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  • Brief Communication
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Red-shifted luciferase–luciferin pairs for enhanced bioluminescence imaging

Abstract

Red-shifted bioluminescence reporters are desirable for biological imaging. We describe the development of red-shifted luciferins based on synthetic coelenterazine analogs and corresponding mutants of NanoLuc that enable bright bioluminescence. One pair in particular showed superior in vitro and in vivo sensitivity over commonly used bioluminescence reporters. We adapted this pair to develop a bioluminescence resonance-energy-based Antares reporter called Antares2, which offers improved signal from deep tissues.

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Figure 1: Bioluminescent reporters based on synthetic substrates and re-engineered luciferases.
Figure 2: Bioluminescence imaging of luciferase–luciferin pairs at superficial sites and in deep tissues of live mice.

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References

  1. Negrin, R.S. & Contag, C.H. Nat. Rev. Immunol. 6, 484–490 (2006).

    Article  CAS  Google Scholar 

  2. Arranz, A. & Ripoll, J. Front. Pharmacol. 6, 189 (2015).

    Article  Google Scholar 

  3. Inglese, J. et al. Nat. Chem. Biol. 3, 466–479 (2007).

    Article  CAS  Google Scholar 

  4. Saito, K. et al. Nat. Commun. 3, 1262 (2012).

    Article  Google Scholar 

  5. Hall, M.P. et al. ACS Chem. Biol. 7, 1848–1857 (2012).

    Article  CAS  Google Scholar 

  6. Stacer, A.C. et al. Mol. Imaging 12, 1–13 (2013).

    Article  Google Scholar 

  7. Adams, S.T. Jr. & Miller, S.C. Curr. Opin. Chem. Biol. 21, 112–120 (2014).

    Article  CAS  Google Scholar 

  8. Evans, M.S. et al. Nat. Methods 11, 393–395 (2014).

    Article  CAS  Google Scholar 

  9. Kuchimaru, T. et al. Nat. Commun. 7, 11856 (2016).

    Article  CAS  Google Scholar 

  10. Inouye, S. & Shimomura, O. Biochem. Biophys. Res. Commun. 233, 349–353 (1997).

    Article  CAS  Google Scholar 

  11. Wu, C., Nakamura, H., Murai, A. & Shimomura, O. Tetrahedr. Lett. 42, 2997–3000 (2001).

    Article  CAS  Google Scholar 

  12. Inouye, S. et al. Biochem. Biophys. Res. Commun. 437, 23–28 (2013).

    Article  CAS  Google Scholar 

  13. Jiang, T., Du, L. & Li, M. Photochem. Photobiol. Sci. 15, 466–480 (2016).

    Article  CAS  Google Scholar 

  14. Nishihara, R. et al. Chem. Commun. (Camb.) 51, 391–394 (2015).

    Article  CAS  Google Scholar 

  15. Inouye, S., Sato, J., Sahara-Miura, Y., Yoshida, S. & Hosoya, T. Biochem. Biophys. Res. Commun. 445, 157–162 (2014).

    Article  CAS  Google Scholar 

  16. Tomabechi, Y. et al. Biochem. Biophys. Res. Commun. 470, 88–93 (2016).

    Article  CAS  Google Scholar 

  17. Chu, J. et al. Nat. Biotechnol. 34, 760–767 (2016).

    Article  CAS  Google Scholar 

  18. Suzuki, K. et al. Nat. Commun. 7, 13718 (2016).

    Article  CAS  Google Scholar 

  19. Dixon, A.S. et al. ACS Chem. Biol. 11, 400–408 (2016).

    Article  CAS  Google Scholar 

  20. Yang, J. et al. Nat. Commun. 7, 13268 (2016).

    Article  Google Scholar 

  21. Schindelin, J. et al. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

  22. Ando, Y. et al. Photochem. Photobiol. 83, 1205–1210 (2007).

    Article  CAS  Google Scholar 

  23. Loening, A.M., Dragulescu-Andrasi, A. & Gambhir, S.S. Nat. Methods 7, 5–6 (2010).

    Article  CAS  Google Scholar 

  24. Ando, Y. et al. Nat. Photonics 2, 44–47 (2008).

    Article  CAS  Google Scholar 

  25. Liu, F., Song, Y. & Liu, D. Gene Ther. 6, 1258–1266 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

pNCS-Antares and pcDNA3-ReNL were gifts from M. Lin and T. Nagai (Addgene plasmids 74279 and 85203), respectively. We thank the University of California, Riverside, the National Institutes of Health (R01GM118675 and R21EB021651), and the National Science Foundation (CHE-1351933) for financial support. We thank T.M. Truong for reading and revising the manuscript.

