Periodic Reporting for period 1 - RNAatHD (Development of a high-resolution method to monitor structural changes of regulatory RNAs and therapeutic oligo-nucleotides directly in-cell.)
Période du rapport: 2017-05-01 au 2019-04-30
Preparation of an in-cell sample, where the AON is successfully delivered into human cells. This also meant that the delivered AON was shown to be functional in the cells and that the cells are viable. We tested functionality by measuring the downregulation of the target mRNAs by qRT-PCR. A very interesting result was that tagged (biotinylated or Cy3-tagged) versions of the AON drug, as used for microscopy, showed significantly less downregulation, i.e. functionality compared to the actual, unmodified drug molecule. Therefore they might not reflect how untagged AON behaves in the cell stressing the need for a tag-free technique to detect (drug-) molecules in cells as developed in this project.
In-cell NMR method: In order to develop a non-invasive in-cell NMR approach we decided to freeze the transfected cells and acquire solid-state dynamic nuclear polarization (DNP) NMR experiments. Cryoprotected, frozen cells stay intact for extended amounts of measurement times and, can be recultivated later, indicating a biologically relevant environment. Applying DNP to frozen cells, we overcome limitations of solution-state in-cell NMR (e.g. size, stability and sensitivity) as well as of visualization techniques since no tagging of the AON is required. Using this “ICE-DNP”(frozen In-Cell Enhanced NMR by DNP) method, we were indeed able to obtain an in-cell NMR signal of the investigated AON. To our knowledge, this is the first time that DNP is used to detect an exogenous oligonucleotide delivered into cells, and also that a transfected functional antisense drug in macromolecular complexes could be detected in intact human cells. The possibility to detect an untagged, active drug, interacting in its natural environment, means that the main objective of the project was reached.
In-vitro versus in-cell: For a complete RNAatHD approach, in parallel to developing an in-cell NMR technique, we also set out to do in-vitro comparison experiments. The idea thereby was to identify and add more and more interaction partners to reconstitute a more realistic in-vitro sample.
Application: The RNAatHD approach with the steps described above was successfully set up with and applied to an AON drug provided by AstraZeneca (as described above). In addition, the approach was also applied to a 13C/15N isotopically labelled miRNA prepared in the host lab.
The results so far were disseminated in two peer reviewed journal publications and presented at various seminars and conferences by the post-doc and the PI of the lab.
In addition, to ensure exploitation of the method we worked closely with pharmaceutical industry (AZ).
The post-doc was also selected as one of 30 “highly promising researchers” to represent 100000 funded fellows. She presented the project at the “Science is wonderful” event at the European Researchers’ Night in Brussels, 2017.