Search

Restriction endonucleases are expressed in all bacteria investigated so far and play an essential role for the bacterial defense against viral infections. Besides their important biological role, restriction endonucleases are of great use for different biotechnological purposes and are indispensable for many cloning and sequencing procedures. Methods for specific detection of restriction endonuclease activities can therefore find broad use for many purposes. In the current study, we demonstrate proof-of-concept for a new principle for the detection of restriction endonuclease activities. The method is based on rolling circle amplification of circular DNA products that can only be formed upon restriction digestion of specially designed DNA substrates. By combining the activity of the target restriction endonuclease with the highly specific Cre recombinase to generate DNA circles, we demonstrate specific detection of selected restriction endonuclease activities even in crude cell extracts. This is, to our knowledge, the first example of a sensor system that allows activity measurements of restriction endonucleases in crude samples. The presented sensor system may prove valuable for future characterization of bacteria species or strains based on their expression of restriction endonucleases as well as for quantification of restriction endonuclease activities directly in extracts from recombinant cells.

Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

This work presents the first reported imbibition mechanism of femtoliter (fL)-scale droplets produced by microchannel cantilever spotting (μCS) of DNA molecular inks into porous substrates (hydrophilic nylon). Differently from macroscopic or picoliter droplets, the downscaling to the fL-size leads to an imbibition process controlled by the subtle interplay of evaporation, spreading, viscosity, and capillarity, with gravitational forces being quasi-negligible. In particular, the minimization of droplet evaporation, surface tension, and viscosity allows for a reproducible droplet imbibition process. The dwell time on the nylon surface permits further tuning of the droplet lateral size, in accord with liquid ink diffusion mechanisms. The functionality of the printed DNA molecules is demonstrated at different imbibed oligonucleotide concentrations by hybridization with a fluorolabeled complementary sequence, resulting in a homogeneous coverage of DNA within the imbibed droplet. This study represents a first step toward the μCS-enabled fabrication of DNA-based biosensors and microarrays into porous substrates.

Malaria is among the major threats to global health with the main burden of disease being in rural areas of developing countries where accurate diagnosis based on non-invasive samples is in high demand. We here present a novel molecular assay for detection of malaria parasites based on technology that may be adapted for low-resource settings. Moreover, we demonstrate the exploitation of this assay for detection of malaria in saliva. The setup relies on pump-free microfluidics enabled extraction combined with a DNA sensor substrate that is converted to a single-stranded DNA circle specifically by topoisomerase I expressed by the malaria causing Plasmodium parasite. Subsequent rolling circle amplification of the generated DNA circle in the presence of biotin conjugated deoxynucleotides resulted in long tandem repeat products that was visualized colorimetrically upon binding of horse radish peroxidase (HRP) and addition of 3,3',5,5'-Tetramethylbenzidine that was converted to a blue colored product by HRP. The assay was directly quantitative, specific for Plasmodium parasites, and allowed detection of Plasmodium infection in a single drop of saliva from 35 out of 35 infected individuals tested. The results could be determined directly by the naked eye and documented by quantifying the color intensity using a standard paper scanner.

With the novel possibilities for detecting molecules of interest with extreme sensitivity also comes the risk of encountering hitherto negligible sources of error. In life science, such sources of error might be the broad variety of additives such as dithiothreitol (DTT) used to preserve enzyme stability during in vitro reactions. Using two different assays that can sense strand interruptions in double stranded DNA, we here show that DTT is able to introduce nicks in the DNA backbone. DTT was furthermore shown to facilitate the immobilization of fluorescent DNA on an NHS-ester functionalized glass surface. Such reactions may in particular impact the readout from single molecule detection studies and other ultrasensitive assays. This was highlighted by the finding that DTT markedly decreased the signal to noise ratio in a DNA sensor based assay with single molecule resolution.

In the current study we describe a novel DNA sensor system that allows the detection of single catalytic DNA integration events mediated by retrovirus encoded integrase (IN) present in viral particles. This is achieved by rolling circle amplification mediated conversion of enzymatic reactions happening within nanometer dimensions to directly detectable micrometer sized DNA products. The system utilizes the unique integration reaction of IN to generate a surface anchored nicked DNA circle that serves as a substrate for rolling circle amplification and allows for specific, quantitative and sensitive detection of purified recombinant IN or virus particles with a detection limit of less than 30 virus particles per μL of sample. Moreover, by modifying the nucleotide sequences of the utilized DNA it was possible to tailor the system to distinguish between the highly pathogenic lentivirus HIV and the gammaretrovirus murine leukemia virus present in a given sample. Infections with HIV remain a major threat to global health with more than 2 million new infections and 1 million deaths each year. The sensitive and specific detection of HIV particles based on IN activity holds promise for the development of a new type of diagnostic tools suitable for early (within hours of infection) detection of HIV, which would be valuable for prevention strategies as well as for efficient treatment.

