RNA

Part:BBa_K3113006

Designed by: Theresa Keil   Group: iGEM19_Munich   (2019-09-04)


MS2-Stemloop

MS2 is a stemloop structure from the MS2 bacteriophage genome. This stemloop is used as an adapter for RNA binding and loading said RNA into extracellular vesicles.

Usage

The informational readout of ALiVE is RNA. To load a specific RNA into the vesicles we fused it to RNA-motifs which are bound specifically by RNA binding proteins. Two combinations of binding protein and motif were tested. One was MCP, the MS2 bacteriophage coat protein, which binds to the MS2 RNA motif.

Biology

The RNA hairpin motif that is recognized by the MS2 capsid protein dimers.[1]


Characterization

qPCR

Using a coiled-coil system allowed us to successfully load the two different RNA binding proteins (RBPs) L7Ae or MCP into exosomes to export FLuc mRNA from HEK293T cells. We used the published parallel heterodimer pair P9SN:P10SN (Ljubetič et al. 2017), where P9SN was fused to the C-terminus of the exosome marker protein CD63 and P10SN N-terminal to the RNA binding proteins L7Ae or MCP. The export efficiencies of FLuc mRNA cargo measured by qPCR proved that coiled-coil mediated targeting of the RBPs L7Ae and MCP into exosomes worked. Exosomes formed from CD63-P9SN and loaded with P10SN-L7Ae or -MCP could export about 800 and 400 transcripts per cell, respectively. This also shows that the two RBPs have different export capabilities, with L7Ae exporting twice as many transcripts per cell compared to MCP.

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Figure: Vesicles can be modularly assembled via coiled-coils. Two different RNA binding proteins (RBP) were fused to the coiled-coil (CC) peptide P10SN and loaded into exosomes displaying the complementary part P9SN. qPCR analysis demonstrated specific export of target FLuc mRNA and a higher efficiency for L7Ae compared to MCP. Quantification was done via standard curve measurements and assuming a confluent well with 350.000 HEK293T cells/cm2 at the time of harvesting. n = 2

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

  1. ↑ Bleckley, Samuel, and Susan J Schroeder. “Incorporating global features of RNA motifs in predictions for an ensemble of secondary structures for encapsidated MS2 bacteriophage RNA.” RNA (New York, N.Y.) vol. 18,7 (2012): 1309-18. doi:10.1261/rna.032326.112
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