Specific detection of topoisomerase I from the malaria causing P. falciparum parasite using isothermal rolling circle amplification.

We present a Rolling-Circle-Enhance-Enzyme-Activity-Detection (REEAD) system with potential use for future point-of-care diagnosis of malaria. In the developed setup, specific detection of malaria parasites in crude blood samples is facilitated by the conversion of single Plasmodium falciparum topoisomerase I (pfTopI) mediated cleavage-ligation events, happening within nanometer dimensions, to micrometer-sized products readily detectable at the single molecule level in a fluorescence microscope. In principle, REEAD requires no special equipment and the readout is adaptable to simple colorimetric detection systems.

Microfluidics-mediated isothermal detection of enzyme activity at the single molecule level.

Conventional analysis of enzymatic activity, often carried out on pools of cells, is blind to heterogeneity in the population. Here, we combine microfluidics with a previously developed isothermal rolling circle amplification-based assay to investigate multiple enzymatic activities in down to single cells. This microfluidics-meditated assay performs at very high sensitivity in picoliter incubators with small quantities of biological materials.

Peptide Inhibition of Topoisomerase IB from Plasmodium falciparum.

Control of diseases inflicted by protozoan parasites such as Leishmania, Trypanosoma, and Plasmodium, which pose a serious threat to human health worldwide, depends on a rather small number of antiparasite drugs, of which many are toxic and/or inefficient. Moreover, the increasing occurrence of drug-resistant parasites emphasizes the need for new and effective antiprotozoan drugs. In the current study, we describe a synthetic peptide, WRWYCRCK, with inhibitory effect on the essential enzyme topoisomerase I from the malaria-causing parasite Plasmodium falciparum.

Detection of single enzymatic events in rare or single cells using microfluidics.

In the present study we demonstrate highly sensitive detection of rare, aberrant cells in a population of wild-type human cells by combining a rolling-circle-enhanced enzyme activity single-molecule detection assay with a custom-designed microfluidic device. Besides reliable detection of low concentrations of aberrant cells, the integrated system allowed multiplexed detection of individual enzymatic events at the single cell level. The single cell sensitivity of the presented setup relies on the combination of single-molecule rolling-circle-enhanced enzyme activity detection with the fast reaction kinetics provided by a picoliter droplet reaction volume and subsequent concentration of signals in a customized drop-trap device.

Strategies for highly sensitive biomarker detection by Rolling Circle Amplification of signals from nucleic acid composed sensors.

The use of nucleic acids as components in highly sensitive biosensors has attracted increasing interest during recent years, not least due to the ease by which nucleic acids can be synthesized, manipulated and signal-amplified. To date, several enzymatic reactions, including Polymerase Chain Reaction, Rolling Circle- and Strand Displacement Amplification for signal enhancement of nucleic acid biosensors, have been presented. Of these, the isothermal Rolling Circle Amplification, in which a small single-stranded DNA circle is replicated to generate long tandem repeat products, presents the advantage of allowing single molecule detection and easy quantification as well as of posing little requirement to equipment and personnel training.

Variant Plasmodium ovale isolated from a patient infected in Ghana.

Recent data have found that Plasmodium ovale can be separated in two distinct species: classic and variant P. ovale based on multilocus typing of different genes. This study presents a P.

Topoisomerase I deficiency results in chromosomal alterations in cervical cancer cells.

Human topoisomerase I has been suggested to be implicated in the maintenance of genomic stability via its ability to regulate genome topology during transcription and replication. In the present study, we demonstrate by whole-genome array comparative genomic hybridization (aCGH) and fluorescence in situ hybridisation (FISH) analysis that topoisomerase I deficiency results in chromosome 5p gain in the cervical cancer cell line, HeLa-CCL2. In contrast, chromosome 5p copy number remained unaffected by topoisomerase I down-regulation in the non-cancer cell line HEK293T, as demonstrated by FISH analysis.

A novel secondary DNA binding site in human topoisomerase I unravelled by using a 2D DNA origami platform.

The biologically and clinically important nuclear enzyme human topoisomerase I relaxes both positively and negatively supercoiled DNA and binds consequently DNA with supercoils of positive or negative sign with a strong preference over relaxed DNA. One scheme to explain this preference relies on the existence of a secondary DNA binding site in the enzyme facilitating binding to DNA nodes characteristic for plectonemic DNA. Here we demonstrate the ability of human topoisomerase I to induce formation of DNA synapses at protein containing nodes or filaments using atomic force microscopy imaging.

Structure of nanoscale truncated octahedral DNA cages: variation of single-stranded linker regions and influence on assembly yields.

The assembly, structure, and stability of DNA nanocages with the shape of truncated octahedra have been studied. The cages are composed of 12 double-stranded B-DNA helices interrupted by single-stranded linkers of thymidines of varying length that constitute the truncated corners of the structure. The structures assemble with a high efficiency in a one-step procedure, compared to previously published structures of similar complexity.

