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Article

A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes

1
Graduate Program of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), 18052-780 São Carlos, São Paulo, Brazil
2
Graduate Program of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCar), 18119-001 Sorocaba, São Paulo, Brazil
3
Department of Physics, Chemistry and Mathematics, Federal University of São Carlos (UFSCar), 18052-780 São Carlos, São Paulo, Brazil
4
Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(1), 303; https://doi.org/10.3390/ijms22010303
Received: 26 November 2020 / Revised: 15 December 2020 / Accepted: 17 December 2020 / Published: 30 December 2020
Beetle luciferases produce bioluminescence (BL) colors ranging from green to red, having been extensively used for many bioanalytical purposes, including bioimaging of pathogen infections and metastasis proliferation in living animal models and cell culture. For bioimaging purposes in mammalian tissues, red bioluminescence is preferred, due to the lower self-absorption of light at longer wavelengths by hemoglobin, myoglobin and melanin. Red bioluminescence is naturally produced only by Phrixothrix hirtus railroad worm luciferase (PxRE), and by some engineered beetle luciferases. However, Far-Red (FR) and Near-Infrared (NIR) bioluminescence is best suited for bioimaging in mammalian tissues due to its higher penetrability. Although some FR and NIR emitting luciferin analogs have been already developed, they usually emit much lower bioluminescence activity when compared to the original luciferin-luciferases. Using site-directed mutagenesis of PxRE luciferase in combination with 6′-modified amino-luciferin analogs, we finally selected novel FR combinations displaying BL ranging from 636–655 nm. Among them, the combination of PxRE-R215K mutant with 6′-(1-pyrrolidinyl)luciferin proved to be the best combination, displaying the highest BL activity with a catalytic efficiency ~2.5 times higher than the combination with native firefly luciferin, producing the second most FR-shifted bioluminescence (650 nm), being several orders of magnitude brighter than commercial AkaLumine with firefly luciferase. Such combination also showed higher thermostability, slower BL decay time and better penetrability across bacterial cell membranes, resulting in ~3 times higher in vivo BL activity in bacterial cells than with firefly luciferin. Overall, this is the brightest FR emitting combination ever reported, and is very promising for bioimaging purposes in mammalian tissues. View Full-Text
Keywords: bioimaging; Far-Red bioluminescence; NIR bioluminescence; luciferin amino-analogs; biophotonics bioimaging; Far-Red bioluminescence; NIR bioluminescence; luciferin amino-analogs; biophotonics
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MDPI and ACS Style

Viviani, V.R.; Bevilaqua, V.R.; de Souza, D.R.; Pelentir, G.F.; Kakiuchi, M.; Hirano, T. A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes. Int. J. Mol. Sci. 2021, 22, 303. https://doi.org/10.3390/ijms22010303

AMA Style

Viviani VR, Bevilaqua VR, de Souza DR, Pelentir GF, Kakiuchi M, Hirano T. A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes. International Journal of Molecular Sciences. 2021; 22(1):303. https://doi.org/10.3390/ijms22010303

Chicago/Turabian Style

Viviani, Vadim R., Vanessa R. Bevilaqua, Daniel R. de Souza, Gabriel F. Pelentir, Michio Kakiuchi, and Takashi Hirano. 2021. "A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes" International Journal of Molecular Sciences 22, no. 1: 303. https://doi.org/10.3390/ijms22010303

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