- fluorogenic, intercalating acridine-tetrazine
- straightforward "mix and measure" assay of DNA synthesis
- synergistic, combinatorial chemotherapeutic approach
This invention describes a new, fluorogenic intercalating agent "PINK" containing a tetrazine unit that selectively reacts with vinyl (H2C=CH-R) groups in nucleic acids to give a highly fluorescent product. Employing a dual enhancement approach, the templated reaction on nucleic acids proceeds with a 60,000-fold kinetic and 71-fold fluorescent increase. The PINK-vinyl reaction product exhibits potent anti-proliferative activities across multiple cancer cell lines. The combined modalities of fluorescent product formation and anti-proliferative activities make PINK a new type of "theranostic" agent.
The acridine-tetrazine conjugate “PINK” constitutes a novel fluorogenic intercalator that exhibits increased fluorescence upon covalently reacting with nucleic acids. Making use of a dual enhancement strategy, the templated reaction on nucleic acids proceeds with a 60,000-fold kinetic and 71-fold fluorescent increase. PINK thus overcomes the limitations of conventional fluorescent tetrazine probes, where the tight packing of cellular chromatin prevents an efficient bioorthogonal reaction. PINK allows for dynamic, long-term experiments in cell culture and enables the first “mix and measure” fluorogenic assay of DNA replication and synthesis in living cells. In addition, we utilized the combination of PINK and 5-vinyl-2′-deoxyuridine (VDU) for a novel combinatorial, chemotherapeutic approach; while both compounds are relatively non-toxic on their own, antiproliferative activity in multiple eukaryotic cell lines is exerted in response to the bioorthogonal reaction between them.
Our invention provides a highly promising diagnostic tool, which allows operationally simple cell cycle analysis by flow cytometry or microscopy. In addition, it constitutes a versatile probe for researchers in the field of nucleic acids. Possible applications include elucidation of DNA repair and replication mechanisms by single-molecule nanoscopy, studying and tracking nucleic acids in whole animal models, or studying mechanisms of viral entry of replication. In addition, a combination of PINK and vinyl-nucleosides constitutes a novel, combinatorial approach, that can be developed into cell-type selective chemotherapy.
Name: Timothy Corkery