Accelerated Photobleaching of a Cyanine Dye in the Presence of a Ternary Target DNA, PNA Probe, Dye Catalytic Complex: A Molecular Diagnostic

Abstract

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.Photobleaching of cyanine dyes is usually seen as an undesirable characteristic which hampers their use in nucleic acid detection. However, we have discovered that in the presence of a target specific peptide nucleic acid (PNA) oligomer probe, the rate of 3,3′-diethylthiacarbocyanine iodide (DiSC 2 (3)) photobleaching is directly related to the amount of target DNA present. We call this type of reaction "smartDNA" and this rapid color loss can be used as a sensitive indicator for the presence of a specific DNA sequence.PNAs, which are used in the current assay as hybridization probes, are oligonucleotide analogues where the negatively charged phosphoribose backbone has been replaced with a neutral N-(2-aminoethyl) glycine group. 1,2 The absence of the negatively charged backbone gives PNAs unique physiochemical properties for binding to nucleic acid targets. PNAs rapidly *Corresponding

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