Chemistry Reference
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-termini by various species: for instance, helix X by Cy3 (donor), helix B by Cy5
(acceptor), and helix Hby biotin (Figure 11.4). In one stacking conformer (isoI), helix
B stacks on helix H, minimizing the inter-
uorophore distance (high FRET), while in
the other conformer (isoII), helix B stacks on helix X, maximizing the distance (low
FRET). These assignments were independently veri
ed by comparison with gel
mobility assays [105]. Three different sequences named junctions 1, 3 and 7 were
used. smFRET time records show two-state
fluctuations between isoI and isoII
(Figure 11.5A). The bias between the two conformers is a function of sequence but
not of ionic conditions [14]. The conformer bias observed in smFRET experiments
(isoI over isoII for junction 1 shown in Figure 11.5B for instance) exactly matched the
bias deduced from the gel mobility assay of unlabeled DNA. In addition, switching
the dye positions or biotin position to different arms did not change the transition
rates [131]. Therefore surface-immobilization and dye-labeling do not signi
cantly
perturb the HJ dynamics.
Figure 11.5 (A) Single-molecule FRET time trace of junction 1.
(B) FRET efficiency histogram from 25 junction-1 molecules.
(C) The sum of the two rates (isoI to isoII and isoII to isoI) for a
variety of ionic conditions.
