RESUMO
Intramolecular G-quadruplexes (G4s) are G-rich nucleic acid structures that fold back on themselves via interrupting loops to create stacked planar G-tetrads, in which four guanine bases associate via Hoogsteen hydrogen bonding. The G4 structure is further stabilized by monovalent cations centered between the stacked tetrads. The G-tetrad face on the top and bottom planes of G4s are often the site of interaction with proteins and small molecules. To investigate the potential impact of interrupting loops on both G4 structure and interaction with proteins/small molecules, we characterized a specific G4 from the 3'-UTR of PITX1 mRNA that contains loops of 6 nucleotides using biophysical approaches. We then introduced mutations to specific loops to determine the impact on G4 structure and the ability to interact with both proteins and a G4-specific ligand. Our results suggest that mutation of a specific loop both affects the global G4 structure and impacts the ability to interact with a G4 binding protein and small molecule ligand.
Assuntos
Quadruplex G , MicroRNAs/química , MicroRNAs/ultraestrutura , Conformação de Ácido Nucleico , Fatores de Transcrição Box Pareados/química , Fatores de Transcrição Box Pareados/ultraestrutura , Sítios de Ligação , Simulação por Computador , MicroRNAs/genética , Modelos Químicos , Modelos Genéticos , Modelos Moleculares , Fatores de Transcrição Box Pareados/genética , Ligação Proteica , Proteínas/química , Proteínas/genética , Proteínas/ultraestrutura , Relação Estrutura-AtividadeRESUMO
We report the use of atomic force microscopy (AFM) to study Sox2-Pax6 complex formation on the regulatory DNA element at a single molecule level. Using an origami DNA scaffold containing two DNA strands with different levels of tensile force, we confirmed that DNA bending is necessary for Sox2 binding. We also demonstrated that two transcription factors bind cooperatively by observing the increased occupancy of Sox2-Pax6 on the DNA element compared to that of Sox2 alone.
