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detection channels, we succeed in measuring Cy5 and Cy5.5 alternatively lighting
up with identical transitions rates as measured via two-color FRET. These anti-
correlated fluctuations of FRET to Cy5 and to Cy5.5 were fully synchronized
within the 20-ms time resolution used, demonstrating that the Cy5 arm is
moving toward the Cy3 arm at the same time as the Cy5.5 arm is moving away
from the Cy3 arm. While this was not a surprising result, such a conclusion
cannot be drawn from regular two-color FRETstudies. In the second experiment,
we moved the dye positions so that strong FRET to Cy5 occurs only if a parallel
conformation is populated (Figure 11.6B). The data show anti-correlated signal
fluctuations of Cy3 and Cy5.5 but no change in Cy5, proving that the parallel
conformations are not even transiently populated within the 6-ms time resolu-
tion used [132].
11.4.3
Spontaneous Branch Migration Observed with a Single Step Resolution
In a more recent study [15], we modi ed the central base sequence of the non-
migratable HJ, junction 7, to obtain a mono-migratable junction with two possible
branch points (Figure 11.7A). In one branch point (
U
) all four arms are 11 bp in
length. In the other branch point (
), the vertical arms are 12 bp long and the
horizontal arms are 10 bp long. In the presence of Mg 2 þ , each branch point would
have two stacking conformations; hence four different states are expected. This was
indeed con rmed from smFRET traces (Figure 11.7B). The molecule shows slow
two-state fluctuations interrupted by a brief period with much more rapid two-state
fluctuations (zoom-in shown in Figure 11.7C). In comparison, we have never
observed more than two FRET states from non-migratable junctions. In the rapid
fluctuation phase E values for the higher FRETstate are lower than those of the slow
M
fluctuation phase (Figure 11.7D). Since simple geometric considerations suggest that
the E in the high FRETstates should be higher for the branch point
U
, we tentatively
assigned the slowly uctuatingmode to
. This
assignment was con rmed by a hydroxyl radical cleavage experiment that located the
dominant branch point. This result shows that we have the FRETresolution to detect
even a single base-pair branchmigration and that multiple conformer changes occur
in each branch point before branch migration. The lifetime of each conformer
decreased at lower Mg 2 þ concentrations with no change in relative bias. Similar
behavior was observed from four other sequences and no parallel states were
observed when dyes weremoved to two opposing arms. Thus thesemono-migratable
HJs behave very much like non-migratable HJs when they are residing in one branch
point, implying that the migratability is only a small perturbation in the dynamic
structural properties of the HJ.
The average number of conformer transitions before a branch migration step
remained constant as we varied Mg 2 þ . Consequently the lifetime of each branch
point is directly proportional to the average lifetime of stacked conformers within that
branch point, indicating that the stability of stacked conformations, determined by
the DNA sequences, governs the branch migration kinetics. Similar observations
U
and the rapidly uctuatingmode to
M
 
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