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DNA topoisomerases are key enzyme responsible for modulating the topological state of the DNA by breaking and rejoining of DNA strand. Characterization of a Gly717Asp mutation in the human topoisomerase was performed using several catalytic assays. The mutant enzyme was shown to have comparable cleavage and fast religation rate as compared to the wild-type protein. Addition of the anticancer drug camptothecin significantly reduced the religation step. The simulative approaches and analysis of the cleavage/religation equilibrium indicate that the mutation is able to modify the architecture of the drug binding site, increasing the persistence of the drug for the enzyme-DNA covalent complex. Taken together these results indicate that the structure modification of the drug binding site is the key reason for the increasing CPT persistence and furthermore provide the possibility for new anti-cancer drug discovery., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Selective targeting is a crucial property of nanocarriers used for drug delivery in cancer therapy. We generated biotinylated octahedral DNA nanocages functionalized with folic acid through bio-orthogonal conjugation chemistry. Molecular modelling indicated that a distance of about 2.5 nm between folic acid and DNA nanocage avoids steric hindrance with the folate receptor. HeLa cells, a folate receptor positive tumour cell line, internalize folate-DNA nanocages with efficiency greater than 40 times compared to cells not expressing the folate receptors. Functionalized DNA nanocages are highly stable, not cytotoxic and can be efficiently loaded with the chemotherapeutic agent doxorubicin. After entry into cells, doxorubicin-loaded nanoparticles are confined in vesicular structures, indicating that DNA nanocages traffic through the endocytic pathway. Doxorubicin release from loaded DNA cages, facilitated by low pH of endocytic vesicles, induces toxic pathways that, besides selectively killing folate receptor-positive cancer cells, leads to cage degradation avoiding nanoparticles accumulation inside cells., (Copyright © 2018. Published by Elsevier Inc.)

Human topoisomerase 1B is a ubiquitous and essential enzyme involved in relaxing the topological state of supercoiled DNA to allow the progression of fundamental DNA metabolism. Its enzymatic catalytic cycle consists of cleavage and religation reaction. A ternary fluorescence resonance energy transfer biosensor based on a suicide DNA substrate conjugated with three fluorophores has been developed to monitor both cleavage and religation Topoisomerase I catalytic function. The presence of fluorophores does not alter the specificity of the enzyme catalysis on the DNA substrate. The enzyme-mediated reaction can be tracked in real-time by simple fluorescence measurement, avoiding the use of risky radioactive substrate labeling and time-consuming denaturing gel electrophoresis. The method is applied to monitor the perturbation brought by single mutation on the cleavage or religation reaction and to screen the effect of the camptothecin anticancer drug monitoring the energy transfer decrease during religation reaction. Pathological mutations usually affect only the cleavage or the religation reaction and the proposed approach represent a fast protocol for assessing chemotherapeutic drug efficacy and analyzing mutant's properties., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A high-sensitivity assay has been developed for the detection of human topoisomerase I with single molecule resolution. The method uses magnetic sepharose beads to concentrate rolling circle products, produced by the amplification of DNA molecules circularized by topoisomerase I and detectable with a confocal microscope as single and discrete dots, once reacted with fluorescent probes. Each dot, corresponding to a single cleavage-religation event mediated by the enzyme, can be counted due to its high signal/noise ratio, allowing detection of 0.3pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples., (Copyright © 2014 Elsevier Inc. All rights reserved.)