I am currently working with a compound which is expected to bind G-quadruplex structure in c-MYC promoter. While I observed very poor affinity between c-MYC quadruplex and the compound in vitro in Isothermal titration calorimetry, I observed significant decrease of promoter activity in c-MYC by the same drug through quadruplex binding in its promoter. The compound has quadruplex-to-duplex selectivity but fails to distinguish different quadruplexes in the cells and non-specifically binds to other oncogene and telomere quadruplex in the cells?
Sometimes binding affinity determined in vitro is not consist with that in cells. There are two unclear issue need to make sure first. 1. “I observed significant decrease of promoter activity in c-MYC by the same drug through quadruplex binding in its promoter ” means decreasing only in luciferase with c-myc promoter containing G4 but not c-myc promoter with mutated G-rich sequence? 2. Some interactions drive by entropy are not determined in ITC, you could try another methods, such as TO displacement or SPR to confirm the in vitro binding affinity.
Best wishes.
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