(415g) Design Strategies for Countering the Effect of Fluorophore-Quencher Labelling on DNA Hairpin Thermodynamics

In this work, we studied the effect of fluorophore modifications on DNA hybridization thermodynamics with a focus on hairpin opening. We observed an extremely low hybridization efficiency of single stranded trigger strand with end-modified hairpins. The hybridization efficiency improved drastically, i.e. up to ca. 40-folds, by incorporating an external hairpin stem structure on the single stranded trigger strand to promote coaxial stacking hybridization. An alternative counter strategy was to modify the dye internally in the hairpin stem to destabilize its closed state. Lastly, the effect of dye modifications on hairpin opening was similarly observed in the more complex molecular operation of Hybridization Chain Reaction (HCR). Overall, the study affirmed that the careful choice of fluorophore-quencher pair, aside from their spectral properties, and their strategic placement should be accounted for in the design of oligo probes which is particularly crucial for probes with hairpin structure.