title: Optimized Detection of Plasmodium falciparum Topoisomerase I Enzyme Activity in a Complex Biological Sample by the Use of Molecular Beacons.
abstract: The so-called Rolling Circle Amplification allows for amplification of circular DNA structures in a manner that can be detected in real-time using nucleotide-based molecular beacons that unfold upon recognition of the DNA product, which is being produced during the amplification process. The unfolding of the molecular beacons results in a fluorescence increase as the Rolling Circle Amplification proceeds. This can be measured in a fluorometer. In the current study, we have investigated the possibility of using two different molecular beacons to detect two distinct Rolling Circle Amplification reactions proceeding simultaneously and in the same reaction tube by measurement of fluorescence over time. We demonstrate the application of this fluorometric readout method, for automated and specific detection of the activity of the type IB topoisomerase from the malaria parasitePlasmodium falciparumin the presence of human cell extract containing the related topoisomerase I from humans. The obtained results point towards a future use of the presented assay setup for malaria diagnostics or drug screening purposes. In longer terms the method may be applied more broadly for real-time sensing of various Rolling Circle Amplification reactions.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark. asgergivskov@gmail.com.
authors.forename: Asger
authors.initials: A
authors.lastname: Givskov
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark. emillk@mbg.au.dk.
authors.forename: Emil L
authors.initials: EL
authors.lastname: Kristoffersen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark. kwp93@live.dk.
authors.forename: Kamilla
authors.initials: K
authors.lastname: Vandsø
authors.affiliation: Division of Biomedical Engineering, Department of Electronic Engineering, The Chinese University of Hong Kong, Shatin, New Territories 999077, Hong Kong, China. ypho@ee.cuhk.edu.hk.
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Ho
authors.affiliation: Department of Pathology, Aarhus University Hospital, 8000 Aarhus C, Denmark. magnus.stougaard@clin.au.dk.
authors.forename: Magnus
authors.initials: M
authors.lastname: Stougaard
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark. brk@mbg.au.dk.
authors.forename: Birgitta R
authors.initials: BR
authors.lastname: Knudsen
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D005470:Fluorometry; D006801:Humans; D008288:Malaria / Q000201:enzymology; D021141:Nucleic Acid Amplification Techniques; D010963:Plasmodium falciparum / Q000201:enzymology*
chemical_list: D004264:DNA Topoisomerases, Type I
keywords: malaria; multiplexed detection; real-time sensing; rolling circle amplification; topoisomerase I