To facilitate the use of fluorescent RNA base analogues in the FRET-based drug screening method developed in this project they needed to be carefully characterized inside RNA. To this end we incorporated the base analogues at various positions in the pre-miRNAs and investigated their influence on the native structure of the pre-miRNA as well as how fluorescence properties depend on their position in the RNA sequence. We found that the modified bases behave structurally as their native counterparts and obtained understanding that helped us in the design of the pre-miRNAs used for screening, i.e. where to place the light emissive reporter RNA base analogues in the pre-miRNAs.
With the knowledge on how to position the fluorescent base analogues in the pre-miRNA we set up an initial qualitative small-molecule screen in which we benchmarked our novel FRET method against existing techniques, such as ITC (calorimetry) and SPR (surface plasmon resonance). We found that our method can report on the binding process of the small molecule to pre-miRNA-21, which is upregulated in various cancers, and that the binding information obtained correlates correctly with currently used techniques.
As the next step, we further developed our method to enable screening of a larger number of molecules and to be quantitative. This enabled determination of the small molecule-pre-miRNA binding constant. Importantly, we were also able to determine the site of binding of a set of small molecules on the pre-miRNA-377 (associated with the etiology of heart failure), which is not obtainable with currently used high-throughput techniques. The knowledge of the nature and the position of the binding site is important in the development of improved drug candidates targeting a certain pre-miRNA. Finally, as a way to understand the details of conformation and dynamics of the pre-miRNA with and without the drug candidate bound to it, we have initialized single-molecule investigations using optical tweezers. Together with similar information obtained from the FRET-measurements of the pre-miRNA using the RNA base analogues and at similar conditions, we, thanks to our new method, help pharmaceutical R&D in the development of improved drug candidates targeting pre-miRNAs.
