Fig 4. Kinetics of PfR6 interaction with HMG-box.

HMG-box^540Q and PfR6^FAM were used to monitor the interaction between the aptamer and the protein in real-time using a stopped-flow apparatus (Applied Photophysics, UK). (A) FRET between HMG-box^540Q and PfR6^FAM. Mixing the labeled protein with the probe-bound aptamer (green) resulted in a decrease of fluorescence over time, indicating that the binding of both components results in the quenching of fluorescein due to its vicinity with the quencher in the protein. FRET assessment controls lacking the aptamer or the quencher Atto-540 in the HMG-box are shown in orange and purple traces respectively. (B) HMG-box ^540Q Pf (green) or HMG-box ^540Q Hs (blue) comparison during the binding with PfR6^FAM at pH 5.5. 7–10 replicates for each reaction were measured and averaged. Continuous lines show the fitting with exponential functions.