DNA offers excellent programming properties for the generation of nanometer-scaled polyhedral structures with a broad variety of potential applications. Translation to biomedical applications requires improving stability in biological fluids, efficient and selective cell binding, and/or internalization of the assembled DNA nanostructures. Here, we report an investigation on the selective mechanism of cellular uptake of pristine DNA nanocages in cells expressing the receptor "oxidized low-density lipoprotein receptor-1" (LOX-1), a scavenger receptor associated with cardiovascular diseases and, more recently, identified as a tumor marker. For this purpose a truncated octahedral DNA nanocage functionalized with a single biotin molecule, which allows DNA cage detection through the biotin-streptavidin assays, was constructed. The results indicate that DNA nanocages are stable in biological fluids, including human serum, and are selectively bound and very efficiently internalized in vesicles only in LOX-1-expressing cells. The amount of internalized cages is 30 times higher in LOX-1-expressing cells than in normal fibroblasts, indicating that the receptor-mediated uptake of pristine DNA nanocages can be pursued for a selective cellular internalization. These results open the route for a therapeutic use of pristine DNA cages targeting LOX-1-overexpressing tumor cells.

We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.

Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment.

We demonstrate temperature-controlled encapsulation and release of the enzyme horseradish peroxidase using a preassembled and covalently closed three-dimensional DNA cage structure as a controllable encapsulation device. The utilized cage structure was covalently closed and composed of 12 double-stranded B-DNA helices that constituted the edges of the structure. The double stranded helices were interrupted by short single-stranded thymidine linkers constituting the cage corners except for one, which was composed by four 32 nucleotide long stretches of DNA with a sequence that allowed them to fold into hairpin structures. As demonstrated by gel-electrophoretic and fluorophore-quenching experiments this design imposed a temperature-controlled conformational transition capability to the structure, which allowed entrance or release of an enzyme cargo at 37 °C while ensuring retainment of the cargo in the central cavity of the cage at 4 °C. The entrapped enzyme was catalytically active inside the DNA cage and was able to convert substrate molecules penetrating the apertures in the DNA lattice that surrounded the central cavity of the cage.

Real-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples such as biopsies. TDP1 removes covalent 3'DNA adducts in DNA single-strand break repair. This enzymatic activity forms the basis of the design of the TDP1-biosensor, which consists of a short hairpin-forming oligonucleotide having a 5'fluorophore and a 3'quencher brought in close proximity by the secondary structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only "signal amplification" the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1 activity in pure enzyme fractions as well as in crude cell extracts. In the present study we demonstrate the specificity of the biosensor, its ability to quantitatively detect up- or down-regulated TDP1 activity, and that it may be used for measuring and for analyzing the mechanism of TDP1 inhibition., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I. The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor is specific for topoisomerase I even in raw cell extracts and presents a simple mean of following enzyme kinetics using standard laboratory equipment such as a qPCR machine or fluorimeter. Human topoisomerase I is a well-known target for the clinically used anti-cancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented DNA sensor, suggesting a potential application of the sensor for first line screening for potential topoisomerase I targeting anti-cancer drugs.

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. By means of a two-dimensional (2D) DNA origami platform, we monitor the interactions between a single human topoisomerase I covalently bound to one DNA fragment and a second DNA fragment protruding from the DNA origami. This novel single molecule origami-based detection scheme provides direct evidence for the existence of a secondary DNA interaction site in human topoisomerase I and lends further credence to the theory of two distinct DNA interaction sites in human topoisomerase I, possibly facilitating binding to DNA nodes characteristic for plectonemic supercoils.

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. Since amplification involves no thermal cycling, each fluorescent rolling circle product, which gives rise to an individual signal upon microscopic analysis, will correspond to a single human topoisomerase I mediated cleavage-ligation event. Regarding sensitivity, speed, and ease of performance, the presented activity assay based on single-molecule product detection is superior to current state of the art assays using supercoiled plasmids or radiolabeled oligonucleotides as the substrate for topoisomerase I activity. Moreover, inherent in the experimental design is the easy adaptation to multiplexed and/or high-throughput systems. Human topoisomerase I is the cellular target of clinically important anticancer drugs, and the effect of such drugs corresponds directly to the intracellular topoisomerase I cleavage-ligation activity level. We therefore believe that the presented setup, measuring directly the number of cleavage-ligation events in a given sample, has great diagnostic potential, adding considerably to the possibilities of accurate prognosis before treatment with topoisomerase I directed chemotherapeutics.