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Authors and Affiliations

Authors

Contributions

H.-w.A. conceived and supervised the entire project. H.-w.A. and M.M.M.-G. supervised the mouse imaging experiments. H.-W.Y. performed synthetic, protein engineering, and in vitro and in cellulo characterization experiments. H.-W.Y. and O.K. performed the mouse imaging experiments. A.J. assisted H.-W.Y. in completing compound synthesis. D.C. assisted in the mouse imaging experiments. H.-W.Y. and H.-w.A. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Hui-wang Ai.

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Competing interests

UC Riverside has filed a provisional patent application in the USA (No. 62/382,255) that is partially based on results described in the manuscript. H.-w.A. and H.-W.Y. are listed as coinventors.

Integrated supplementary information

Supplementary Figure 1 Chemical structures of coelenterazine (CTZ) and CTZ analogs.

Supplementary Figure 2 Sequence alignment between NanoLuc and NanoLuc mutants described in this work.

teLuc mutations are highlighted in teal fonts and gray background, whereas all yeLuc mutations are highlighted in yellow fonts and gray background. Residues in this figure are numbered according to Protein Date Bank (PDB) ID 5B0U.

Supplementary Figure 3 Illustration of the binding pocket of NanoLuc and the residues mutated to derive teLuc and yeLuc.

This model was created by docking the CTZ substrate into the crystal structure of the apo form of NanoLuc (PDB ID 5B0U) using the AutoDock Vina software. Since CTZ is quite flexible, the exact binding mode might not be accurate. Nevertheless, the model shows the rough enzyme-substrate binding site. NanoLuc residues mutated in teLuc or yeLuc are presented in sticks: teal for mutated residues in teLuc only, yellow for mutated residues in yeLuc only, and green for mutated residues in both teLuc and yeLuc.

Supplementary Figure 4 Illustration of the process used to engineer teLuc and yeLuc.

Supplementary Figure 5 Bioluminescence decay kinetics for pure enzymes.

The final concentrations of all enzymes and substrates were 100 pM and 30 μM, respectively. Measurements were taken every 60 s after substrate addition. Individual data points from three independent measurements are presented.

Supplementary Figure 6 Substrate titrations with pure enzymes to determine apparent Michaelis constants (Km).

The final concentrations of all enzymes were 100 pM. Substrate concentrations varied from 0.78 to 50 μM, and peak bioluminescence intensities at individual substrate concentrations were used to fit the Michaelis-Menten equation. Individual data points from three independent measurements at each substrate concentration are presented.

Supplementary Figure 7 Domain arrangement and spectral overlap of Antares2.

(a) Primary structural arrangement of Antares and Antares2. (b) Fluorescence and bioluminescence profiles of CyOFP1, NanoLuc, and teLuc, showing a better BRET spectral overlap between teLuc and CyOFP1 than between NanoLuc and CyOFP1. The BRET efficiency increased from 67% in Antares to 71% Antares2 by comparing the intensities of Antares, Antares2, NanoLuc, and teLuc at the same concentrations (also see Fig. 1b).

Supplementary Figure 8 Representative pseudocolored images and quantifications of FLuc-expressing HEK 293T cells.

Images were acquired without a filter (a) or with a 695±25 nm NIR emission filter (c). Panels b and d are quantification results for Panels a and c, respectively. All values were normalized to the intensities of FLuc/D-luciferin 50 μM) under the same imaging conditions. Individual data points and mean with s.d. from three independent measurements are presented. The bioluminescence intensity of FLuc with millimolar D-luciferinor AkaLumine-HCl was still 1-2 orders of magnitude lower than that of teLuc or Antares2 in the presence of micromolar DTZ (also see Fig. 1c-f).

Supplementary Figure 9 Comparison of the bioluminescence of FLuc and FLuc2 in vitro and in live mammalian cells.

