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Green fluorescent protein (GFP) messenger RNA is a synthetic or in vitro transcribed (IVT) nucleic acid molecule that encodes the GFP protein, originally derived from the jellyfish Aequorea victoria (Shimomura et al., 1962). It serves as a ubiquitous research tool and a gold-standard reporter in the fields of molecular biology, gene therapy, and vaccine development. When introduced into eukaryotic cells, the mRNA is recognized by the cellular translation machinery, leading to the synthesis of the GFP protein, which emits bright green fluorescence upon excitation without the need for additional cofactors (Tsien, 1998). This property makes it ideal for visualizing and quantifying the efficiency of various delivery systems, such as lipid nanoparticles (LNPs) and polymers, as well as for studying promoter activity and cellular processes (Sahin et al., 2014). While GFP mRNA itself is not a therapeutic agent for human disease, its use is critical for the preclinical optimization of mRNA-based platforms, allowing researchers to assess the kinetics of protein expression and the biodistribution of the delivery vehicle (Pardi et al., 2018).
The mechanism involves the translation of the mRNA sequence by the host cell's ribosomal machinery into the Green Fluorescent Protein (GFP). Following translation, the protein undergoes a spontaneous, oxygen-dependent cyclization and oxidation of the Ser65-Tyr66-Gly67 tripeptide to form the fluorescent chromophore (Tsien, 1998).
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