Sensitive fluorescence-based thermodynamic and kinetic measurements of DNA hybridization in solution

摘要: Kinetic and thermodynamic constants associated with DNA hybridization were determined in solution using fluorescence measurements complementary fluorophore-labeled oligomers. One oligomer was labeled a 5'-terminal fluorescein, the other 3'-terminal rhodamine. The juxtaposition of two labels double-stranded complexes results strong quenching fluorescein emission, thereby providing means for distinguishing single-stranded from DNA. Since based on fluorescence, denaturation association could be monitored routinely at strand concentrations 100-1000-fold lower than permitted by absorbance hypochromicity measurements. To determine if mirrored base pair formation, temperature profiles dissociation constructed both number different concentrations. Analyses these "all-or-none" model provided data which statistically indistinguishable between measurement methods, thus validating use studies effects fluorophore attachment properties strands investigated analyzing melting curves combinations unlabeled presence found to stabilize about -1.5 kcal delta G degrees 298, primarily due label. Association rate temperatures, linear Arrhenius plots obtained. unique "label dilution" method measuring oligomers upon dynamic constant temperature. simplified since relatively low mixed, reducing rates eliminating need rapid mixing techniques.