FLuc2, also known as luc2, is an engineered and codon-optimized mutant of FLuc. FLuc and FLuc2 were cloned from pGL2 and pGL4 plasmids (Promega), respectively. Pure proteins were prepared for enzyme assays (a) or the genes were inserted into pcDNA3 for head-to-head comparison in mammalian cells (b). FLuc2 only slightly enhanced bioluminescence in mammalian cells, mainly due to its high protein levels (also see Supplementary Figure 10).The bioluminescence intensity of FLuc2 was still 1–2 orders of magnitude lower than that of teLuc or Antares2 (also see Fig. 1c-f). Individual data points from three independent measurements at each substrate concentration are presented.

Supplementary Figure 10 Examination of the effect of luciferase protein levels on bioluminescence intensities.

FLuc2, also known as luc2, is an engineered and codon-optimized mutant of FLuc. FLuc and FLuc2 were cloned from pGL2 and pGL4 plasmids (Promega), respectively. Pure proteins were prepared for enzyme assays (a) or the genes were inserted into pcDNA3 for head-to-head comparison in mammalian cells (b). FLuc2 only slightly enhanced bioluminescence in mammalian cells, mainly due to its high protein levels (also see Supplementary Figure 10).The bioluminescence intensity of FLuc2 was still 1–2 orders of magnitude lower than that of teLuc or Antares2 (also see Fig. 1c-f). Individual data points from three independent measurements at each substrate concentration are presented.

Supplementary Figure 11 Bioluminescence decay kinetics in intact HEK 293T cells and HEK 293T cell lysates.

(a,b) The assay was performed with 30 μM substrates in a 96-well plate format containing ~5,000 luciferase-expressing HEK 293T cells (a) or cell lysates (b). Bioluminescence intensities were measured at 60 s intervals after substrate addition.Individual data points from three independent measurements are presented.

Supplementary Figure 12 Evaluation of signal-to-background ratios (S/B) of various luciferase—luciferin pairs in HEK 293T cell—based assays.

The addition of DTZ to untransfected HEK 293T cells generated signals close to instrumental noises. S/B ratios were 2.5 × 104 for 30 μM DTZ and 5.0 × 104 for 100 μM DTZ in the presence or absence of teLuc, and 136 for 2 mM D-luciferin in the presence or absence of FLuc. Individual data points and mean with s.d. from three independent experiments are presented.

Supplementary Figure 13 Evaluation of the cytotoxicity of various luciferins.

(a)Viability of HEK 293T cells determined using RealTime-GloMT Cell Viability Assay (Promega) after incubation with individual luciferin substrates for 24 h at 37°C. Individual data points from three independent measurements are presented. (b) Evaluation of cell morphology and staining of dead cells. Cells incubated with the indicated luciferins for 24 h at 37°C were stained with propidium iodide (PI), a fluorescent dye for dead cells. The cytotoxicity of STZ, furimazine, and AkaLumine was further confirmed by the red fluorescence of PI. These experiments were independently repeated three times.

Supplementary Figure 14 Bioluminescence kinetics of intraperitoneally injected luciferins (3.3 μmol each) in hydrodynamically transfected mice.

Values are normalized to the starting intensities (t = 180 s post-injection) of each group and shown as individual data points of three independent experiments.

Supplementary Figure 15 Representative images of BALB/c mice with intravenously injected luciferase-expressing HEK 293T cells and intraperitoneally injected luciferin substrates.

3.3 μmol D-luciferin, 0.3 μmol DTZ, and 0.3 μmol furimazine were used in these experiments. Since the bioluminescence of FLuc/D-luciferin was very low after injecting 1×105 or 5×105 cells, the corresponding bioluminescence images are also shown in the bottom row after a 100- or 10-fold upscaling. The minimal threshold for the detection by FLuc/D-luciferin was ~ 5×105 cells. In contrast, the signals from 1×105 cells expressing teLuc, Antares, or Antares2 were well above the background. To evaluate the background signal from auto-oxidation, DTZ was intraperitoneally injected to mice containing intravenously injected HEK 293T cells that were previously transfected with an empty pcDNA3 vector, and no background signal above the camera noise was detected. These experiments were independently repeated three times.

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Yeh, HW., Karmach, O., Ji, A. et al. Red-shifted luciferase–luciferin pairs for enhanced bioluminescence imaging. Nat Methods 14, 971–974 (2017). https://doi.org/10.1038/nmeth.4400

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