Abstract
We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely rotating conformation and experience a fluorescence increase when restricted in the planar conformation. We hypothesized that intercalation of a molecular rotor between DNA base pairs would result in a fluorescence turn-on signal. Upon displacement by a DNA binding protein, measurable loss of signal would facilitate use of the molecular rotor in the fluorescent intercalator displacement (FID) assay. A panel of probes was interrogated using the well-established p53 model system across various DNA response elements. A novel, readily synthesizable molecular rotor incorporating an acridine orange DNA intercalating group (AO-R) outperformed other conventional dyes in the FID assay. It enabled relative measurement of p53 sequence-specific DNA interactions and study of the dominant-negative effects of cancer-associated p53 mutants. In a further application, AO-R also proved useful for staining apoptotic cells in live zebrafish embryos.
Highlights
Interactions between proteins and DNA are essential cellular processes
The two units chosen, pyrene (Py-R) and acridine orange (AO-R), have flat aromatic structures likely to facilitate intercalation into the DNA helix (Fig. 1, Scheme 1). Utility of these rotors was compared against common probes used in fluorescent intercalator displacement (FID) assays, ethidium bromide (EtBr) and thiazole orange (TO-C), as well as commercial acridine orange (AO-C)
We studied the use of two minor groove binders with the carbazole motif (Carb-2 and Carb-3) for use in FID (Fig. 1)
Summary
Interactions between proteins and DNA are essential cellular processes. There exists a requirement for robust assays enabling fundamental understanding of molecular interactions, high-throughput identification of compounds that restore correct DNA-binding in compromised cellular targets, and assessment of DNA binding proteins in clinical diagnostics[2]. The p53 protein is a tumor suppressor crucial in preventing cancer through the maintenance of cellular homeostasis and genomic integrity[3]. It senses intracellular disturbances, those that promote tumorigenesis (radiation damage, hypoxia, glucose starvation, oncogene activation), and functions to limit damage by augmenting sophisticated cellular responses that include cell-cycle arrest, DNA repair, apoptosis and cellular senescence[3]. Mutations in p53 have been found in more than half of human cancers[4] and typically result in the translation of full-length mutant proteins defective in DNA binding
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
