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A 'light-up' intercalator displacement assay for detection of triplex DNA stabilizers.

ABSTRACT: Here, we developed a coralyne-based, 'light-up' intercalator displacement assay to identify molecular stabilizers of triplex DNA using a sequence from a chromosomal breakpoint hotspot in the human c-MYC oncogene. Its potential to identify triplex DNA ligands was demonstrated using BePI and doxorubicin. Identification of triplex-interacting ligands may allow the regulation of genetic instability in human genomes.

SUBMITTER: Del Mundo IMA

PROVIDER: S-EPMC7323859 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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A 'light-up' intercalator displacement assay for detection of triplex DNA stabilizers.

Del Mundo Imee M A IMA Cho Eun Jeong EJ Dalby Kevin N KN Vasquez Karen M KM

Chemical communications (Cambridge, England) 20200201 13

Here, we developed a coralyne-based, 'light-up' intercalator displacement assay to identify molecular stabilizers of triplex DNA using a sequence from a chromosomal breakpoint hotspot in the human c-MYC oncogene. Its potential to identify triplex DNA ligands was demonstrated using BePI and doxorubicin. Identification of triplex-interacting ligands may allow the regulation of genetic instability in human genomes. ...[more]

PMID: 31960843

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Project description:Thermodynamic studies on the interactions between intercalator-neomycin conjugates and a DNA polynucleotide triplex [poly(dA).2poly(dT)] were conducted. To draw a complete picture of such interactions, naphthalene diimide-neomycin (3) and anthraquinone-neomycin (4) conjugates were synthesized and used together with two other analogues, previously synthesized pyrene-neomycin (1) and BQQ-neomycin (2) conjugates, in our investigations. A combination of experiments, including UV denaturation, circular dichroism (CD) titration, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC), revealed that all four conjugates (1-4) stabilized poly(dA).2poly(dT) much more than its parent compound, neomycin. UV melting experiments clearly showed that the temperature (T(m3-->2)) at which poly(dA).2poly(dT) dissociated into poly(dA).poly(dT) and poly(dT) increased dramatically (>12 degrees C) in the presence of intercalator-neomycin conjugates (1-4) even at a very low concentration (2 muM). In contrast to intercalator-neomycin conjugates, the increment of T(m3-->2) of poly(dA).2poly(dT) induced by neomycin was negligible under the same conditions. The binding preference of intercalator-neomycin conjugates (1-4) to poly(dA).2poly(dT) was also confirmed by competition dialysis and a fluorescent intercalator displacement assay. Circular dichroism titration studies revealed that compounds 1-4 had slightly larger binding site size ( approximately 7-7.5) with poly(dA).2poly(dT) as compared to neomycin ( approximately 6.5). The thermodynamic parameters of these intercalator-neomycin conjugates with poly(dA).2poly(dT) were derived from an integrated van't Hoff equation using the T(m3-->2) values, the binding site size numbers, and other parameters obtained from DSC and ITC. The binding affinity of all tested ligands with poly(dA).2poly(dT) increased in the following order: neomycin < 1 < 3 < 4 < 2. Among them, the binding constant [(2.7 +/- 0.3) x 10(8) M(-1)] of 2 with poly(dA).2poly(dT) was the highest, almost 1000-fold greater than that of neomycin. The binding of compounds 1-4 with poly(dA).2poly(dT) was mostly enthalpy-driven and gave negative DeltaC(p) values. The results described here suggest that the binding affinity of intercalator-neomycin conjugates for poly(dA).2poly(dT) increases as a function of the surface area of the intercalator moiety.

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A 'light-up' intercalator displacement assay for detection of triplex DNA stabilizers.

Publications

A 'light-up' intercalator displacement assay for detection of triplex DNA stabilizers.

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