Team:Munich/Parts

Alive

Parts Overview

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Our best basic part is a modified version of the exosomal membrane protein CD63. Our goal for this part was to insert a His-tag in its unresolved extracellular loop. Together with Prof. Dr. Hagn, we analyzed its sequence and modeled its structure based on the known structure of a similar protein, CD81. Based on his feedback we designed the His-tagged loop and evaluated the resulting purification capabilities. Our purification results confirmed the parts functionality. Therefore, our His-tagged CD63 enables affinity-based exosome purification for the first time, making it our best basic part.
By making our purification construct accessible to everyone, we aim to simplify previously resource-intensive exosome purification for researchers everywhere.

Best Composite Part

ALiVE's toolbox holds many innovations like the coiled-coil loading system or the novel exosome purification construct. We tested many combinations of our parts and found a winning combination: Fusing the VLP-specific protein Gag directly to the RNA-binding protein L7Ae with the addition of the HiBiT tag showed the best vesicle secretion efficiency as well as the highest extracted RNA amount while being easily analysable due to the HiBiT tag. Therefore, it is our best composite part, allowing efficient vesicle secretion and RNA loading with just one construct.

Best 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 for 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.

Vesicle Constructs

Vesicle-specific Proteins

The vesicle-specific marker proteins are our part collection's first layer. One can choose between ALiVE's two vesicles, exosomes and Virus-Like particles (VLPs). Choosing exosomes means using CD63, an exosomal membrane protein, as one's base construct. For VLPs, the viral structural protein HIV-Gag (Group-specific antigen) builds ALiVE's first layer.

Loading Mechanism

After choosing the vesicle type, the next step is to select the desired loading mechanism to target a RNA-binding protein into the vesicle. The classic method is to fuse the proteins directly. We designed a more modular approach using coiled-coil protein interactions. Small peptides are fused to the vesicle-specific marker protein as well as the cargo to mediate vesicle loading via highly specific peptide-peptide interactioins. We designed two variants of the system, based on two different coiled-coil sets, P9SN interacting with P10SN, and DHD154a interacting with DHD154b.

Analytic Tag

To enable the verification of vesicle formation and loading, we integrated one half of a split luciferase tag (HiBiT) into our marker constructs. On the one hand, this allows us to confirm the formation of intact vesicles, as the HiBiT tag faces the lumen and is thus not accessible for a luciferase complementation assay, unless the vesicles are broken. At the same time, we can confirm adapter loading, as only vesicles containing the tagged marker protein will show a luminescence signal.

Vesicle Formation Analysis

Vesicle Loading Analysis

RNA-Binding Proteins

Finally, we tested two RNA-binding proteins, which we either loaded over the coiled-coil system or direct fusion. One can choose between L7Ae, an archaic ribosomal protein, and MCP (Major Capsid Protein from the MS2 phage), a viral protein. Both were successfully used for RNA export.

Gene Reporter Parts

Structural Motifs

The gene exporters have two main aspects. The first one, the structural motif, an RNA secondary loop structure, is necessary for the RNA-binding proteins to interact with the RNA and, therefore, for its export. Here the choice is between the C/D-Box-loop, which interacts with the RNA-binding protein L7Ae, and the MS2-loop that acts with the RNA-binding protein MCP. Therefore the structural motif decision is dependent on the RNA-binding protein of choice.