Figure 5.

Studying synaptic complex formation using TFM-FRET. (A) Cartoon. A 1 kb DNA is labeled with Cy5 and Cy3B, 10 bp and 990 bp from the biotin end, respectively. The substrate DNA displays a broad Cy3B FIW. Upon addition of Cre, synaptic complex formation between loxP sites located close to the labeling positions leads to a looping of the DNA and a decrease in FIW, as well as bringing the Cy3B and Cy5 fluorophores into close proximity, resulting in FRET. (B) Representative time trace of reversible synaptic complex formation in the presence of Cre, with the intensities corresponding to the emission of the donor, in green (light gray), and acceptor, in red (darker), under 532 nm continuous excitation. Synapsis is evident between 110 and 135 s. (C) Histogram of FRET efficiency, measured within the looped complex formed, with mean E^∗ = 0.299 ± 0.004, determined using an unweighted least-squares fit to the data. (D) Histogram of dwell times of individual looping events, fit using maximum likelihood to a one parameter single exponential with a dwell time of 32 ± 4 s. The uncertainty in fit parameters is defined by the 1-σ confidence interval of the fit (n = 52). Movies were taken with a laser power of 1 mW and a frame rate of 10 Hz. To see this figure in color, go online.