Construction of Messenger RNA (mRNA) Probes Delivered By Lipid Nanoparticles to Visualize Intracellular Protein Expression and Localization at Organelles

Abstract

Organelles are specialized compartments, where various proteins reside and play crucial roles to maintain essential cellular structures and functions in mammalian cells. A comprehensive understanding of protein expressions and subsequent localizations at each organelle is of great benefit to the development of organelle‐based therapies. Herein, a set of single or dual organelle labeling messenger RNAs (SOLAR or DOLAR) is designed as novel imaging probes, which encode fluorescent proteins with various organelle localization signals. These mRNA probes enable to visualize the protein localizations at different organelles and investigate their trafficking from ribosomal machinery to specific organelles. According to the in vitro results, SOLAR probes show organelle targeting capabilities consistent with the design. Moreover, DOLAR probes with different linkers display distinct targeting properties depending on different organelle localization signals. Additionally, these mRNA probes also exhibit organelle labeling ability in vivo when delivered by lipid nanoparticles (LNPs). Therefore, these mRNA‐based probes provide a unique tool to study cell organelles and may facilitate the design of organelle‐based therapies.

Rational Design of Polymeric mRNA Delivery Vectors to Achieve Excellent Room-Temperature Storage Stability and Delivery Efficiency

“…However, their applications are limited by potential high toxicity, a risk of altering the host’s DNA, and difficulties of production . On the other hand, nonviral delivery vectors like lipid nanoparticles (LNPs) ,− and cationic polymers, which have a relatively higher biosafety, are employed to avoid these limitations, but they are less effective and less stable than the viral ones. LNPs are commonly used for mRNA delivery that can be manufactured on a large scale. , However, LNPs are limited by the production mode and storage conditions, which require complicated microfluidic equipment to achieve more uniform particles , and cryogenic storage. − Another commonly used delivery vector is cationic polymers which condense negatively charged mRNA into nanocomplexes (polyplexes) to protect mRNA against degradation. , Since the components of polyplexes are much simpler and the complexation procedure is much more straightforward compared with LNPs, polyplexes could be a promising candidate to replace LNPs in the future .…”

Integration of Hybridization Chain Reaction and Protein-Binding Amplification for Long-Term Imaging of Intracellular mRNA: Avoiding Signal Fluctuation

“…Messenger RNA (mRNA) is a principled member of the RNA family, responsible for carrying genetic information and playing an important role in protein synthesis. − The expression of mRNA differs between physiological and pathological conditions. For example, the level of NSD2 mRNA expression in colon cancer tissues is typically higher than that in normal tissues, which can be attributed to the involvement of NSD2 mRNA in the proliferation, migration, and invasion of colon cancer cells .…”