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Nucleic acids are often covalently modified with fluorescent reporter molecules to create a hybridization state-dependent optical signal. Designing such a nucleic acid reporter involves selecting a fluorophore, quencher, and fluorescence quenching design. This report outlines the effect that these choices have on the DNA hybridization characteristics by examining six fluorophores in four quenching schemes: a quencher molecule offset from the fluorophore by 0, 5, or 10 bases, and nucleotide quenching. The similar binding characteristics of left-handed L-DNA were evaluated in comparison with right-handed DNA to quantify the effect of each quenching scheme. These results were applied to the Adaptive PCR method, which monitors fluorescently-labeled L-DNA as a sentinel for analogous unlabeled D-DNA in the reaction. All of the tested fluorophores and quenching schemes increased the annealing temperature of the oligonucleotide pairs by values ranging from 0.5 to 8.5 °C relative to unlabeled oligonucleotides. The design with the smallest increase (0.5 °C) was a sense strand with a FAM fluorophore and an anti-sense strand with Black Hole Quencher 2 offset by 10 bases from the FAM. An identical design that did not offset the quencher molecules resulted in a shift in annealing temperature of 5 °C. PCR was performed using temperature switching based on each of these L-DNA designs, and efficiency was significantly increased for the 10-base offset design, which had the smallest shift in annealing temperature. These results highlight the importance of selecting an appropriate fluorescence quenching scheme for nucleic acid optical signals.

Fluorophore-quencher interactions effect on hybridization characteristics of complementary oligonucleotides