RESUMO
RNA G-quadruplexes (rG4) have recently emerged as major regulatory elements in both mRNA and non-coding RNA. In order to investigate the biological roles of rG4 structures, chemists have developed a variety of highly specific and potent ligands. All of these ligands bind to the rG4s by stacking on top of them. The binding specificity is demonstrated by comparison to other structures such as duplex or three-way junctions. It remains unclear whether rG4-ligands merely stabilize fully formed rG4 structures, or if they actively participate in the folding of the rG4 structure through their association with an unfolded RNA sequence. In order to elucidate the innate steps of ligand-rG4 associations and mechanisms robust in vitro techniques, including FRET, electrophoretic mobility shift assays and reverse transcriptase stalling assays, were used to examine the capacity of five well-known G4 ligands to induce rG4 structures derived from either long non-coding RNAs or from synthetic RNAs. It was found that both PhenDC3 and PDS induce rG4 formation in single RNA strands. This discovery has important implications for the interpretation of RNA-seq experiments. Overall, in vitro data that can assist biochemists in selecting the optimal G4-ligands for their RNA cellular experiments are presented, and the effects induced by these ligands on the rG4s are also considered.
