Figure 6
Distance changes determined via FRET in the presence of PIFE.
FRET between Cy3/ATTO647N attached to a 40 bp-dsDNA is probed simultaneously to PIFE that occurred between Cy3 and different restriction enzymes in R1 = 1 bp distance to the donor. (A) Schematic of DNA constructs probing FRET distances in 13, 18, 23 and 40 bp between ATTO647N and Cy3(B) in the presence of BamHI, respectively in 18, 23, 28 and 40 bp in the presence of EcoRV. While BamHI does not interfere with the 3D structure of the DNA, EcoRV is reported to introduce a 50° kink. (B) 1D ALEX histograms showing 500 nM BamHI bound to 1bp-Cy3-BamHI-dsDNA. Binding is detected as constant shift in S from ~0.3 (free dsDNA, black) to ~0.4 (green). BamHI introduces a conformational change in the dsDNA seen by smaller FRET values. (C) Differences in Stoichiometry ΔS(EPr) and R0-corrected FRET ΔER0 between free and BamHI-bound DNA. While binding is observed via PIFE of Cy3 (green, triangle), it is undetected for Cy3B (green, square). The conformational change in FRET is also observed for Cy3B (black, square). (D) 1D ALEX histograms showing 50 nM EcoRV bound to 1bp-Cy3-EcoRV-dsDNA. Binding is observed as a constant shift in S from 0.29 to 0.38. EcoRV is reported to kink dsDNA, which is observed by increased FRET values. (E) Differences in Stoichiometry ΔS(EPr) and R0-corrected FRET ΔER0 between free and EcoRV-bound DNA labelled with Cy3 (triangle) or Cy3B (square). While distance changes in R2 for different FRET distances are observed for both donor fluorophores (black panel), binding is only observed via PIFE to Cy3 (green, triangle) and undetected for Cy3B (green, square). Error bars were obtained from at least n = 2 experimental repeats. Full data sets and controls provided in Fig. S10.
