Principles of Fluorescence Spectroscopy

782 SINGLE-MOLECULE DETECTION
Figure 23.44. Top: Structure of wild-type DHOD from E. coli.
Bottom: Environment of FMN in the catalytic site. Reprinted with
permission from [80]. Copyright © 2004, American Chemical
Society.
23.10.2. Single-Molecule Molecular Beacons
We started this chapter with a schematic of a single-molecule
molecular beacon (Figure 23.1). In fact, such an experiment
has been reported. 81 Figure 23.46 shows a surfacebound
molecular beacon. This sequence has a biotin on one
end and the fluorophore MR121 at the opposite end. The
sequence is designed so that a guanine residue is next to
MR121 when the beacon is in the hairpin conformation. In
solution this beacon displays a sixfold increase in intensity
when hybridized with the complementary oligo.
The lower panels in Figure 23.46 show confocal images
of surface-bound beacons in the absence (left) and
presence (right) of the complementary oligo. Hybridization
with the complementary oligo results in an increase in the
number of observable spots. The actual increase in intensity
is larger than it appears because the scale on the left
image is four times smaller than the right image. This
approach allows detection of single hybridization events.
Figure 23.45. Emission intensities of FMN bound to dihydroorotate
dehydrogenase in the absence (top) and presence (bottom) of substrates.
The top panel shows traces for three different molecules. The
protein was immobilized in an agarose gel. Reprinted with permission
from [80]. Copyright © 2004, American Chemical Society.
23.10.3. Conformational Dynamics of a
Holliday Junction
Single-molecule FRET has been extensively useful in studies
of the conformational dynamics of DNA and RNA. 82–88
One example is a study of the structural dynamics of a Holliday
junction. 83 Genetic recombination is an important
component of genetic diversity and evolution. Recombination
occurs when sections of DNA are exchanged between
chromosomes. This recombination occurs at sites that are
called Holliday junctions. These junctions or sections of
DNA form a four-way cross (Figure 23.47). These junctions
are formed and break when DNA strands are exchanged.
The Holliday junction in Figure 24.47 contains four
DNA oligomers. One oligomer was synthesized with a donor
(Cy3) and a second oligomer was synthesized with an
acceptor (Cy5) both on the 5' ends of the DNA strands. A
third strand was labeled with biotin for surface immobilization.
The four-way junction was expected to change its con-