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Visualization of nitric oxide in living cells by a copper-based fluorescent probe

Abstract

Nitric oxide (NO) serves as a messenger for cellular signaling. To visualize NO in living cells, we synthesized a turn-on fluorescent probe for use in combination with microscopy. Unlike existing fluorescent sensors, the construct—a Cu(II) complex of a fluorescein modified with an appended metal-chelating ligand (FL)—directly and immediately images NO rather than a derivative reactive nitrogen species. Using spectroscopic and mass spectrometric methods, we established that the mechanism of the reaction responsible for the NO-induced fluorescence involves reduction of the complex to Cu(I) with release of the nitrosated ligand, which occurs irreversibly. We detected NO produced by both constitutive and inducible NO synthases (cNOS and iNOS, respectively) in live neurons and macrophages in a concentration- and time-dependent manner by using the Cu(II)-based imaging agent. Both the sensitivity to nanomolar concentrations of NO and the spatiotemporal information provided by this complex demonstrate its value for numerous biological applications.

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Figure 1: The fluorescence response of CuFL to NO.
Figure 2: Specificity of CuFL for NO over other reactive nitrogen and oxygen species.
Figure 3: CuFL detection of NO produced by cNOS.
Figure 4: CuFL detection of NO produced by iNOS.
Figure 5: NO detection in SK-N-SH and Raw 264.7 cells by CuFL.

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Acknowledgements

This work was supported by grant CHE-0234951 from the US National Science Foundation (NSF). Spectroscopic instrumentation at the Massachusetts Institute of Technology Department of Chemistry Instrumentation Facility is maintained with funding from US National Institutes of Health Grant 1S10RR13886-01 and NSF Grants CHE-9808063, DBI9729592 and CHE-9808061. M.H. Lim thanks the Martin Family Society at the Massachusetts Institute of Technology for partial fellowship support. We thank A.Y. Ting and C.-W. Lin for assistance with epifluorescence microscopy, D.G. Nocera, D. Song and E.M. Nolan for helpful discussions, and C.D. Novina and D.M. Dykxhoorn for a gift of plasmid pcDNA3.1-Zeo(–)-U6 used in the RNAi experiments.

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M.H.L. and S.J.L. initiated, designed and performed the project. The RNAi experiment was carried out by D.X.

Corresponding author

Correspondence to Stephen J Lippard.

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The authors declare no competing financial interests.

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Lim, M., Xu, D. & Lippard, S. Visualization of nitric oxide in living cells by a copper-based fluorescent probe. Nat Chem Biol 2, 375–380 (2006). https://doi.org/10.1038/nchembio794

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