Structure-based Discovery Of A Fluorescent Probe Targeting C-MYC G-quadruplex With High Selectivity And Dynamic Recognition

Green Fluorescent Protein and its derivatives have revolutionized protein imaging research.The spatial and temporal dynamics of RNA could be imaged by the tagged aptamer and its fluorescent probes,while the G-quadruplex structures were found to be the recognition core for these probes.G-quadruplexes(G4)are four-stranded structures formed by guanine-rich DNA or RNA sequences.G-quadruplex forming sequences are widely distributed in eukaryotic telomeres and the promoter regions.The use of fluorescent probe molecules to identify G-quadruplex structures can be used to study the effects of G-quadruplex formation and disassembly on gene transcription and translation processes.However,the lack of fluorescent probes selectively recognizing a specific G-quadruplex structure presents challenges to precisely locate a specific DNA and RNA in cells.By exploiting the unique cleft formed in the c-MYC Pu22 G-quadruplex structure,we have discovered the fluorescent probe 9CI(6-(2-(anthracen-9-ylmethylene)hydrazinyl)-N~2,N~4-diphenyl-1,3,5-triazine-2,4-diamine)to recognize c-MYC Pu22 G-quadruplex with high selectivity in vitro and in vivo,through structure-based screening and optimization.Interestingly,molecular dynamics study explores that the entire recognition process consists of the first contact,dynamic interaction,and the final stacking.This process contributes to the high selectivity and strong fluorescence response.The results were listed below in details:1、Discovery of a fluorescent probe targeting c-MYC G-quadruplex with high selectivity and the Optimization1)Based on the unique cleft formed in the c-MYC Pu22 G-quadruplex,virtual screening was carried out to identify fluorescent probes which can selectively recognize the c-MYC Pu22 G-quadruplex;2)Through bioisosteric displacement,9CI compound with strong selectivity was found;3)The selectivity of 9CI for c-MYC Pu22 G-quadruplex in vitro was measured;4)The selectivity of 9CI for c-MYC Pu22G-quadruplex in live cell was analysed.2、Analysis for the recognition mechanisms between 9CI and c-MYC Pu22G-quadruplex1)Job plot experiments showed that the stoichiometric ratio of 9CI to c-MYC Pu22 G-quadruplex was 1:1;2)ITC and fluorescence titration experiments showed that 9CI binds with c-MYC Pu22 G-quadruplex with weak affinity;3)1D NMR analysis and the fluorescence response of 9CI to the mutant c-MYC Pu22 sequences indicated that 9CI interacted with the 3’terminal pocket of the c-MYC Pu22G-quadruplex;4)Molecular docking and molecules dynamic simulation studies discovered that the entire identification process of 9CI and c-MYC Pu22G-quadruplex consisted of three processes:initial contact,dynamic interaction,and stable accumulation.This process plays an important role in the recognition between9CI and c-MYC Pu22 G-quadruplexThis study could benefit the understanding of the recognition mechanisms between G-quadruplex and the fluorescent probes,and guide the design of ligands which recognize G-quadruplexes with high selectivity.