Targeting a G-quadruplex from let-7e pre-miRNA with small molecules and nucleolin

Let-7e precursor microRNA has the potential to adopt a G-quadruple x (rG4) structure and recently, its roles in oncology have been the focus of much attention, as it is now known that let-7e pre-miRNA is frequently dys-regulated in cancers. Therefore, it is crucial to unvei l and fully characterize its ability to adopt a rG4 structure, which could be stabilized or destabilized by sma l l molecules and proteins such as nucleolin, a protein that is deeply associated with miRNA biogenesis. Herein, by combining a set of different methods such as circular di-chroism (CD), nuclear magnetic resonance (NMR), UV spectroscopy (thermal difference spectra (TDS) and isothermal difference spectra (IDS)) and polyacrylamide gel electrophoresis (PAGE), we demonstrate the for-mation of the rG4 structure found in let-7e pre-miRNA sequence in the presence of K+ (5'-GGGCU-GAGGUAGGAGG-3'). The ability of eight sma l l molecules (or ligands) to bind to and stabilize this rG4 structure was also fully assessed. The dissociation constants for each RNA G-quadruplex/ligand complex, determined by surface plasmon resonance (SPR), ranged in the 10-6 to 10-9 M range. Lastly, the binding of the rG4 structure to nucleolin in the presence and absence of ligands was evaluated via CD, SPR, PAGE and confocal microscopy. The sma l l molecules 360 A and PDS demonstrated attractive properties to targetthe rG4 structure of let-7e pre-miRNA and control its biology. Ou r findings also highlighted that the interaction of TMPyP4 with the G-quad-ruplex of let-7e precursor miRNA cou l d block the formation of the comple x between the rG4 and nucleolin. Overall, this study introduces an approach to target the rG4 found in let-7e pre-miRNA which opens up a new opportunity to control the microRNA biogenesis.

Automated summary

The study demonstrated the potential of let-7e precursor microRNA to adopt a rG4 structure, which can be influenced by various small molecules and proteins. By using multiple methods, the presence of rG4 structure and its interactions with other molecules were determined, providing a new approach to control microRNA biogenesis.