RNAatHD · Development of a high-resolution method to monitor structural changes of regulatory RNAs and therapeutic oligo-nucleotides directly in-cell.

Development of a high-resolution method to monitor structural changes of regulatory RNAs and therapeutic oligo-nucleotides directly in-cell.

MicroRNAs (miRs) are targeting different mRNAs, and thereby affect many biological processes and are involved in various human diseases such as cardiac diseases, Alzheimer’s, diabetes or cancer. This fact is making them prime drug targets and candidates for molecular diagnostics, however, this opportunity can only be fully exploited by a better comprehension of how and when each mRNA is targeted by a miRNA, a fact currently still poorly understood. Besides regulatory RNAs, synthetic antisense oligonucleotides (AONs), which also bind segments of mRNAs, are currently tested as cancer drugs. Progress has however been slow due to unspecific off-target-effects, difficulty to quantify the cellular uptake and again, the lack of understanding the mechanism of AONs targeting mRNAs. NMR is the method of choice to obtain high-resolution information of biomolecules and therefore to shine a light on this targeting process. While in-vitro experiments can give detailed in-sight on structure and dynamics of an oligonucleotide in a reconstituted minimal complex, a clear limitation is that simplified aqueous solutions do not allow full comprehension of the cellular environment and how it influences the behavior of oligonucleotides in the living cell. In contrast, functional data is routinely obtained in tissues or cells using visualization techniques, e.g. confocal microscopy that requires tagging for visualization by chemical modification of the molecule of interest. Thus, while the context in which data is acquired is highly relevant, the tagged system may exhibit altered behaviour and function. As a consequence, classical structural biology research needs to interface more with cellular biology, as it is crucial for the structural data obtained in vitro to be validated within the cellular context. Within this project we developed exactly this missing tool allowing us to carry out structural biology of macromolecules and complexes directly in their natural, most relevant environment, in intact human cells, and to overcome this lack of understanding of the mRNA targeting process. Combining the knowledge of the host (Petzold Lab) in structural and molecular biology of miRNAs with the experience in methods development in Nuclear Magnetic Resonance (NMR) of the experienced researcher (ER), an in-cell NMR method was successfully developed, using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP). Using this new In-Cell Enhanced DNP (“ICE-DNP”) NMR approach, we were able to detect oligonulceotides directly in intact frozen cells. With these methods it is possible to address differences between in-cell and in vitro experiments and to determine interaction partners, which have to be used to reconstitute a more realistic in-vitro sample. The scope of the method was demonstrated on a miRNA, miR-34a, as well as a synthetic oligonucleotide drug candidate in collaboration with AstraZeneca, to show the value and insight this method can add to the drug discovery process.

Data: CORDIS, © European Union