tethering. (l) Imaging of mitochondrial localized mRNA labeled with Riboglow and Pepper620. The COS-7 cells co-transfected with TOMM20-tdMCP-BFP and ACTB-4Pepper or ACTB-(A)4x expression plasmids were incubated with HBC620 or loaded with Cbl-5xPEG-ATTO 590, respectively. Scale bar, 10 µm. (m) Quantification of the fluorescence of Pepper620, Riboglow and BFP in the outer membrane of mitochondrial and cytosol. Statistical comparison was performed by two-tailed t test. Data represent the mean ± s.d. (N = 50 cells). For b, d-g, i and l, at least two independent experiments were carried out with similar results.

Supplementary Figure 10 Live cell super-resolution imaging of RNA aptamers.

(a)-(c), Photostability of different RMFPs and FPs in live 293T/17 cells. (a) Cells expressing Pepper, Broccoli, mCherry-H2B, mVenus-H2B, UnaG-H2B, EGFP-H2B, or ECFP-H2B were imaged using a confocal laser scanning microscope with a 561 nm laser for Pepper599, Pepper620 and mCherry, a 488 nm laser for Pepper525, Pepper530, mVenus-H2B, UnaG-H2B and EGFP-H2B, a 458 nm laser for Pepper514, Pepper508, Pepper497, Pepper485, Broccoli-DFHBI-1T and ECFP-H2B. The curves were normalized to spectra of the RNA-fluorophore complexes or fluorescent proteins. (b) The same cells in (a) were imaged using a 1040 nm two-photon excitation for Pepper599, Pepper620 and mCherry, a 976 nm two-photon excitation for Pepper525, Pepper530, mVenus-H2B, UnaG-H2B and EGFP-H2B, a 900 nm two-photon excitation for Pepper514, Pepper508, Pepper497, Pepper485, Broccoli-DFHBI-1T and ECFP-H2B. (c) Photoswitching properties of Broccoli, Pepper497, Pepper508, Pepper 514, Pepper525, Pepper530 in live cells. Cells expressing Pepper or Broccoli were labeled by their cognate ligands and imaged with five cycles of continuous 458 nm or 488 nm laser scanning for 10 seconds (solid lines in the white regions), with 60 seconds of darkness between cycles (gray regions). The percent recovery (%REC) of the peak initial fluorescence was almost 100% for both Broccoli and Peppers, for according to the equation: \(\% REC = \frac{{f_r - f_{bl}}}{{f_0 - f_{bl}}}\), where f0 is the peak initial fluorescence, fbl is the post-bleach fluorescence, and fr is the post-dark recovery fluorescence8. All data in (a-c) are normalized to the initial image intensity (at time 0), N=10 cells. (d-n) HeLa cells expressing Pepper tethered to H2B in nuclei (d-g) or TOMM20 on the outer mitochondrial membrane (k-m) were studied by widefield and 3D-SIM imaging in the presence of HBC620. The lateral and axial spatial resolutions of SIM imaging were 116-nm and 350-nm, respectively. (d, k) Wide-field imaging showing co-localization of BFP and Pepper620. (e) Apical section taken from the surface of the nuclear envelope close to the coverslip. (f) Images of H2B-tethered Pepper in mid-cross-section. 3D-SIM images reveal a punctated pattern of regions devoid of Pepper620 fluorescence. (g) An x-z planar cross-section through the dashed line shown in (f). (h-j) Raw images of Broccoli-DFHBI-1T, Pepper530 and Pepper620 for SIM imaging. HeLa cells expressing 2Broccoli-MS2 (h) or 4Pepper-MS2 and tdMCP-tagBFP-H2B (i, j) were imaged by structured illumination microscopy (SIM) imaging in the presence of 20 μM DFHBI-1T (h), 2 μM HBC (i) or 1 μM HBC620 (j). (l) Images of TOMM20-tethered Pepper shown in maximum-intensity projection along the z dimension through the cell volume. (m) One x–z planar cross-section through the dashed line shown in (l). (n) 3D SIM versus conventional wide-field with depth encoded in color. This `ure shows the maximum-intensity projection of a HeLa cell expressing 4Pepper-MS2 and TOMM20-tdMCP-tagBFP, and is mostly the same as (l) except that different colors are used to indicate the axial position, or depth, of the Pepper620. The depth-to-color map is shown at the bottom right corner. Scale bars in (d-f), 5 μm; (h-l) and (n), 10 μm; (g) and (m), 1 μm. For d-n, at least two independent experiments were carried out with similar results.

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Supplementary Figs. 1–10, Tables 1–3 and Notes 1–3.

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Chen, X., Zhang, D., Su, N. et al. Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. Nat Biotechnol 37, 1287–1293 (2019). https://doi.org/10.1038/s41587-019-0249-1

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