Our design was focused on making a protein nanocompartment (PNC) toolkit to better facilitate research as a whole. Our primary focus was to make the VINCEnT toolkit:

While virus-like particles are useful components of VINCEnT, we also wanted to develop a non-immunogenic PNC with RNA packaging capabilities. Such a tool could potentially enable simpler transfection of mammalian cell lines for fellow iGEMers and other researchers.

Arc is an activity-regulated cytoskeletal-associated protein that has recently been recognized as a repurposed Ty3/Gypsy retrotransposon. A bi-lobar domain within Arc has significant homology to Gag proteins, which are the major capsid proteins of many viruses including Human Immunodeficiency Virus type 1 (HIV-1) and Rous-Sarcoma Virus (RSV). In response to synaptic activity in neurons, Arc proteins self-assemble via this Gag domain (similar to the related viral particles) to encapsulate Arc mRNA and shuttle it to neighbouring cells (Pastuzyn et al., 2018; Ashley et al., 2018).

For RNA encapsulation, we take advantage of the self-mRNA encapsulation strategy employed by Arc proteins in vivo. Based on sequence homology with HIV-1 Gag, the predicted mRNA encapsulation sequence is located within the N-lobe of the Arc protein and so fusion of cargo RNA to the Arc mRNA (or this short predicted consensus sequence) should enable encapsulation (Clever et al., 1995). However, Arc PNCs also readily encapsulate nearby non-specific RNA molecules lacking this consensus sequence in vitro (Pastuzyn et al., 2018). To test RNA encapsulation efficiency, Clover and mRuby RNA with or without the predicted Arc encapsulation sequence was incubated with Arc proteins in vitro before application to various cell cultures.

To ensure we would not retain any native Arc functionality that might impact cellular activity in culture, we also designed a “minimal” Arc Gag protein based on homology with other known Gag domains, including HIV-1 and RSV. We used template-based structural predictions to model this minimal Arc Gag and its predicted assembly into higher-order structures. We expected this minimal Arc PNC to perform similarly to the full-length Arc PNC.

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