Principles of Fluorescence Spectroscopy

726 DNA TECHNOLOGY
Figure 21.44. Structure of a fluorescein-labeled aptamer specific for
PDGF. The lower panel shows the fluorescein anisotropy. Revised and
reprinted with permission from [138]. Copyright © 2001, American
Chemical Society.
potential protein marker for cancer diagnosis. PDGF is typically
detected using ELISA or radiotracer methods. Figure
21.44 shows a fluorophore-labeled aptamer specific for
PDGF. Upon addition of PDGF the anisotropy increases
more than twofold. 138 This result shows that other fluorescence
parameters can be used with an aptamer, not just
RET. The twofold increase in anisotropy is probably larger
than could be obtained with a fluorescein-labeled antibody.
The molecular weight of IgG is near 150,000, and an Fab
fragment has a molecular weight near 50,000. For proteins
of this size the fluorescein anisotropy would be near its
maximal value before binding to PDGF. The smaller size of
the aptamers and their high degree of flexibility in the
absence of ligand should result in similar anisotropy
changes in other aptamers.
21.5.1. DNAzymes
The uses of aptamers have been extended to include DNA
sequences that have enzymatic activity, 139–140 analogous to
the activity displayed by ribozymes. A combination apta-
Figure 21.45. Detection of Pb 2+ using a DNAzyme. The fluorophore
is TAMRA and the quencher is dabcyl. From [142].
mer-DNAzyme was developed for detection of lead
ions. 141–142 The aptamer contained two parts that were
labeled with a TAMRA donor or a dabcyl acceptor. These
two oligomers spontaneously hybridized, which resulted in
quenching of TAMRA by RET (Figure 21.45). Upon addition
of Pb 2+ the DNAzyme undergoes autocatalytic cleavage
to release the fragments of the cleaved oligomer. The
donor intensity increases when the quencher oligomer is
released, which can be used to perform assays for lead.
Aptamer technology may evolve to create a new class of
sensors with high specificity and enzymatic activity.
21.6. MULTIPLEXED MICROBEAD ARRAYS:
SUSPENSION ARRAYS
In Section 21.9 we will describe detection of DNA sequences
using two-dimensional arrays of capture oligomers