Simple fluorimetric method for probing AT-rich regions in DNA based on the photoisomerization of distilbazolium ligand Original Research Article

A simple fluorimetric assay has been developed for probing AT-rich regions in DNA. A fluorescent ligand, 1,4-bis[2-(1-methylpyridinium-4-yl)vinyl] benzene diiodide (pMPVB), binds to GC and AT sequences with comparable affinity and the only fluorescent species is trans-pMPVB/AT complex. Since the ligand undergoes photoisomerization, the system is initially photoequilibrated to reach a photostationary state (pss), which contains a high fraction of the non-fluorescent cis-isomer. Subsequent “analytical” irradiation at the wavelength of isosbestic point (357 nm) leads to a new pss with higher content of the fluorescent trans-isomer (in situ generation of trans-isomer). An increase in fluorescence, recorded during the analytical irradiation, is considered as an analytical signal that is proportional to the content of A and T bases arranged in continuous sequences of 4 bp or more (AT quartets). The calibration graph, plotted for mixtures of homopolymers [poly(dA-dT)]2 and [poly(dG-dC)]2 at varied molar ratios, was linear over the range of 0.1–0.7 molar fraction of AT quartets with the detection limit (3σ) of 0.03. The method has been proved experimentally on two natural DNA samples, calf thymus and T4-phage DNA, giving molar fractions of AT quartets of 0.17 and 0.26, respectively, in good agreement with expected results calculated from the published genomic sequences. The relative standard deviation (n=3) was within 7%.