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.

Figure 1

(A) UV–vis spectra of N-3,3′-alkyl and tricationic dyes in the presence of 1 μM PNA–DNA (PD) oligomer duplex in pH 5 homopipes buffer, with dye final concentrations 9 μM. PNA used here is TB23, sequence shown in S1 in the Supporting Information. DiSC₂(3) alone in the same buffer is also depicted as no PD. (B) CD spectra of DiSC₂(3) in the presence of PNA, DNA, or PNA–DNA duplex, (left ordinate scale). The PNA used in the study is TB23, with its cDNA oligo. The mixture had final concentrations of 9 μM dye, 1 μM PNA, 1 μM DNA, or 1 μM PNA–DNA duplex. UV–vis spectra of dye with the same PNA–DNA duplex is included for comparison (right ordinate scale).

Figure 2

smartDNA assay run in a gel. See S1 (Supporting Information) for oligonucleotide and PNA sequences.

Figure 3

The initial (first 4 min) photobleaching rate of smartDNA reactions with 2 ng of DNA from M. tuberculosis (MTB) or from non-M. tuberculosis species in the presence and absence of M. tuberculosis specific PNA probe. The reaction was performed in 10 mM homopipes buffer, pH 5.0, with 0.05% Tween-80, and PNA probe used was TB19; the final concentration of PNA in the 50 μL reaction is 160 nM. The inlay shows the dose response curve of TB14 against M. tuberculosis genomic DNA. The photobleaching rate (milliabsorbance units/min) was calculated for each genomic DNA concentration and plotted.

Figure 4

Comparison of the photobleaching rate of the thiacarbocyanine dyes with substituents of ethyl, propyl, butyl, pentyl, and pyridinium, (corresponding to DiSC₂(3), DiSC₃(3), DiSC₄(3), DiSC₅(3), and DiSC_py(3), respectively). The label on the X axis shows the amount of M. tuberculosis (MTB) genomic DNA in each reaction. The PNA probe used here is TB14, sequence shown in S1. The final concentration of PNA in the 50 μL reaction is 160 nM. All dyes had a final concentration of 9 μM. The buffer is 10 mM homopipes, pH 5.0, 0.05% Tween 80.

Figure 5

Proposed mechanism of accelerated photobleaching based on the generation of singlet oxygen.

Chart 1

Chemical Structures of the 3,3′-Dialkylthiacarbocyanine Dyes Used in the Study^{a a} (A) n = 1 for DiSC₂(3), 3,3′-diethylthiadicarbocyanine, n = 2 for DiSC₃(3) 3,3′-dipropylthiadicarbocyanine, n = 3, for DiSC₄(3) 3,3′-dibutylthiadicarbocyanine, n = 4 for DiSC₅(3) 3,3′-dipentylthiadicarbocyanine; (B) tricationic thiacarbocyanine (3,3′-di(3-propylpyridinium)thiacarbocyanine) DiSC_py(3); and (C) structure of a photooxygenation product.