Multiplexed detection of site specific recombinase and DNA topoisomerase activities at the single molecule level.

We previously demonstrated the conversion of a single human topoisomerase I mediated DNA cleavage-ligation event happening within nanometer dimensions to a micrometer-sized DNA molecule, readily detectable using standard fluorescence microscopy. This conversion was achieved by topoisomerase I mediated closure of a nicked DNA circle followed by rolling circle amplification leading to an anchored product that was visualized at the single molecule level by hybridization to fluorescently labeled probes (Stougaard et al. ACS Nano 2009, 3, 223-33).

Single-molecule detection of human topoisomerase I cleavage-ligation activity.

In the present study, we demonstrate the conversion of a single human topoisomerase I mediated DNA cleavage-ligation event happening within nanometer dimensions to a micrometer-sized DNA molecule, readily detectable using standard fluorescence microscopy. This conversion is achieved by topoisomerase I mediated closure of a nicked DNA dumbbell structure, followed by rolling circle amplification. The resulting product consists of multiple tandem repeats of the DNA dumbbell and can subsequently be visualized by annealing to fluorescently labeled probes.

Identification of a minimal functional linker in human topoisomerase I by domain swapping with Cre recombinase.

Cellular forms of type IB topoisomerases distinguish themselves from their viral counterparts and the tyrosine recombinases to which they are closely related by having rather extensive N-terminal and linker domains. The functions and necessity of these domains are not yet fully unraveled. In this study we replace 86 amino acids including the linker domain of the cellular type IB topoisomerase, human topoisomerase I, with four, six, or eight amino acids from the corresponding short loop region in Cre recombinase.

Assembly and structural analysis of a covalently closed nano-scale DNA cage.

The inherent properties of DNA as a stable polymer with unique affinity for partner molecules determined by the specific Watson-Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA-interacting enzymes. More recently, strategies for synthesis of more complex two-dimensional (2D) and 3D DNA structures have emerged.

Tryptophane-205 of human topoisomerase I is essential for camptothecin inhibition of negative but not positive supercoil removal.

Positive supercoils are introduced in cellular DNA in front of and negative supercoils behind tracking polymerases. Since DNA purified from cells is normally under-wound, most studies addressing the relaxation activity of topoisomerase I have utilized negatively supercoiled plasmids. The present report compares the relaxation activity of human topoisomerase I variants on plasmids containing equal numbers of superhelical twists with opposite handedness.

Resolution of Holliday junction substrates by human topoisomerase I.

Prompted by the close relationship between tyrosine recombinases and type IB topoisomerases we have investigated the ability of human topoisomerase I to resolve the typical intermediate of recombinase catalysis, the Holliday junction. We demonstrate that human topoisomerase I catalyzes unidirectional resolution of a synthetic Holliday junction substrate containing two preferred cleavage sites surrounded by DNA sequences supporting branch migration. Deleting part of the N-terminal domain (amino acid residues 1-202) did not affect topoisomerase I resolution activity, whereas a topoisomerase I variant lacking both the N-terminal domain and amino acid residues 660-688 of the linker domain was unable to resolve the Holliday junction substrate.

Locus control region elements HS2 and HS3 in combination with chromatin boundaries confer high-level expression of a human beta-globin transgene in a centromeric region.

Expression constructs are subject to position-effects in transgenic assays unless they harbour elements that protect them from negative or positive influences exerted by chromatin at the site of integration. Locus control regions (LCRs) and boundary elements are able to protect from position effects by preventing heterochromatization of linked genes. The LCR in the human beta-globin gene locus is located far upstream of the genes and composed of several erythroid specific DNase I hypersensitive (HS) sites.

Regions within the N-terminal domain of human topoisomerase I exert important functions during strand rotation and DNA binding.

The human topoisomerase I N-terminal domain is the only part of the enzyme still not crystallized and the function of this domain remains enigmatical. In the present study, we have addressed the specific functions of individual N-terminal regions of topoisomerase I by characterizing mutants lacking amino acid residues 1-202 or 191-206 or having tryptophane-205 substituted by glycine in a broad variety of in vitro activity assays. As a result of these investigations we find that mutants altered in the region 191-206 distinguished themselves from the wild-type enzyme by a faster strand rotation step, insensitivity towards the anti-cancer drug camptothecin in relaxation and the inability to ligate blunt end DNA fragments.

Recombinogenic flap ligation mediated by human topoisomerase I.

Aberration of eukaryotic topoisomerase I catalysis leads to potentially recombinogenic pathways by allowing the joining of heterologous DNA strands. Recently, a new ligation pathway (flap ligation) was presented for vaccinia virus topoisomerase I, in which blunt end cleavage complexes ligate the recessed end of duplex acceptors having a single-stranded 3'-tail. This reaction was suggested to play an important role in the repair of topoisomerase I-induced DNA double-strand breaks.

The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation.

Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis.