Fluorescence Reporting Based on FRET Between Conjugated Polyelectrolyte and Organic Dye for Biosensor Applications - Advanced Fluorescence Reporters in Chemistry and Biology - page 427

When the target DNA is complementary to the PNA probe, the DNA/PNA duplexes
remain after S1 nuclease digestion, which allows TO intercalation to give fluores-
cence. Addition of CCP to this solution results in enhanced TO emission due to
FRET from 1Br to TO. In contrast, in the presence of even one base mismatched
DNA or dsDNA molecules, S1 nuclease can effectively digest the DNA strands into
small fragments and thus no dye intercalation occurs, leading to very weak dye
emission upon excitation of 1Br. This method allows visual detection of target
DNA with a detection limit of 5

M according to the fluorescence color of the
solution, which paves the way for the future exploration of real-time instrument-
free SNP diagnosis.
DNA hybridization assays in heterogeneous formats have become increasingly
common and powerful concomitant with the advancement of gene chip and DNA
microarray technologies [ 60 ]. These systems allow for high-throughput screening
of hundreds to thousands of genes in a single experiment [ 61 ]. Scheme 3 thus was
applied in the solid state using a yellow light-emitting CCP (2) with two absorption
bands centered at 330 and 444 nm as the light-harvesting donor [ 62 ]. This polymer
can be excited at 488 nm and thereby is compatible with the excitation source used
in commercial DNA microarray readers. The assay scheme was carried out with the
PNA-modified surfaces (PNA 2 :NH 2 -O-O-TCC ACG GCA TCT CA) which were
treated with Cy5-labeled complementary ssDNA (ssDNA 6 -Cy5: 5 0 -Cy5-TGA GAT
GCC GTG GA-3 0 ) and Cy5-labeled noncomplementary ssDNA (ssDNA 7 -Cy5:
5 0 -Cy5-ATC TTG ACT GTG TGG GTG CT-3 0 ), respectively. Treatment of the
PNA-containing surface with complementary ssDNA results in an increase of the
surface-negative charge, allowing 2 to bind to the surface. Excitation of the
polymer thus induces FRET to the reporter dye, leading to strong Cy5 emission.
In contrast, in the presence of the noncomplementary ssDNA, the reporter dye is not
incorporated onto the surface, ultimately giving rise to neglectable Cy5 emission. In
addition to this electrostatic interaction-based strategy, Kim's group has reported a
kind of DNA-chips fabricated by DNA synthesis on a thin film of organosoluble
CPs [ 63 ]. In these chips, the CP thin film serves as a signal amplifying surface to
enhance the fluorescence of dye-labeled ssDNA. This method has been proven
equally effective for the label-free DNA assay using SYBR green I as the inter-
calating dye [ 64 ].
Similar to the use of dye-labeled PNA or DNA as a signaling probe, dye-labeled
RNA has also been used to study the interaction between tectoRNA molecules [ 65 ].
In addition, specific RNA/peptide interaction has been utilized for HIV-1 study
[ 66 ]. The concept has been illustrated using the binding of the transactivator (Tat)
peptide to the transactivation responsive element RNA sequence (TAR RNA) of
HIV-1. In one study, the RNA and protein chosen were the HIV-1 mRNA fragment,
TAR RNA, and its specific Tat polypeptide [ 66 ]. The bulge structure in TAR RNA
is essential for Tat binding. A nonspecific polypeptide sequence labeled with Fl at
the N-terminus (SH3-Fl*) and a three-base mutation dTAR RNA were also chosen
for comparison. When 1I serves as the donor, intense Fl emission is only observed
for Tat-Fl*/TAR RNA pair, indicating good selectivity of the assay for RNA
detection.

m