Development of a diagnostic tool for the detection of the activity of the human topoisomerase IB

DNA topoisomerases are essential enzymes in the control and in the maintenance of the topological state of the DNA. One of the best studied topoisomerases is the human Topoisomerase IB (hTop1). This enzyme has gained great interest because it is the only target of the anti-cancer drugs of the camptothecin (CPT) family. HTop1 accomplishes its function by breaking and rejoining the DNA backbone to solve torsional stresses occurring during essential DNA metabolic processes, such as transcription and replication. The efficiency of CPT is dependent on the intracellular hTop1 activity. For this reason, the detection of hTop1 activity possesses a great clinical potential in the cancer diagnostics and therapy. The standard assays for the detection of the activity of hTop1 do not permit to measure the activity of low concentration of enzyme, such as in clinical samples. In this thesis, two different novel techniques to detect the activity of hTop1 at very low concentration are presented. First, we improved the Rolling Circle Amplification (RCA)-based assay, using magnetic sepharose beads in order to concentrate the rolling circle products, produced by the amplification of DNA substrates circularized by hTop1 and detected by annealing with fluorescent probes. The sensitivity of the assay was considerably increased, with a detection limit of 0.3 pM hTop1. Second, a graphene-based biosensor was developed. The DNA substrates were designed to decouple the cleavage and the religation steps. The activity of 300 pM hTop1 can be detected by measuring electrically the sensor response. This method is label-free, fast and permit to measure the activity of hTop1 in real-time.