G-quadruplex deconvolution with physiological mimicry enhances primary screening: Optimizing the FRET Melt2 assay

Publication date: Available online 28 December 2019Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory MechanismsAuthor(s): Rhianna K. Morgan, Alexandra Psaras, Quinea Lassiter, Kelsey Raymer, Tracy A. BrooksAbstractNon-B-DNA G-quadruplex (G4) structures have shown promise as molecular targets. Modulating G4 stability for oncogenic transcriptional control is a promising avenue for the development of novel therapeutics. Extracellularly, G4 stabilization can be mediated by alkali cations, modifying water content, or with molecular crowding. Intracellularly, G4 formation is mediated by negative superhelicity and transcriptional activity, and can be stabilized with small molecules or oligonucleotides. Numerous G4-stabilizing compounds have been identified that impact promoter activity in plasmids. These compounds, however, infrequently show activity in cells, are found to have non-G4-mediated mechanisms of action, or do not demonstrate activity in vivo. The G4 field requires enhanced predictive screening methods to identify compounds with G4-mediated in vitro activity and in vivo efficacy. Using the best characterized promoter G4 to date, MYC, we examined the effects of varying annealing conditions (rate of cool down and number of heat/cool cycles), co-solvents (glucose, acetonitrile, polyethylene glycol, dextran sulfate, sucrose, ficoll-70, glycerol) and nucleoplasm on G4 formation and compound screening. We observed a marked decrease in hit rates when shifting from si...
Source: Biochimica et Biophysica Acta (BBA) Gene Regulatory Mechanisms - Category: Genetics & Stem Cells Source Type: research