Fluorescence assays reveal DNA ligands
Stopping the formation of telomeres — repetitive DNA sequences at the ends of chromosomes — likely will prevent cancer from surviving indefinitely. Because in vitro evidence suggests that telomere formation can be blocked by DNA structures called G-quadruplexes and because anything that obstructs telomere development probably will fight cancer, ligands that promote the assembly of G-quadruplexes could serve as cancer drugs. The structures consist of four DNA strands, stabilized by four guanines from the ends of the telomeres. The strands can come from one, two or four DNA molecules.
Besides cancer, G-quadruplexes have been implicated in other diseases, and they have been found in chromosomal regions other than telomeres.
Drugs that potentially could act on G-quadruplexes can be discovered using fluorescence-based DNA melting assays, as expounded in a review by researchers from Muséum National d’Histoire Naturelle and Collège de France, both in Paris. Melting refers to the separation of DNA strands by heat, and it often is followed by cooling that causes the strands to rejoin.
The most popular melting assays use fluorophores that interact via Förster resonance energy transfer (FRET). When the strands are together, energy transfer causes fluorescence quenching. When they are melted, energy transfer no longer occurs, and fluorescence increases.
One can monitor fluorescence with spectrometry, but real-time polymerase chain reaction (PCR) apparatuses enable simultaneous measurement of multiple samples because they use multiwell plates.
The reviewers detail how to design DNA melting assays based on FRET using real-time PCR machines, discussing protocols, analysis methods and additional considerations, as well as providing examples. They explain that controlling the experimental conditions in these assays is particularly important because even slightly distinct experimental conditions can change ligand binding dramatically. (Methods, June 2007, 183-195.)
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