Figure 5.

Monitoring the real-time interaction of quencher-modified molecules with surface-immobilized fluorescent molecules: a complex of DNA polymerase with DNA. (A and B) Illustration of the KF-DNA binary complex (PDB: 1L3U) using structural data from Bacillus stearothermophilus DNA polymerase, a KF homolog (32). (A) View of the DNA binding pocket between the fingers (top left) and thumb (top right). The DNA helix of the original crystal structure was extended manually and is shown in tubes and schematic bases. (B) View of the labeled positions on KF and DNA. The 44 Å distance corresponds to KF binding to the DNA hairpin at position +1 (see numbering of basepairs in panel C). The distances were measured from the C_β atom of the lysine residue and the phosphate atom of each DNA base. (C) Sequence of the DNA hairpin oligonucleotide used in this study. In the folded hairpin, the top strand in panel C is referred to as the primer strand and the bottom strand as the template strand. Numbering of the bases is based on the 3′-end of the primer strand. The hairpin is biotinylated at the hairpin loop (position −18, primer strand) and labeled with Cy3B (position −11, template strand), which acts as a donor in the Cy3B-QSY7 FRET pair. (D–F) Typical fluorescence time-traces of Cy3B attached to DNA ($F_{G_{exc}}^{G_{em}}$) for DNA-only samples (D), samples with 15 nM KF^Q in solution (E), and samples with 30 nM KF^Q in solution (F). Histograms of the intensity values are projected on the right-hand side. Data bins after photobleaching are not projected. The quenched states (lower dotted line) correspond to binary complexes of KF^Q and DNA (∼90% quenching). The unquenched states (upper dotted line) correspond to free DNA. (G–J) Dwell-time distributions of the quenched (G and I) and the unquenched (H and J) states for samples with KF^Q. The distributions were fitted with a monoexponential function F (F (t) = A exp(− t/τ)) with A and τ as the fitted parameters. K_d values were calculated from the fitted parameters τ_bound and τ_free and the concentration of KF^Q in solution according to Eq. S2 in the Supporting Material.