Use of pteridine nucleoside analogs as hybridization probes

The pteridine nucleoside analog 3-methyl iso-xanthopterin (3-MI) is highly ¯uorescent, with a quantum yield of 0.88, and it can be synthesized as a phosphoramidite and incorporated into oligo-nucleotides through a deoxyribose linkage. Within an oligonucleotide, 3-MI is intimately associated with native bases and its ¯uorescence is variably quenched in a sequence-dependent manner. Bend-ing, annealing, binding, digestion or cleavage of ¯uorophore-containing oligonucleotides can be detected by monitoring changes in ¯uorescence properties. We developed a single step method for detecting annealing of complementary DNA sequences using 3-MI-containing oligonucleotides as hybridization probes. One of the complementary strands contains the ¯uorophore as an insertion and when annealing occurs, the ¯uorophore bulges out from the double strand, resulting in increased ¯uorescence intensity. We have examined the sequence dependency, optimal strand length and impact of multiple ¯uorophores per strand in terms of brightness and impact on the annealing process. We describe the application of this technique to the detection of positive PCR products using an HIV-1 detection system. This sequence-dependent hybrid-ization technique can result in ¯uorescence inten-sity increases of up to 27-fold. Fluorescence intensity increases are only seen upon speci®c binding to bulge-generating complements, remov-ing issues of high background from non-speci®c binding