Firefly Luciferase-Based Commercial Reagent Screening of Messenger RNA Nanocomplex Transfection Efficiency in vitro

Abstract

Messenger RNA (mRNA) has recently emerged as a revolutionary, novel nucleic-acid based therapeutic class with great potential in a broad range of applications. Nanoparticles loaded with mRNA have been transfected into cells for transient translation of the corresponding protein. However, substantial advancements have been met with challenges such as instability and immune response activation. In this study, two types of nucleotide-modified Firefly Luciferase mRNA, Trilink (5-Methoxy Uridine) and Drew Weissman (N1-Methyl Pseudouridine), were complexed as nanoparticles with four commercially available transfection reagents, Lipofectamine MessengerMAX (LFMM), Trans-IT (TIT), TransMessenger (TM), and JetMessenger (JM), for in vitro delivery into two mammalian cell lines, HEK293 and A549, to determine transfection efficiency (TE) while also assessing the stability of fresh vs. frozen lysates. Here we show the amount of Firefly Luciferase (FLuc) and cell viability of each mRNA-reagent nanocomplex. We found JM to produce the highest TE and TM and JM to result in the least cell death. Frozen lysates were concluded to produce a lower amount of FLuc compared to fresh lysates suggesting instability over freeze-thaw cycles Furthermore, it can be concluded that TIT results in the greatest amount of cytotoxicity as determined by microscopy and MTT assay. Future implications include determining the source of user variability and optimizing plating conditions/density.