Accelerated photobleaching of a cyanine dye in the presence of a ternary target DNA, PNA probe, dye catalytic complex: a molecular diagnostic.
In many settings, molecular testing is needed but unavailable due to complexity and cost. Simple, rapid, and specific DNA detection technologies would provide important alternatives to existing detection methods. Here we report a novel, rapid nucleic acid detection method based on the accelerated photobleaching of the light-sensitive cyanine dye, 3,3'-diethylthiacarbocyanine iodide (DiSC(2)(3) I(-)), in the presence of a target genomic DNA and a complementary peptide nucleic acid (PNA) probe. On the basis of the UV-vis, circular dichroism, and fluorescence spectra of DiSC(2)(3) with PNA-DNA oligomer duplexes and on characterization of a product of photolysis of DiSC(2)(3) I(-), a possible reaction mechanism is proposed. We propose that (1) a novel complex forms between dye, PNA, and DNA, (2) this complex functions as a photosensitizer producing (1)O(2), and (3) the (1)O(2) produced promotes photobleaching of dye molecules in the mixture. Similar cyanine dyes (DiSC(3)(3), DiSC(4)(3), DiSC(5)(3), and DiSC(py)(3)) interact with preformed PNA-DNA oligomer duplexes but do not demonstrate an equivalent accelerated photobleaching effect in the presence of PNA and target genomic DNA. The feasibility of developing molecular diagnostic assays based on the accelerated photobleaching (the smartDNA assay) that results from the novel complex formed between DiSC(2)(3) and PNA-DNA is under way.
Wang
M
M
Investigen Inc., Hercules, California, USA.
Holmes-Davis
R
R
Rafinski
Z
Z
Jedrzejewska
B
B
Choi
K Y
KY
Zwick
M
M
Bupp
C
C
Izmailov
A
A
Paczkowski
J
J
Warner
B
B
Koshinsky
H
H
D052160:Benzothiazoles / Q000737:chemistry*; D002232:Carbocyanines / Q000737:chemistry*; D002384:Catalysis; D002942:Circular Dichroism; D004396:Coloring Agents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D025202:Molecular Diagnostic Techniques; D015345:Oligonucleotide Probes / Q000737:chemistry*; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D038761:Photobleaching; D017422:Sequence Analysis, DNA / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D052160:Benzothiazoles; D002232:Carbocyanines; D004396:Coloring Agents; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D004247:DNA; C072030:3,3'-diethylthiacarbocyanine iodide