CD63-L7Ae--exosomal membrane fusion protein for RNA packaging

L7Ae (Archaeoglobus fulgidus) is an RNA binding protein that can specifically bind to C/D box structure on mRNA. L7Ae is linked to the C-terminus of CD63, and mRNA is packaged during the formation of exosome.

characterization 2019 by XJTLU2019

In this part of experiments, we investigated the function of L7Ae, which may binds the C/D Box on our designed mRNA, and anchor the therapeutic mRNA on the inner face of exosomes. The wet lab focused on two impacts L7Ae may bring to the exosome medicine: 1. The production of exosome in exosome donor (HEK293T cells) 2. The RNA anchored in the exosome

Experiment data

1. Nanoparticle Tracking Assay

After the engreen co-transfection of L7Ae with C/D Box-nluc/nluc/empty pcDNA3.1(+), we conducted NTA to detect if the transfection may influence the exosome production, and ensure that the concentration reached 2x105 /ul, which is the recommended exosome concentration in exosome transfection (Exo-fect) in the sample ultracentrifugated. We distributed the transfected HEK293T cells in a 12-well plate as below:

We sent 50 ul of exosome isolated from each set of cells, the results were sent back and listed below:

The original concentration of particles in three groups were all measured to be 1.3x1010 particles/ml, which is approximately 50x recommended concentration for exosomes to be transfected by Exo-fect.

For the particles with different diameters, the distribution of their concentration in each set of samples are listed as below:

Figure 1: The distribution of particles with different diameters measured by NTA in a 50 ul ultracentrifuged exosome solution of cells those transfected with (1) nluc+L7Ae (2) pcDNA3.1(+)+L7Ae (3) C/D Box-nluc+L7Ae, the solution was diluted with a factor of 300x.

These results shows that the co-transfection and expression of the plasmids listed would not affect the production of exosome production, the concentration of harvested exosomes with ~150nm diameter is about 1.08 x 109/ml.

2. qRT-PCR

In the qRT-PCR experiment, we selected hsa-miR-23a-3p as the endogenous reference gene. We selected nluc as our target gene to test if the L7Ae may help to enlarge the volume of mRNA anchored on the inner surface of exosomes. We calculated the ΔCt values using the Ct of target gene (nluc) to be divided by Ct of endogenous reference gene.

The distribution of 96-well plate in qRT-PCR is displayed as below:

After the qRT-PCR, we did not get any normal signal from the group of pcDNA3.1(+)+L7Ae, nluc targeted group. In terms of the rest two groups, we analyzed theΔCt as below:

Figure 2: The ΔCt values calculated in the groups with and without C/D Box transfected.

This results indicated that the transfection of L7Ae may slightly increase the RNA anchored on the exosomes, but the difference inΔCt between the C/D Box transfected and untransfected were not significant. The characterization of L7Ae and C/D Box will be repeated in future experiments.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 736
Illegal XhoI site found at 747
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Part:BBa_K2619101