mRNA expressing Firefly Luciferase protein
Firefly luciferase mRNA encodes a 550-amino acid oxidoreductase enzyme derived from the North American firefly Photinus pyralis that catalyzes the ATP- and oxygen-dependent oxidative decarboxylation of its substrate D-luciferin, producing oxyluciferin and a characteristic yellow-green bioluminescent emission at ~560 nm. Unlike fluorescent reporters such as eGFP and mCherry, firefly luciferase generates its own light signal without the need for external excitation, resulting in an exceptionally low background signal and an extremely wide dynamic range — spanning up to seven orders of magnitude. In its native form, firefly luciferase is targeted to peroxisomes via a C-terminal serine-lysine-leucine (SKL) tripeptide targeting signal (Gould et al., 1989). In this construct, SKL has been replaced with IAV, abolishing peroxisomal import and resulting in uniform cytosolic distribution of the translated protein. The mRNA sequence includes a codon-optimized coding sequence bearing a 5' Cap1 structure and poly(A) tail to support efficient translation in both cell-free and cellular systems. Because the reaction requires exogenous luciferin substrate, luciferase activity can be precisely controlled, enabling real-time, substrate-gated detection of mRNA translation.
Applications:
1) de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., & Subramani, S. (1987). Firefly luciferase gene: structure and expression in mammalian cells. Molecular and Cellular Biology, 7(2), 725–737
2) Gould, S. J., Keller, G. A., Hosken, N., Wilkinson, J., & Subramani, S. (1989). A conserved tripeptide sorts proteins to peroxisomes. Journal of Cell Biology, 108(5), 1657–1664
3) Evers, M. J. W., Du, W., Yang, Q., Kooijmans, S. A. A., Vink, A., van Steenbergen, M., Vader, P., de Jager, S. C. A., Fuchs, S. A., Mastrobattista, E., Sluijter, J. P. G., Lei, Z., & Schiffelers, R. (2022). Delivery of modified mRNA to damaged myocardium by systemic administration of lipid nanoparticles. Journal of Controlled Release, 343, 207–216
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