Principles of Fluorescence Spectroscopy

808 FLUORESCENCE CORRELATION SPECTROSCOPY
Figure 24.11. Effect of cryptophycin on the normalized autocorrelation
function of TAMRA-labeled tubulin dimer. Revised and reprinted
with permission from [38]. Copyright © 2003, American Chemical
Society.
increase in the diffusion time of tubulin. Such a large
increase in τ D indicates that tubulin must aggregate into
larger particles than dimers. This increase in τ D was roughly
consistent with the increase in hydrodynamic radius
expected for self-association to an octamer. 38
24.4.3. DNA Applications of FCS
As might be expected, FCS has been applied to DNA analysis.
Surprisingly, a relatively small number of papers have
appeared on hybridization of oligomers of similar size. 30 A
large number of papers have appeared where one of the
DNA strands was much larger than the other or using dualcolor
FCS, which is discussed later in this chapter. FCS has
also been used to study the interaction of DNA with proteins,
39–42 DNA condensation, 43–44 or binding of DNA
oligomers to larger RNA targets. 45
FCS can be effectively used to monitor DNA
hybridization when there is a large change in diffusion coefficient.
46–48 Figure 24.12 shows the normalized autocorrelation
functions for a rhodamine-labeled 18-mer during
hybridization with M13 DNA, which contains about 7250
base pairs (specifically M13mp18). Because of the large
change in effective molecular weight the shift in G(τ) is
dramatic. There was no change in brightness of the labeled
oligomer upon binding to M13, so that the fractions bound
and free could be calculated using eq. 24.17.
Figure 24.12. Hybridization of a rhodamine-labeled 18-mer to M13
DNA containing the appropriate complementary sequence. M13 DNA
has about 7250 bases. Revised from [47].
FCS has also been used to study degradation of DNA
by enzymes. One example is shown in Figure 24.13 for
double-stranded DNA with a 500-base-pair oligomer. 47–48
The 500-mer was randomly labeled at low density with a
tetramethylrhodamine-labeled nucleotide, TMR-dUTP. The
oligomer was progressively digested from the 3' end by T7
exonuclease. As the reaction proceeds the amplitude of G(τ)
decreases, reflecting the increased number of diffusing
species. Examination of the normalized curves (insert)
shows a progressive shift to shorter diffusion times as the
DNA is progressively degraded. This shift is expected given
the small size of a labeled nucleotide relative to a 500-basepair
oligomer. In principle the τ = 0 intercepts of G(τ) can
be used to recover the number of diffusing species. However,
for such an analysis it is necessary to know the relative
brightness of each species (eq. 24.20). For the case shown
in Figure 24.13 there are two dominant species: free TMRd-UTP
and the residual section of the 500-mer. The relative
brightness of the species will be approximately proportional
to the number of fluorophores per particle. In the initial
stages of the reaction one could probably assume just two
species: a dim monomer that contains one TMR and a
bright oligomer that contains many TMRs. As the reaction
proceeds it will become progressively more difficult to
resolve the population of the various sized DNA fragments.
FCS was also used to measure the appearance of
labeled DNA fragments during polymerase chain reaction
(PCR) and to characterize the size of the fragments. 31 The
fluorophore TMR-dUTP was incorporated into the PCR
products. FCS analysis was performed following removal
of the free TMR-dUTP, which otherwise would decrease
the amplitude of G(τ). Incorporation of TMR-dUTP into a
217-mer was easily observed from the shift in G(τ) (Figure