Principles of Fluorescence Spectroscopy

718 DNA TECHNOLOGY
Figure 21.25. Pyrene-labeled oligonucleotide probes. Modified from
[96].
hybridization to be determined using wavelength-ratiometric
measurements.
21.3.2. DNA Hybridization Measured by
Excimer Formation
DNA hybridization can also be detected by pyrene excimer
formation. 94–96 DNA probes were synthesized with pyrene
attached to the 5' and 3' ends (Figure 21.25). It is well
known that one excited pyrene molecule can form an excit-
Figure 21.26. Principle of the excimer-forming DNA hybridization
array. Modified from [96].
ed-state complex with another ground-state pyrene, forming
an excimer. This complex displays an unstructured
emission near 500 nm as compared to the structured emission
of pyrene monomer near 400 nm. The use of excimer
formation to detect DNA hybridization is shown in Figure
21.26. The assay requires two DNA probes that bind to
adjacent sequences on the target DNA. In this example the
correct target DNA is a 32-mer oligonucleotide. If both the
5'- and 3'-pyrene probe bind to target DNA, the pyrene
Figure 21.27. Effect of target DNA (32-mer) and mismatched target DNA (33- and 34-mer) on the emission from DNA probes labeled with pyrene
at the 3' and 5' ends. The target DNA 32-mer and the mismatched target DNA have the sequence 5'-AGAGGGCACGGATACC*GCGAGGTGGAGC-
GAAT-3', where the asterisk denotes the location of one or two extra thymine residues in the 33- and 34-mer, respectively. Modified from [96].