Figure 1.
Experimental setup for telomere DNA GQ single molecule FRET measurements.
(A) Planar telomere DNA G-quartet structure with coordinated monovalent cation. (B) Grey rectangles represent planar G-quartets and backbone polarity (5’à3’) is indicated by black arrowheads. Molecules are immobilized on a microscope slide and FRET is measured as the energy transfer between the donor (Cy3) and acceptor (Cy5) dye. (C) Human telomere G-quadruplex structures formed by Tel23 sequence.
Figure 2.
Single-molecule FRET histograms for telomere DNA GQ constructs.
Gaussian fits to the data are shown in black. (A)Tel23 thermally annealed in 100 mM KCl. (B) Tel23 in situ refolded in 100 mM KCl.
Figure 3.
Single-molecule FRET histograms of Tel23 in situ refolded in 100 mM KCl for the indicated period of time.
Figure 4.
Single-molecule FRET histograms of Tel23 in situ refolded at the indicated KCl concentrations.
Figure 5.
Structural assignments of distinct FRET states.
(A) CD spectra of Tel23 thermally annealed in 100 mM KCl (open circles), Tel23 in 100 mM NaCl (asterisks), and Hybrid Mutant in 100 mM KCl (closed circles). (B) The smFRET distribution of Tel23 thermally annealed in 100 mM NaCl fit with Gaussian functions. Gaussian fits to the data are shown in black. (C) The smFRET distribution of Hybrid Mutant thermally annealed in 100 mM KCl fit with Gaussian functions. Gaussian fits to the data are shown in black.
Figure 6.
A qualitative energy landscape model for kinetic partitioning during telomere DNA GQ folding.
A lower energy barrier between the low-FRET (unfolded) and the high-FRET (folded) state creates a kinetic trap during the early stages of folding. To escape the kinetic trap, the molecule must unfold and then re-fold into one of the more energetically stable mid-FRET (folded) states. This process is facilitated during thermal annealing at higher temperatures, or during in situ folding by low ionic strength or prolonged incubation times.