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 parasite Plasmodium falciparum in 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.

Interlinked DNA nano-circles for measuring topoisomerase II activity at the level of single decatenation events

“…This suggests that unlinking of the B-circle and nicking support RCA as expected from the assay outline depicted in Figure 1B . The data also illustrate that the two readout methods can be used to detect the results form a single reaction mixture in either a quick convenient (fluorometric readout ( 20 , 64 )) or a highly sensitive (microscopic readout ( 59 , 66 )) manner. Note that in the sample with only Nt.BbvCI added, a small but consistent increase in signal compared to the sample with only TADA-complex was observed (compare columns 3 and 2 in Figures 2E and F ).…”

“…The samples were placed in the qPCR machine (Mx3000P, Agilent Technologies, Inc.) and incubated at 30°C for up to 4 h. The samples were analyzed by measuring FAM fluorescence emission over time (every 1 min) essentially as described in (59). Data from the run were processed in Microsoft Excel and unless stated otherwise, increases in fluorescence over time (as an estimate of reaction rate) were calculated from raw data generated between 80–120 min.…”

“…7.5 μl of samples containing released and nicked B-circle, prepared as described above, were moved to quantitative PCR (qPCR) tubes and the volume adjusted to 10 μl of 1× RCA buffer (33 mM Tris-acetate, pH 7.9, 10 mM MgCl 2 , 66 mM KCl, 1% (v/v) Tween 20, and 10 mM DTT) with 1 mM dNTP, 0.1 μg/μl BSA and 1 μM of the ID33 molecular beacon before Phi29 polymerase (1.5 nM, final concentration) was added. The samples were placed in the qPCR machine (Mx3000P, Agilent Technologies, Inc.) and incubated at 30°C for up to 4 h. The samples were analyzed by measuring FAM fluorescence emission over time (every 1 min) essentially as described in ( 59 ). Data from the run were processed in Microsoft Excel and unless stated otherwise, increases in fluorescence over time (as an estimate of reaction rate) were calculated from raw data generated between 80–120 min.…”

“…Then, the volume was adjusted to 10 μl of 1xRCA buffer (33 mM Tris-acetate, pH 7.9, 10 mM MgCl 2 , 66 mM KCl, 1% (v/v) Tween 20 and 10 mM DTT) with 1 mM dNTP, 0.1 μg/μl BSA and 1 μM of the ID33 and ID16 molecular beacons with a FAM and CAL-Fluor-Red-590 (TAMRA analog) fluorophore, respectively, before Phi29 polymerase (1.5 nM, final concentration) was added to generate RCPs. The RCPs were hybridized to capture oligonucleotide printed Epoxy slides as described before ( 30 , 59 ). Finally, the slides were mounted with 2.5 μl Vectashield (Vector Laboratories) and a cover glass before analyses using the 63× objective of an Olympus IX73 fluorescence microscope.…”

Rolling Circle RNA Synthesis Catalysed by RNA

“…In biology, both viroids and Hepatitis D virus (HDV) replication proceeds through RCS on circular RNA genomes mediated by proteinaceous RNA polymerases but RCS has also been reported for circular DNA templates and proteinaceous DNA polymerases in nature (Wawrzyniak et al, 2017) and in biotechnology (Daubendiek et al, 1995; Givskov et al, 2016; Kristoffersen et al, 2017; Mohsen and Kool, 2016). dsDNA persistence length is somewhat shorter than dsRNA (dsDNA: 45-50 nm (140-50 nt) vs. dsRNA 60 nm (200 nt) and stacking interactions weaker than in dsRNA (Kebbekus et al, 1995; Svozil et al, 2010), therefore dsDNA may more readily adopt a circular shape or kinks to alleviate build-up of strain or to adopt strong bends (Wolters and Wittig, 1989), we would nevertheless expect the a similar strand displacement effect would play part.…”