Abstract
Applying rational design, we developed 17 kDa cyanobacteriochrome-based near-infrared (NIR-I) fluorescent protein, miRFP718nano. miRFP718nano efficiently binds endogenous biliverdin chromophore and brightly fluoresces in mammalian cells and tissues. miRFP718nano has maximal emission at 718 nm and an emission tail in the short-wave infrared (SWIR) region, allowing deep-penetrating off-peak fluorescence imaging in vivo. The miRFP718nano structure reveals the molecular basis of its red shift. We demonstrate superiority of miRFP718nano-enabled SWIR imaging over NIR-I imaging of microbes in the mouse digestive tract, mammalian cells injected into the mouse mammary gland and NF-kB activity in a mouse model of liver inflammation.
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Data availability
The data supporting the findings of this study are available within the article and its Supplementary Information. All other data that support the findings of the study are available from the corresponding author upon request. The main plasmids constructed in this study, their maps and sequences are deposited to Addgene. For the structure of miRFP670nano: PDB ID 6MGH, https://www.rcsb.org/structure/6mgh. For the structure of miRFP709 PDB ID: 5VIQ, https://www.rcsb.org/structure/5VIQ.
Code availability
The miRFP718nano nucleotide sequence in GenBank is MW627296.1, https://www.ncbi.nlm.nih.gov/nuccore/MW627296.1. The miRFP718nano structural data were deposited at the Protein Data Bank (PDB ID 7LSD), https://www.rcsb.org/structure/7LSD.
Change history
02 February 2023
A Correction to this paper has been published: https://doi.org/10.1038/s41592-023-01798-y
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Acknowledgements
We thank N. Peitsaro and N. Aarnio from the Flow Cytometry Core Facility of the University of Helsinki for the technical assistance. This work was supported by grants from the US National Institutes of Health (NIH) (grant nos. GM122567, EB028143, NS111039 and NS115581), Chan Zuckerberg Initiative (grant no. 226178), Cancer Foundation Finland and Magnus Ehrnrooth Foundation. S.P. was supported in part by the NIH Intramural Research Program for the Vaccine Research Center of the National Institute of Allergy and Infectious Diseases.
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Contributions
O.S.O. developed and characterized the protein in vitro and in cultured cells. C.M. developed the SWIR imaging system, performed imaging and analyzed the data. C.T. developed the SWIR imaging system and measured fluorescence emission. H.S. performed animal surgeries. S.P. designed structural biology experiments and analyzed the data. M.B. purified the recombinant proteins and prepared cells for SWIR imaging. J.Y. planned and supervised the SWIR imaging experiments and analyzed the data. V.V.V. conceived, planned and supervised the whole project and together with J.Y., O.S.O. and S.P. wrote the manuscript. All authors reviewed the manuscript.
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Nature Methods thanks Takeharu Nagai and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.
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Extended data
Extended Data Fig. 1 Structure of miRFP718nano and its red-shifted chromophore.
Overall structures of (a) miRFP670nano (PDB ID: 6MGH), (b) miRFP718nano, and (c) miRFP709 (PDB ID: 5VIQ). The biliverdin (BV) chromophores are shown in magenta. The PAS and GAF domains of miRFP709 are in cyan and yellow, respectively. The BV chromophores in (d) miRFP670nano, (e) miRFP718nano, and (f) miRFP709 bound to the respective Cys residues and their chemical formulas. Carbon, nitrogen, oxygen, and sulfur atoms are in white, blue, red, and yellow, respectively. Sticks representations show only rings A and B of the chromophores and Cys residues. In miRFP670nano, the BV chromophore (d) is bound to Cys86 via the C31 atom, miRFP718nano (e) and miRFP709 (f) have the same chromophore species bound to the Cys57 and Cys20, respectively. (g) Superposition of miRFP670nano (green) and miRFP718nano (yellow) structures. (h) miRFP718nano hydrogen bond network around the chromophores. (i) Stacking interactions between the chromophores and the surrounding residues in miRFP718nano. (j) The chromophores of miRFP718nano bound to the respective Cys57 residues in the 2Fo-Fc electron density map. The map is countered at 2.0σ-levels. (k) Superposition of the chromophores in miRFP670nano (green) and miRFP718nano (yellow). (l) Stabilizing mutations and hydrophobic clusters in miRFP718nano. The residues forming H-bonds are shown in green, hydrophobic clusters (one formed by residues Leu8, Ile11, Val12, Val26, Ile104, Leu114, Met140 and the other by Val15, Phe18, Leu19, Trp128, Phe132, Leu133) are in cyan and magenta.
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Supplementary Notes 1 and 2, Figs. 1–16, Tables 1–4 and References.
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Oliinyk, O.S., Ma, C., Pletnev, S. et al. Deep-tissue SWIR imaging using rationally designed small red-shifted near-infrared fluorescent protein. Nat Methods 20, 70–74 (2023). https://doi.org/10.1038/s41592-022-01683-0
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DOI: https://doi.org/10.1038/s41592-022-01683-0
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