The ALiVE Part Collection
Team Munich proudly presents the ALiVE part collection. With ALiVE, we developed an open-access toolbox for scientists to allow live-cell monitoring bypassing the information content limitations of fluorescent-based methods or the destructive nature of transcriptomics. We created 14 basic parts that were further combined to over 30 composite parts. This comprehensive collection enables the community to tailor their mammalian systems achieving time-resolved and minimal-invasive monitoring of living cells based on vesicular RNA export. The two vesicles Virus-Like Particles (VLPs) and Exosomes can be fine-tuned by adapting to the biological question, e.g., cell line, specific RNA loading or unspecific transcriptome loading. For the first time, we demonstrated with our best basic part the affinity purification of exosomes, as well as a new method of vesicular protein loading over our modular coiled-coil system.
The Markers
RNA is an instable molecule with a half-life of 30 minutes to 10 hours depending on its protective characteristics. Therefore, we engineered protective cargo vesicles for exported RNA, formed continuously over time.
size: 30 - 100 nm
endogenous
minimal cell stress
As a first option, we hijacked the endogenous exosomal pathway implicated in intercellular communication and immune regulation (Batista and Melo 2019). By fusing RNA-binding proteins to the exosomal marker CD63, cytosolic RNA of interest will be enriched in exosomes and consequently exocytosed. We further engineered CD63 with an internal 6xHis-tag that faces the outside of the vesicle to purify exosomes without the need for expensive kits. Sensitive luciferase reporters allow the quantification of vesicle secretion efficiency and collateral transfection.
size: 100 - 140 nm
orthogonal export mechanism
higher RNA loading capacity
As a second option, we included an orthogonal export mechanism using Virus-Like Particles (VLPs). These can be secreted by human cells when expressing the HIV’s group antigen (Gag) protein (Eyckerman et al. 2016; Titeca et al. 2017). Gag itself is harmless as it exclusively forms the viral matrix, capsid, and nucleocapsid (Langer et al. 2017). To enable RNA loading, we fused RNA-binding proteins to the Gag monomers, leading to higher amounts of RNA-binding proteins per vesicle in comparison to the exosomes.
The Adapters
To introduce modularity into the system, interacting protein domains were fused to CD63 and Gag such that vesicle loading could be tuned at will. Two sets of alpha-helical heterodimerizing peptides were tested for this purpose.To specifically load RNA into the vesicles two RNA binding proteins were tested.
The 50S ribosomal protein L7Ae from Archaeoglobus fulgidus is a well characterized RNA-binding protein, that interacts with a 30 nucleotide C/D-box RNA structure. This system has already been used in exosomes to deliver therapeutic mRNAs from one cell type to another (Kojima et al. 2018).
The MS2 bacteriophage coat protein (MCP) is a RBP which interacts with a stem-loop (MS2) motif from the phage genome and has been shown to be able to target RNA to VLPs (Prel et al. 2015).
The first one consists of the 32 aa long coiled-coil domains P9SN and P10SN, which bind to each other in parallel orientation (N-terminus to N-terminus; from Ljubetič et al. 2017).
The second set is composed of the double-helical domains DHD154a and DHD154b from Chen et al. 2019. Each domain consists of two alpha-helical domains linked by a turn, which upon dimerization form a four-helix bundle.
The RNA Motifs
Specific RNA sequences are known to form RNA secondary structures. Their interaction with RNA-binding proteins (RBPs) with high affinity can be exploited to direct RNA from the cytosol to cellular membranes.
The Extra
A HiBiT peptide tag fused to the markers of the vesicles effectively shows the export of the vesicles or the efficiency of the purification.
We have equipped our vesicles with a luminescence-based tag: Promega's 11 amino acids long High Bit (HiBiT) peptide (Dixon et al. 2016; Promega®). This short sequence is able to reconstitute a functional NanoLuc luciferase enzyme when bound to the complementing polypeptide Large BiT (LgBiT), to which it has a high affinity (Kd ~1nM). The HiBiT tag is fused to the C-terminus of both CD63 and Gag, such that it is only present inside the exosomes or VLPs respectively.
References
- Chen, Z., Boyken, S.E., Jia, M., Busch, F., Flores-Solis, D., Bick, M.J., Lu, P., VanAernum, Z.L., Sahasrabuddhe, A., Langan, R.A., et al. (2019). Programmable design of orthogonal protein heterodimers. Nature 565, 106–111.
- Dixon, A.S., Schwinn, M.K., Hall, M.P., Zimmerman, K., Otto, P., Lubben, T.H., Butler, B.L., Binkowski, B.F., MacHleidt, T., Kirkland, T.A., et al. (2016). NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem. Biol. 11, 400–408.
- Eyckerman, S., Titeca, K., Van Quickelberghe, E., Cloots, E., Verhee, A., Samyn, N., De Ceuninck, L., Timmerman, E., De Sutter, D., Lievens, S., et al. (2016). Trapping mammalian protein complexes in viral particles. Nat. Commun. 7, 11416.
- Kojima, R., Bojar, D., Rizzi, G., Hamri, G.C.-E., El-Baba, M.D., Saxena, P., Ausländer, S., Tan, K.R., and Fussenegger, M. (2018). Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nat. Commun. 9, 1305.
- Langer, S., and Sauter, D. (2017). Unusual fusion proteins of HIV-1. Front. Microbiol. 7, 2152.
- Ljubetič, A., Lapenta, F., Gradišar, H., Drobnak, I., Aupič, J., Strmšek, Ž., Lainšček, D., Hafner-Bratkovič, I., Majerle, A., Krivec, N., et al. (2017). Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo. Nat. Biotechnol. 35, 1094–1101.
- Prel, A., Caval, V., Gayon, R., Ravassard, P., Duthoit, C., Payen, E., Maouche-Chretien, L., Creneguy, A., Nguyen, T.H., Martin, N., et al. (2015). Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles. Mol. Ther. - Methods Clin. Dev. 2, 15039.
- Promega® - Quantifying Protein Abundance at Endogenous Levels
