Chemistry Reference
In-Depth Information
In this chapter, I will discuss two different biological systems that we have been
studying using smFRET over the last few years, the DNA unwinding enzyme,
helicase, and the key intermediate in DNA combination known as Holliday
junction.
11.2
Single-molecule FRET
FRET ef
ciency is given by I
A
/(I
A
I
D
) where I
A
is the sensitized emission
intensity of the acceptor, I
D
is the donor intensity, and
þ h
is a parameter representing
relative detection ef
ciencies and quantum yields of the two dyes, and can be
determined from photobleaching events [43]. Our data using Cy3 and Cy5 show
that
h
I
D
) is an excellent approximation for FRET ef
ciency
and all our data are presented in this form. While this FRET value cannot be smaller
than 0 or larger than 1 in principle, in practice I
A
can be very small for FRET
approaching 0 and noise around the background level can make I
A
negative after
background subtraction, leading to calculated FRET values smaller than 0. For the
same reason FRET values larger than 1 can be obtained if it is close to 1. For this
reason our FRET histograms sometimes contain data points below 0 or above 1, but
this is simply due to a
finite signal-to-noise ratio. In almost all of our studies, we do
not need absolute distance information because other control experiments can
unambiguously assign FRET values to corresponding states. For certain cases, it
can be ambiguous, evenwith control experiments, whether an observed FRETchange
is due to distance change or the change in dipole orientations of the dyes, hence in
h
1. Thus E
I
A
/(I
A
þ
2
.
In such cases we canmeasure polarization anisotropy of the dyes at the same time as
FRET from single molecules so that we can determine whether or not the FRET
changes are mere artifacts [44, 45]. Therefore in all of our experiments, we do not
need to convert E to distance in order to answer questions of biological interest. The
distance information can still play a supporting role to determine whether the signal
we obtain is consistent with what is known about the structural properties of the
molecules [37, 38].
k
11.2.1
Non-perturbative Immobilization: BSA and PEG Surfaces, and Vesicle Encapsulation
To study the dynamic changes of individual molecules over extended time periods,
molecules need to be localized in space. This is often achieved by surface immobili-
zation (Figure 11.1). An ideal surface would allow speci
c immobilization of nucleic
acids or proteins while rejecting non-speci
c adsorption. For nucleic acids studies,
we prefer to use a quartz slide coated with biotinylated BSA and streptavidin because
of the simplicity of this system. We can immobilize DNA and RNA with high
speci
city (
500 : 1, compared to control experiments that exclude biotin or strepta-
vidin) and were able to faithfully reproduce their bulk solution activities [19, 20, 31].
This is likely because all three surface constituents (BSA, streptavidin and quartz) are
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