Fast and easy colorimetric tests for single mismatch recognition by PNA–DNA duplexes with the diethylthiadicarbocyanine dye and succinyl-β-cyclodextrin

Abstract

The 3,3′-diethylthiacarbocyanine (DiSC 2 (5)) dye is able to aggregate on full matched PNA-DNA duplexes by changing its absorption properties, which are manifested by an instantaneous colour shift from blue to purple. However the spontaneous aggregation of the dye also on mismatched duplexes and even on free PNA strands makes the test quite aspecific. Here it is demonstrated that the addition of succinyl-β-cyclodextrin (Succ-β-CyD) to the solutions containing PNA-DNA duplexes and the dye strongly enhances the specificity of the colour shift, allowing for a fast, very specific and extremely sensitive visual recognition of mismatches in DNA strands by using PNA probes in combination with the DiSC 2 (5) dye. The phenomenon has been studied by CD and NMR spectroscopies. The method has been optimized and preliminarily applied for the recognition of an apoE gene mutation in human DNA samples.

Food analysis and food authentication by peptide nucleic acid (PNA)-based technologies

“…This feature, which could potentially lead to the development of colorimetric tests for directly visualizing the specific PNA-DNA interaction, is nevertheless made less specific from the tendency of the DiSC 2 (5) dye to aggregate even on free PNA molecules, introducing a strong bias to the colorimetric analysis which can lead to many false positives, especially when excess PNA is to be used, as it is frequent in the case of post-PCR confirmative analyses. 40 However, when observed by spectropolarimetry, the dye aggregate on the PNA-DNA duplex gives rise to a very strong exciton coupling effect at the same wavelength, which could be easily visualized by CD, since the helical chirality of the duplex is transferred to the dye aggregate. The dye being achiral, the free dye in solution does not give rise to any CD signal and, what is most interesting for the robustness of the method, even the dye-free PNA aggregate is ''invisible'' when observed by the CD technique, standard PNAs also being non-chiral molecules.…”

Colorimetric Sensing by Using Allosteric‐DNAzyme‐Coupled Rolling Circle Amplification and a Peptide Nucleic Acid–Organic Dye Probe

“…To alleviate this problem, we measured the absorbance of the dye with the RCA product/PNA duplex in the presence of succinyl-b-cyclodextrin (Succ-b-CyD). [18] This reagent has been shown to interrupt the binding of the dye to PNA alone without interfering with its ability to bind to the RCA product/PNA duplex. [18] Therefore, the ratio of the absorbance of the dye at 535 nm to that at 647 nm can be related directly to the amount of RCA product produced at different concentrations of ATP.…”

“…[18] This reagent has been shown to interrupt the binding of the dye to PNA alone without interfering with its ability to bind to the RCA product/PNA duplex. [18] Therefore, the ratio of the absorbance of the dye at 535 nm to that at 647 nm can be related directly to the amount of RCA product produced at different concentrations of ATP. Indeed, the CR profile of the RCA reaction mixtures at tested concentrations of ATP (Figure 4 c) reflects the increasing intensity of the purple color of relevant solutions (Figure 4 b).…”

Colorimetric Sensing by Using Allosteric‐DNAzyme‐Coupled Rolling Circle Amplification and a Peptide Nucleic Acid–Organic Dye Probe

“…As mentioned earlier, a long incubation of DiSC2 with PNA alone can produce a purple color. To alleviate this problem, we measured the absorbance of the dye with the RCA product/PNA duplex in the presence of succinyl‐β‐cyclodextrin (Succ‐β‐CyD) 18. This reagent has been shown to interrupt the binding of the dye to PNA alone without interfering with its ability to bind to the RCA product/PNA duplex 18.…”

“…To alleviate this problem, we measured the absorbance of the dye with the RCA product/PNA duplex in the presence of succinyl‐β‐cyclodextrin (Succ‐β‐CyD) 18. This reagent has been shown to interrupt the binding of the dye to PNA alone without interfering with its ability to bind to the RCA product/PNA duplex 18. Therefore, the ratio of the absorbance of the dye at 535 nm to that at 647 nm can be related directly to the amount of RCA product produced at different concentrations of ATP.…”