Firefly luciferase (FLuc) is one of the most commonly used reporter genes in research and diagnostics.
The test involves inserting a hairpin upstream of the RLuc cistron, dramatically reducing RLuc synthesis. This result indicates IRES activity when observed with a bona fide EMCV IRES control.
What Is FLuc mRNA Testing?
FLuc mRNA testing is an in vitro translation assay using a modified firefly luciferase gene version to monitor protein expression. The mRNA encodes an amino-terminal segment of firefly luciferase (NFluc) with a complementary carboxy-terminal segment of CFluc directly downstream so that if ribosomes move out of frame during translation, elongated polypeptides containing both in-frame and +1 frameshifted NFluc residues can be produced. The elongated polypeptides produce a measurable signal that can be detected by fluorescence.
In vivo evaluation of mRNA-LNP delivery was carried out in BALB/c mice. Mice were intramuscularly injected with 10 mg of naked mRNA or encapsulated in 4N4T-LNPs. Luminescence signals were normalized to total body weight and presented as mean + standard deviation.
The results demonstrated that the mRNA-packed lipid nanoparticles efficiently co-delivered mRNA to cells and induced significantly higher gene expression than the mRNA alone, peaking at 24 hours after application. Moreover, the mRNA-packed lipid nanoparticles did not clump together upon incubation with Triton-X or other detergents that disrupt lipid membranes.
How Does FLuc mRNA Testing Work?
In vivo, the FLuc mRNA test is performed by intramuscularly injecting 4N4T-LNPs encapsulating GFP or the firefly luciferase (Fluc) mRNA into a mouse. Fluorescence from the mRNA is measurable in various tissues and organs, including the injection site muscle, draining lymph nodes, liver, and spleen. The amount of mRNA encapsulated in each tissue is directly proportional to the fluorescence intensity measured. The measurements can be automated, and data are obtained within a few hours of injection.
GFP or FLuc expression was assessed by flow cytometry and bioluminescence imaging, respectively. Phosphate-buffered saline was employed as a negative control for both tests.
SDS-polyacrylamide gel electrophoresis autoradiographs show that the [35S] methionine-labeled elongated polypeptides produced by the translation of unmodified Fluc mRNA and Fluc+1FS2 mRNA are similar. However, adding 1-methylPs significantly increased ribosomal +1 frameshifting to about 8% of the in-frame protein, and this effect was lost when the slippage sites were mutant.
What Are the Advantages of FLuc mRNA Testing?
The encapsulation of FLuc mRNA into LNPs significantly increased its in vivo expression compared to naked mRNA. In addition, a strong signal was detected even at distal sites within the mouse abdomen (e.g., liver and spleen), which may indicate that lipid-LNP complexes can efficiently transport the mRNA into the organs for local expression.
The results of the analysis of a few different lipid-LNP formulations, including those used in the COVID-19 vaccine clinical trials (MC3, KC2, and L319), showed that their particle sizes and encapsulation efficiency varied depending on the mRNA concentration, the lipid:mRNA ratio, and the mixing conditions. The MC3 and L319 LNPs had the largest particles and demonstrated the best encapsulation efficiency, while the KC2 LNPs displayed a smaller particle size and the weakest encapsulation efficiency.
To further demonstrate that lipid-LNPs could facilitate the translation of mRNA into functional proteins, we employed IVT mRNAs encoded for both the amino- and carboxy-terminal segments of the firefly luciferase protein (NFluc) and its complementing CFluc, encoded in the +1 reading frame. These mRNAs produce catalytically inactive truncated NFluc if translated commonly. Still, if ribosomes move out of the reading frame during translation, elongated CFluc polypeptides can be produced.
What Are the Disadvantages of FLuc mRNA Testing?
The FLuc mRNA encodes a firefly luciferase protein that emits bioluminescence upon interaction with its substrate, luciferin. While this approach can be used to screen mRNA expression in various tissue types, it does have some limitations. For example, the luminescence signal from a luciferin-stimulation experiment may be obscured by background signals from other proteins and cellular activities. In addition, the FLuc mRNA must be injected into cells in a manner that can be monitored by the luminescence signal to determine if a desired effect is being produced.
To overcome these limitations, researchers have developed an alternative method for analyzing mRNA expression in vivo using a dual-luciferase imaging strategy. This approach uses two different mRNAs, one expressing Rluc and the other encoding a fluorescent reporter gene (Fluc). By comparing the kinetics of light production from each mRNA in the same cell population, researchers can monitor changes in mRNA expression over time.
This approach inserts an mRNA-encoding RLuc mini-ORF into the test 5′-end with a FLuc ORF and a promoter. The result is a bicistronic mRNA that the same mRNA-dependent translation system can transcribe as the mRNA encoding the test target.
Injections of the naked mRNA into muscle cells yielded a fluorescent signal that disappeared within two h, consistent with rapid degradation of extracellular mRNA in vivo. However, injection of the mRNA encapsulated into MC3, L319, or KC2 LNPs resulted in significant FLuc expression that lasted at least 48 hours, with the signal detected not only at the site of injection but also throughout the muscle, popliteal, and axillary lymph nodes, and other organs.
