RNA Packaging

Introduction to L7Ae & C/D Box

L7Ae and the C/D box used in our project are a group of archaeal RNA-protein complexes with a dissociation constant of about 1 nM (Saito et al., 2009). L7Ae, a component of the archaeal ribosome, is able to bind to a kink-turn RNA motif called the C/D box (Saito et al., 2009). The C/D box contains two sets of conserved RNA sequence motifs termed the box C (consensus sequence: RUGAUGA ) and box D (consensus sequence: CUGA) (Saito et al., 2009).

Structural organization of L7Ae and the C/D box (Gagnon et al., 2012)

Design overview

We inserted the C/D box to the 3’-UTR of the cargo mRNA, which can bind to the archaeal ribosomal protein L7Ae. In addition, L7Ae was conjugated to the C-terminus of a transmembrane protein CD63 which anchored inside the exosomal membrane.

Aim of the design

We designed to package a specific mRNA, which encodes a protein with therapeutic value, into our engineered exosomes. L7Ae is anchored to the interior of the exosome membrane by fusing with CD63. Then the therapeutic mRNA together with the C/D box gains the ngability to bind to L7Ae and in this sense, the mRNA is loaded into the engineered exosomes during the biogenesis process (Kojima et al., 2018).

Test of the construct

We constructed three plasmids pcDNA3.1-CD63-L7Ae, pcDNA3.1-nanoluc-C/Dbox (nanoluc encoding nanoluciferase), and pcDNA3.1-nanoluc to test the efficiency of the loading of the target mRNA into exosomes.

1. The pcDNA3.1-CD63-L7Ae plasmid and pcDNA3.1-nanoluc plasmid were co-transfected into HEK293T cells as a negative control.

2. The pcDNA3.1-CD63-L7Ae plasmid and pcDNA3.1-nanoluc-C/Dbox plasmid were co-transfected into HEK293T cells as a test group.

The exosomes in these two groups were isolated from the medium of the transfected cells by ultracentrifugation and the quantity of packaged mRNA were measured by qRT-PCR.

Similarly, we chose another group of RNA-protein complex to deliver our cargo mRNA to exosomes. (Why another construct?)

Introduction to MS2 & 3 loops

The MS2 coat protein and the MS2 3X loops in our project is a group of bacteriophage RNA-protein complexes. They come from MS2 bacteriophage, a single-stranded RNA phage (Wang et al., 2018). The MS2 gene encodes a bacteriophage coat protein which can recognize and bind to an RNA hairpin structure, the MS2 stem loop, which is present at the beginning of the replicase gene to modulate the expression of that gene (Peabody, 1990).

Lethbridge iGEM 2017 used MS2 and its corresponding MS2 stem loop for RNA isolation (BBa_K2443038). They employed two MS2 stem loops in their construction. According to a previous research, the MS2 loop tandem trimer (termed 3X loops) has a higher mRNA binding efficiency compared with other numbers of loop tandem repeat. Furthermore, a U to C mutation was introduced at position 13 in the wild type MS2 loop eresulting in a higher affinity e f(Hung and Leonard, 2016). Therefore, we utilized the higher affinity MS2 loop tandem trimer (3X MS2 loops) to achieve a high therapeutic mRNA encapsulation efficiency.

Design overview

We dinserted the y3X MS2 loops downstream of the 3’-UTR of cargo mRNA, which can bind to the cognate bacteriophage coat protein MS2 (Hung and Leonard, 2016). In addition, MS2 is conjugated to the C-terminus of the transmembrane protein CD63, which can allow the fusion protein to localize inside exosomes.

Aim of the design

MS2 is anchored to the interior of the exosome membrane by means of CD63. Then the therapeutic mRNA with the 3X MS2 loops gains the capacity to bind to MS2 coat protein, and in this sense the mRNA is loaded into the engineered exosomes during biogenesis process.

Test of the construct

We constructed three plasmids pcDNA3.1-CD63-MS2, pcDNA3.1-nanoluc-3X MS2 loop (nanoluc encoding nanoluciferase) and pcDNA3.1-nanoluc to test the efficiency of this design.

The pcDNA3.1-MS2 plasmid and pcDNA3.1-nanoluc plasmid were co-transfected into HEK293T cells as a negative control. The pcDNA3.1-MS2 plasmid and pcDNA3.1-nanoluc-3X MS2 loops plasmid were co-transfected into HEK293T cells as the test group.

The exosomes in these two groups were isolated by ultracentrifugation of the medium of the transfected cells and the quantity of packaged mRNA was assessed by qRT-PCR.

References

Gagnon, K., Biswas, S., Zhang, X., Brown, B., Wollenzien, P., Mattos, C. and Maxwell, E. (2012). Structurally Conserved Nop56/58 N-terminal Domain Facilitates Archaeal Box C/D Ribonucleoprotein-guided Methyltransferase Activity. Journal of Biological Chemistry, 287(23), pp.19418-19428.

Hung, M. and Leonard, J. (2016). A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery. Journal of Extracellular Vesicles, 5(1), p.31027.

Kojima, R., Bojar, D., Rizzi, G., Hamri, G., El-Baba, M., Saxena, P., Ausländer, S., Tan, K. and Fussenegger, M. (2018). Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nature Communications, 9(1).

Saito, H., Kobayashi, T., Hara, T., Fujita, Y., Hayashi, K., Furushima, R. and Inoue, T. (2009). Synthetic translational regulation by an L7Ae–kink-turn RNP switch. Nature Chemical Biology, 6(1), pp.71-78.Peabody, D.(1990). Translational repression by bacteriophage MS2 coat protein expressed from a plasmid. A system for genetic analysis of a protein-RNA interaction. Journal of Biological Chemistry, 265, pp.5684-5689

Wang, Y., Araud, E., Shisler, J., Nguyen, T. and Yuan, B. (2018). Influence of algal organic matter on MS2 bacteriophage inactivation by ultraviolet irradiation at 220 nm and 254 nm. Chemosphere, 214, pp.195-202.