RESUMEN
P38γ (MAPK12) is predominantly expressed in triple negative breast cancer cells (TNBC) and induces stem cell (CSC) expansion resulting in decreased survival of the patients due to metastasis. Abundance of G-rich sequences at MAPK12 promoter implied the functional probability to reverse tumorigenesis, though the formation of G-Quadruplex (G4) structures at MAPK12 promoter is elusive. Here, we identified two evolutionary consensus adjacent G4 motifs upstream of the MAPK12 promoter, forming parallel G4 structures. They exist in an equilibria between G4 and duplex, regulated by the binding turnover of Sp1 and Nucleolin that bind to these G4 motifs and regulate MAPK12 transcriptional homeostasis. To underscore the gene-regulatory functions of G4 motifs, we employed CRISPR-Cas9 system to eliminate G4s from TNBC cells and synthesized a naphthalene diimide (NDI) derivative (TGS24) which shows high-affinity binding to MAPK12-G4 and inhibits MAPK12 transcription. Deletion of G4 motifs and NDI compound interfere with the recruitment of the transcription factors, inhibiting MAPK12 expression in cancer cells. The molecular basis of NDI-induced G4 transcriptional regulation was analysed by RNA-seq analyses, which revealed that MAPK12-G4 inhibits oncogenic RAS transformation and trans-activation of NANOG. MAPK12-G4 also reduces CD44High/CD24Low population in TNBC cells and downregulates internal stem cell markers, arresting the stemness properties of cancer cells.
Asunto(s)
G-Cuádruplex , Proteína Quinasa 12 Activada por Mitógenos , Neoplasias de la Mama Triple Negativas , Humanos , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Neoplasias de la Mama Triple Negativas/genética , Proteína Quinasa 12 Activada por Mitógenos/genéticaRESUMEN
The structural diversity and functional relevance of nucleic acids (NAs), mainly deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are indispensable for almost all living organisms, with minute aberrations in their structure and function becoming causative factors in numerous human diseases. The standard structures of NAs, termed canonical structures, are supported by Watson-Crick hydrogen bonding. Under special physiological conditions, NAs adopt distinct spatial organisations, giving rise to non-canonical conformations supported by hydrogen bonding other than the Watson-Crick type; such non-canonical structures have a definite function in controlling gene expression and are considered as novel diagnostic and therapeutic targets. Development of molecular probes for these canonical and non-canonical DNA/RNA structures has been an active field of research. Among the numerous probes studied, probes with turn-on fluorescence in the far-red (600-750 nm) region are highly sought-after due to minimal autofluorescence and cellular damage. Far-red fluorescent probes are vital for real-time imaging of NAs in live cells as they provide good resolution and minimal perturbation of the cell under investigation. In this review, we present recent advances in the area of far-red fluorescent probes of DNA/RNA and non-canonical G-quadruplex structures. For the sake of continuity and completeness, we provide a brief overview of visible fluorescent probes. Utmost importance is given to design criteria, characteristic properties and biological applications, including in cellulo imaging, apart from critical discussion on limitations of the far-red fluorescent probes. Finally, we offer current and future prospects in targeting canonical and non-canonical NAs specific to cellular organelles, through sequence- and conformation-specific far-red fluorescent probes. We also cover their implications in chemical and molecular biology, with particular focus on decoding various disease mechanisms involving NAs.
RESUMEN
Intercalation by threading is anticipated to feature in DNA-binding molecules for developing DNA-targeted diagnostics and therapeutics. We investigated the role of an imidazolyl moiety in threading intercalators of DNA by employing a number of imidazolyl-naphthalenediimide conjugates. Threading intercalation was studied by UV spectroscopy, competitive binding fluorescent dye displacement, circular dichroism, isothermal calorimetry, and computational analysis. NIm6 was found to be a strong candidate, with good half-life, as revealed by dissociation kinetic analysis. Computational studies supported intercalation of the naphthalene core between base pairs and binding of the imidazolyl moieties in the adjacent grooves (threading mechanism) through electrostatic and hydrogen-bonding interactions. The interaction of the positively charged imidazolium moieties with the negatively charged phosphate backbone of DNA contributed to the favorable enthalpy change, as revealed by the experimental and computational data. Threading intercalation by NIm6 caused significant retardation of DNA in an electrophoretic mobility shift assay. The biological significance of potent imidazolyl naphthalenediimide conjugates was demonstrated by the inhibition of topo- isomeraseâ I activity and cytotoxicity against HeLa cells.
Asunto(s)
ADN/química , Imidazoles/química , Imidas/química , Sustancias Intercalantes/química , Naftalenos/química , Unión Competitiva , Calorimetría , Supervivencia Celular/efectos de los fármacos , Dicroismo Circular , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/metabolismo , Diseño de Fármacos , Ensayo de Cambio de Movilidad Electroforética , Colorantes Fluorescentes/química , Semivida , Células HeLa , Humanos , Sustancias Intercalantes/metabolismo , Sustancias Intercalantes/toxicidad , Cinética , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
In this Communication, a molecular beacon-based DNA switch (LMB) is developed as an efficient and reversible pH sensing probe. Remarkably, LMB exhibited reversible structural transition between the closed (molecular beacon) and open (A-motif) states very efficiently in synthetic vesicles and live cells without the need for any transfection agents.