Figure 5

Validation of the PIFE-FRET correction procedure in ALEX (A) Data correction process, to obtain R₀-corrected 2D-histograms. Four dsDNAs with identical sequence (Fig. S4A) are labelled with two different FRET pairs: Cy3(B)/ATTO647N and mixed together. The donor is attached at the 5′-end; the difference in brightness between Cy3 (black) and Cy3B (green) separates the four populations into two groups according to S(E_Pr). The acceptor fluorophore ATTO647N is positioned on the complementary DNA strand in 13 and 23 bp distance; the two distances are deciphered via two different E_Pr values per subgroup. (B) By correcting each fluorophore pair with its corresponding gamma factor γ_Cy3 or γ_Cy3B, accurate FRET values E for each population are obtained. The mean accurate FRET values for 13 or 23 bp differ between the two FRET-pairs due a difference in Förster radius R₀. (C) The proposed R₀-correction allows to convert all accurate FRET values on one common R₀-axis. (D) ALEX-data for 8, 13, 18, 23, 28 and 33 bp after the 3 correction steps, against background, gamma and R₀. Error bars were obtained from n = 4 experimental repeats.