Probing DNA sequences in solution with a monomer-excimer fluorescence color change

Abstract

The use of a simple fluorescent nucleoside analogue in detection of point mutations by hybridization in solution is described. Pyrene is placed at 3' and 5' ends of a pair of oligodeoxynucleotide probes via a phosphoramidite derivative of deoxyribose with this fluorophore attached at the 1' position, replacing a DNA base. Adjacent binding of dual probes containing this fluorophore to a complementary target sequence results in a pronounced spectral change from blue pyrene monomer emission (lambdamax= 381 398 nm) to green-white excimer emission (lambdamax= 490 nm). Optimization of the relative binding positions of the two probes shows that the greatest spectral change occurs when they bind with partial end overlap. In optimum orientation, the monomer emission band for the probes decreases intensity by as much as a factor of seven and the excimer band increases up to 40-fold on binding a complementary target. Application to the detection of a single-base point mutation in solution is described.