Figure 4. Cdc13 and CST can bind and unfold higher order G-tail structure.

(A) A schematic depiction of reaction steps for single molecule FRET experiments. (B) Single molecule FRET efficiency histograms in the presence of the indicated concentrations of Cdc13. (C) Single molecule FRET efficiency histograms in the presence of the indicated concentrations of CST. (D–F) Representative single-molecule FRET-time traces of the 48-nt G-tail construct in 3 mM MgCl₂ and 100 mM NaCl before adding any protein. A 532 nm laser was used for excitation. (G) A representative single-molecule FRET-time trace of the 48-nt G-tail construct in 3 mM MgCl₂ and 100 mM NaCl immediately after adding 1 nM CST, showing a CST binding event at ∼19 sec (the black arrow). A 532 nm laser was used for Cy3 excitation throughout the entire course of data acquisition. A 633 nm laser was briefly turned on for direct Cy5 excitation at ∼86 sec in order to confirm that Cy5 is still active rather than photobleached.