RESUMEN
G-quadruplexes (G4) are special nucleic acid structures with diverse conformational polymorphisms. Selective targeting of G-quadruplex conformations and regulating their biological functions provide promising therapeutic intervention. Despite the large repertoire of G4-binding tools, only a limited number of them can specifically target a particular G4 conformation. Here, we introduce a novel method, G4-SELEX-Seq and report the development of the first L-RNA aptamer, L-Apt12-6, with high binding selectivity to parallel G4 over other nucleic acid structures. Using parallel dG4 c-kit 1 as an example, we demonstrate the strong binding affinity between L-Apt12-6 and c-kit 1 dG4 in vitro and in cells, and notably report the applications of L-Apt12-6 in controlling DNA replication and gene expression. Our results suggest that L-Apt12-6 is a valuable tool for targeting parallel G-quadruplex conformation and regulating G4-mediated biological processes. Furthermore, G4-SELEX-Seq can be used as a general platform for G4-targeting L-RNA aptamers selection and should be applicable to other nucleic acid structures.
Asunto(s)
Aptámeros de Nucleótidos , G-Cuádruplex , Ácidos Nucleicos , Aptámeros de Nucleótidos/químicaRESUMEN
Ligand-Induced duplex-quadruplex transition within the c-MYC promoter region is one of the most studied and advanced ideas for c-MYC regulation. Despite its importance, there is a lack of methods for monitoring such process in cells, hindering a better understanding of the essence of c-MYC G-quadruplex as a drug target. Here we developed a new fluorescent probe ISCH-MYC for specific c-MYC G-quadruplex recognition based on GTFH (G-quadruplex-Triggered Fluorogenic Hybridization) strategy. We validated that ISCH-MYC displayed distinct fluorescence enhancement upon binding to c-MYC G-quadruplex, which allowed the duplex-quadruplex transition detection of c-MYC G-rich DNA in cells. Using ISCH-MYC, we successfully characterized the induction of duplex to G-quadruplex transition in the presence of G-quadruplex stabilizing ligand PDS and further monitored and evaluated the altered interactions of relevant transcription factors Sp1 and CNBP with c-MYC G-rich DNA. Thus, our study provides a visualization strategy to explore the mechanism of G-quadruplex stabilizing ligand action on c-MYC G-rich DNA and relevant proteins, thereby empowering future drug discovery efforts targeting G-quadruplexes.
Asunto(s)
G-Cuádruplex , Proteínas Proto-Oncogénicas c-myc , ADN/química , ADN/genética , Ligandos , Hibridación de Ácido Nucleico , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
RNA G-quadruplex (rG4) structures in the 5' untranslated region (5'UTR) play crucial roles in fundamental cellular processes. ADAR is an important enzyme that binds to double-strand RNA and accounts for the conversion of Adenosine to Inosine in RNA editing. However, so far there is no report on the formation and regulatory role of rG4 on ADAR expression. Here, we identify and characterize a thermostable rG4 structure within the 5'UTR of the ADAR1 mRNA and demonstrate its formation and inhibitory role on translation in reporter gene and native gene constructs. We reveal rG4-specific helicase DHX36 interacts with this rG4 in vitro and in cells under knockdown and knockout conditions by GTFH (G-quadruplex-triggered fluorogenic hybridization) probes and modulates translation in an rG4-dependent manner. Our results further substantiate the rG4 structure-DHX36 protein interaction in cells and highlight rG4 to be a key player in controlling ADAR1 translation.
Asunto(s)
G-Cuádruplex , Regiones no Traducidas 5' , ARN Mensajero/metabolismoRESUMEN
G-quadruplexes are non-canonical nucleic acid structures formed by guanine-rich DNA or RNA sequences. In recent decades, an increasing number of evidences have suggested that G-quadruplexes are associated with a wide range of biological events. Therefore, to further study the function and distribution of G-quadruplexes, developing new probes is an essential part of G-quadruplexes research. In the review, several examples of chemical fluorescent probes for G-quadruplexes developed in recent years are highlighted. These probes are promising tools for future G-quadruplex studies, which display outstanding selectivity and capability of cell imaging.
Asunto(s)
Colorantes Fluorescentes/química , G-Cuádruplex , Animales , ADN/química , Humanos , Imagen Óptica/métodos , ARN/químicaRESUMEN
Because of the absence of methods for tracking RNA G-quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G-quadruplexes is so far limited. Herein, we report a new red-emitting fluorescent probe, QUMA-1, for the selective, continuous, and real-time visualization of RNA G-quadruplexes in live cells. The applications of QUMA-1 in several previously intractable applications, including live-cell imaging of the dynamic folding, unfolding, and movement of RNA G-quadruplexes and the visualization of the unwinding of RNA G-quadruplexes by RNA helicase have been demonstrated. Notably, our real-time results revealed the complexity of the dynamics of RNA G-quadruplexes in live cells. We anticipate that the further application of QUMA-1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G-quadruplexes will uncover more information about the biological roles of RNA G-quadruplexes.