Figure 4. FoxP3 multimers exhibit ultrastability across diverse DNA copy numbers.
A. TnG-TnG “nunchuck” DNA construct used in the single molecule analysis. The construct consists of two dsDNA regions with identical TnG repeat sequences––(T2G)24, (T3G)18, (T4G)15––were connected by an 18-atom hexa-ethyleneglycol spacer (iSp18). Each TnG repeat is labeled with Cy3 or Cy5. FoxP3-induced DNA bridging between the Cy3 and Cy5-labeled repeats was measured by Cy3-Cy5 FRET.
B. Distributions of FRET efficiencies for TnG-TnG with an increasing concentration of FoxP3. Stability of bridged DNA complex was measured by washing with buffer lacking FoxP3 for 45 min.
C. Effect of F331D mutation on FRET for different TnG repeats.
D. Higher-order multimerization of FoxP3 on immobilized TnG-TnG nunchucks in the presence of free Cy7-labeled DNA. Cy7-DNA, containing either TnG repeats or random sequences, was added to immobilized nunchucks along with FoxP3 (300 nM), and were washed with buffer lacking FoxP3 for the indicated times. FoxP3-induced FRET (Figure S5C) and recruitment of Cy7-DNA were measured. The fraction of bound Cy7-DNAs was plotted against washing time.
E. Single-molecule pulldown of TnG repeat DNA. Each DNA was labeled with LD655 and biotin. DNA was incubated with FoxP3 (1 μM) in solution prior to immobilization on streptavidin-coated surface. Number of DNA molecules in each complex was measured as a function of LD655 photobleaching counts.
F. Distribution of the photobleaching counts for different TnG repeats in the absence (left) and presence (right) of FoxP3.
See also Figure S5.