(174ay) Investigation of Arc Protein-mRNA Binding Dynamics Via Surface Plasmon Resonance Biosensors

Messenger RNA (mRNA) has emerged as a novel class of therapeutic agents for the prevention and treatment of a number of diseases (SARS-CoV-2). To function in vivo, the cargo mRNA requires delivery systems that are not only safe and effective but also allow its cellular uptake and release. The currently available mRNA delivery methods are either unsafe—like viral vectors—or have a limited ability to deliver mRNA in vivo—like lipid nanoparticles. Activity-regulated cytoskeleton-associated (Arc) protein is a Gag (Group-specific antigen) homolog, like HIV-1, that self-oligomerizes and encapsulates its mRNA (Arc mRNA) within virus-like proteinaceous shells or capsids to transfer information between neurons in the mammalian brain as extracellular vesicles (EVs). These capsids formed by Arc protein make them a promising vehicle for therapeutic mRNA delivery as they possess in vivo targeting capabilities like viral vectors and biocompatibility as they are endogenous. However, the mechanism of mRNA binding by Arc during capsid formation is unclear. Knowledge of Arc-mRNA interaction is critical to harness it as a promising mRNA delivery agent for efficacious and selective delivery of mRNA in vivo.

Here, we report the development of a fast and high-capacity surface plasmon resonance (SPR) biosensor platform to monitor real-time and label-free protein-mRNA binding interactions. We used a custom-built 6-channel SPR biosensor where the gold-coated SPR chips were functionalized with a self-assembled monolayer (SAM) of carboxy and hydroxy-terminated alkanethiols. Proteins were then chemically immobilized on the surface of the functionalized SPR chip and different mRNA sequences were flown in the 6-channel flow cell for detection. Subsequently, proteins were also flown over the mRNA-bound surface as these sandwich assays allow the investigation of whether these proteins have multiple mRNA binding sites. Wildtype and engineered rat Arc (rArc) proteins were produced using an E. coli expression system and their purity and identity were confirmed by SDS-Page and Western Blot, respectively. Different mRNAs, namely the rArc 5' untranslated region (A5U) mRNA and an exogenous Green Fluorescent Protein (GFP) mRNA were synthesized using in vitro transcription (IVT), followed by gel electrophoresis and a bio-fragment analyzer to characterize the mRNA. In comparison, we also produced HIV-1 RNA binding domain protein, synthesized HIV-1 5' untranslated region (H5U) mRNA and investigated their interactions. We varied protein surface coverage density, mRNA concentration and mRNA buffer conditions to study the protein-mRNA interaction and evaluated the kinetic (k_on and k_off) and thermodynamic (K_D) binding parameters. We found that protein domains and the mRNA sequence and secondary structure play a critical role and affect the protein-mRNA binding dynamics. Hence, these results shed light on the design of the Arc protein-based mRNA delivery system. Moreover, the development of this fast and high-capacity SPR-based platform for examining protein-mRNA binding interactions is noteworthy in molecular biology research.