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Here we present a split-enzyme sensor approach for the sequence-specific detection of metal-based drug adducts of DNA. Split β-lactamase reporters were constructed using domain A of the High Mobility Group Box 1 protein (HMGB1a) in conjunction with zinc finger DNA-binding domains. As a proof of concept, the sensors were characterized with the well-known drug cisplatin, which forms 1,2-intrastrand crosslinks with DNA that are recognized by HMGB1a. After promising results with cisplatin, five ruthenium-based drugs were studied, four of which produced significant signal over background. These results highlight the utility of our approach for rapid screening of novel metal-based chemotherapeutic drug candidates and provide evidence that HMGB1a likely binds to DNA adducts formed by NAMI-A (imidazolium trans -tetrachlorodimethylsulfoxideimidazoleruthenate(III)), KP1019 (indazolium trans -tetrachlorodiindazoleruthenate(III)), KP418 (imidazolium trans -tetrachlorodiimidazoleruthenate(III)), and RAPTA-C (dichloro(η6- p -cymene)(1,3,5-triaza-7-phosphaadamantane)ruthenium(II)). These results thus imply a potential biologically relevant mode of action for the ruthenium-based drugs investigated herein. Split-enzyme sensors with the capacity to detect metal-based adducts of DNA have been developed. The sensors use a split β-lactamase reporter coupled to High Mobility Group Box 1 protein domain A (HMGB1a) partnered with a zinc finger DNA binding domain. Several dsDNA adducts of cisplatin and ruthenium-based drugs were investigated. [Display omitted] • Domain A of the High Mobility Group Box 1 protein (HMGB1a) binds to cisplatin-modified DNA. • Split protein sensors using HMGB1a can detect modification of DNA by cisplatin. • Sensors for cisplatin-modified DNA are modular and can detect various dsDNA targets. • Sensors that detect cisplatin-modified DNA also respond to DNA reacted with Ru drugs. • HMGB1a likely binds to DNA modified by some ruthenium-based drugs. [ABSTRACT FROM AUTHOR]

The ruthenium-based anticancer agent NAMI-A (ImH[ trans -RuCl 4 (dmso)(Im)], where Im = imidazole) has been shown to interact with RNA in vivo and in vitro. We hypothesized that the similarly structured drug KP1019 (IndH[ trans -RuCl 4 (Ind) 2 ], where Ind = indazole) binds to RNA as well. Fluorescence spectroscopy was employed to assay the interactions between either NAMI-A or KP1019 and tRNA Phe through an intrinsic fluorophore wybutosine (Y) base and by extrinsic displacement of the intercalating agent ethidium bromide. In both the intrinsic Y-base and extrinsic ethidium bromide studies, KP1019 exhibited tighter binding to phenylalanine-specific tRNA (tRNA Phe ) than NAMI-A. In the ethidium bromide study, reducing both drugs from Ru III to Ru II resulted in a significant decrease in binding. Our findings suggest that the relatively large heteroaromatic indazole ligands of KP1019 intercalate in the π-stacks of tRNA Phe within structurally complex binding pockets. In addition, NAMI-A appears to be sensitive to destabilizing electrostatic interactions with the negative phosphate backbone of tRNA Phe . Interactions with additional tRNA molecules and other types of RNA require further evaluation to determine the role of RNA in the mechanisms of action for KP1019 and to better understand how Ru drugs fundamentally interact with biomolecules that are more structurally sophisticated than short DNA oligonucleotides. To the best of our knowledge, this is the first study to report KP1019 binding interactions with RNA. [ABSTRACT FROM AUTHOR]