Fluorescence Reporting Based on FRET Between Conjugated Polyelectrolyte and Organic Dye for Biosensor Applications - Advanced Fluorescence Reporters in Chemistry and Biology - page 423

Fig. 1 (A) Absorption (( a ) green and ( c ) orange ) and emission (( b ) blue and ( d ) red ) spectra of
CCP 1I and PNA 1 -Fl, respectively. Fluorescence was measured by exciting at 380 and 480 nm, for
1I and PNA 1 -Fl, respectively. (B) PL spectra of PNA-C* in the presence of complementary (( a )
red ) and noncomplementary (( b ) black ) DNA by excitation of CCP 1I. Conditions are in water at
pH ¼ 5.5. The spectra are normalized with respect to the emission of CCP 1I [ 43 ]

the presence of organic solvent weakens hydrophobic interactions between PNA 1 -
F1 and CCP, consequently reducing Fl emission by a factor of 3 in the presence of
ssDNA 2 , making the signal almost undetectable with a standard fluorometer.
Scheme 3 was also extended for single nucleotide polymorphisms (SNPs)
detection with the aid of S1 nuclease enzyme by Bazan's group in 2005 [ 50 ]. The
detection relies on the fact that PNA molecules are inherently resistant to nucleases
and proteases, and PNA can protect the complementary DNA from S1 nuclease
digestion. In this case, S1 nuclease could digest ssDNA, overhanging ssDNA
segments that extend beyond the region of PNA/DNA duplex, and any unbound
mismatches within a duplex. The targeted SNP was responsible for an arginine to
tryptophan substitution at amino acid position 406 in the microtubule-associated
protein tau. The mutant was linked to frontotemporal dementia. The probe strand
was PNA 2 -F1 (5 0 -Fl-OO-TCC ACG GCA TCT CA-EE-3 0 ) that targets the R406W
mutation. The detection was carried out by the addition of CCP 1Br into the
solution of the PNA/DNA solutions after enzymatic reaction as shown in Scheme 4 .
Excitation of 1Br at 380 led to the selective FRET for only the perfectly comple-
mentary pair, while no obvious FRET occurred for samples containing even one
mismatch (R406W mutation) because of the loss of electrostatic attraction afforded
by the negatively charged DNA target after digestion. This method allows for
straightforward discrimination of strand-specific SNP sites on short and long
DNA target sequences.
Scheme 3 was subsequently modified to use chromophore-labeled ssDNA
(ssDNA-C*) as the signaling probe, instead of PNA-C*, considering that ssDNA-C*
is more common and cost-effective than PNA-C* [ 51 ]. In this case, ssDNA 3 -F1