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. However, the building of such structures is still in progress and more experiences from different research groups and different fields of expertise are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnology. Here we present the design, construction and structural analysis of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double-stranded DNA helices that assembles from eight oligonucleotides with a yield of approximately 30%. As demonstrated by Small Angle X-ray Scattering and cryo-Transmission Electron Microscopy analyses the eight-stranded DNA structure has a central cavity larger than the apertures in the surrounding DNA lattice and can be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-molecules to enable their investigation in certain harmful environments or even allow their organization into higher order structures.

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. The inability of the double deleted variant to mediate resolution correlated with the inability of this enzyme to introduce concomitant cleavage at the two preferred cleavage sites in a single Holliday junction substrate, which is a prerequisite for resolution. As determined by the gel electrophoretic mobility of native enzyme or enzyme crosslinked by disulfide bridging, the double deleted mutant existed almost entirely in a dimeric form. The impairment of this enzyme in performing double cleavages on the Holliday junction substrate may be explained by only one cleavage competent active site being formed at a time within the dimer. The assembly of only one active site within dimers is a well-known characteristic of the tyrosine recombinases. Hence, the obtained results may suggest a recombinase-like active site assembly of the double deleted topoisomerase I variant. Taken together the presented results consolidate the relationship between type IB topoisomerases and tyrosine recombinases.

DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed. While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent on interdomain communication. We have characterized a human topoisomerase IIalpha enzyme with a two-amino acid insertion at position 351 in the transducer domain. The mutation specifically abolishes the DNA strand passage event of the enzyme, probably because of a sterical hindrance of T-segment transport. Thus, the enzyme fails to decatenate and relax DNA, even though it is fully capable of ATP hydrolysis, closure of the N-terminal clamp, and DNA cleavage. The cleavage activity is increased, suggesting that the transducer domain has a role in regulating DNA cleavage. Furthermore, the enzyme has retained a tendency to increase DNA cleavage upon nucleotide binding and also responds to DNA with elevated ATP hydrolysis. However, the DNA-mediated increase in ATP hydrolysis is lower than that obtained with the wild-type enzyme but similar to that of a cleavage-deficient topoisomerase IIalpha enzyme. Our results strongly suggest that the strand passage event is required for efficient DNA stimulation of topoisomerase II-mediated ATP hydrolysis, whereas the stimulation occurs independent of the DNA cleavage reaction per se. A comparison of the strand passage deficient-enzyme described here and the cleavage-deficient enzyme may have applications in other studies where a clear distinction between strand passage and topoisomerase II-mediated DNA cleavage is desirable.

We have previously shown [Straub et al. (1998) J. Biol. Chem. 273, 26261] that the pyrimidine tract binding protein associated splicing factor PSF/p54(nrb) binds and stimulates DNA topoisomerase I. Here we show that cleavage and religation half-reactions of topoisomerase I are unaffected by PSF/p54(nrb), whereas the propensity of the enzyme to jump between separate DNA helices is stimulated. To demonstrate such an effect, topoisomerase I was first captured in suicidal cleavage of an oligonucleotide substrate. Subsequently, a cleavage/ligation equilibrium was established by adding a ligation donor under conditions allowing recleavage of the ligated substrate. Finally, a second oligonucleotide was added to the mixture, which also allowed suicidal cleavage by topoisomerase I, but did not accommodate the ligation donor of the first oligonucleotide. Thus, topoisomerase I was given the choice to engage in repeated cleavage/ligation cycles of the first oligonucleotide or to jump to the second suicide substrate and get trapped. PSF/p54(nrb) enhanced the cleavage rate of the second oligonucleotide (11-fold), suggesting that it stimulates the dissociation of topoisomerase I after ligation. Thus, stimulation of topoisomerase I catalysis by PSF/p54(nrb) seems to be affected by mobilization of the enzyme.

Eukaryotic topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics. Central to the activities of the enzyme is its ability to introduce transient double-stranded breaks in the DNA helix, where the two subunits of the enzyme become covalently attached to the generated 5'-ends through phosphotyrosine linkages. Here, we demonstrate that human topoisomerases IIalpha and IIbeta are able to cleave ribonucleotide-containing substrates. With suicide substrates, which are partially double-stranded molecules containing a 5'-recessed strand, cleavage of both strands was stimulated approximately 8-fold when a ribonucleotide rather than a deoxyribonucleotide was present at the scissile phosphodiester of the recessed strand. The existence of a ribonucleotide at the same position in a normal duplex substrate also enhanced topoisomerase II-mediated cleavage, although to a lesser extent. The enzyme covalently linked to the 5'-ribonucleotide in the cleavage complex efficiently performed ligation, and ligation occurred equally well to acceptor molecules terminated by either a 3'-ribo- or deoxyribonucleotide. Besides the enhanced topoisomerase II-mediated cleavage of ribonucleotide-containing substrates, cleavage of such substrates could be further stimulated by ATP or antitumor drugs. In conclusion, the observed in vitro activities of the human topoisomerase II isoforms indicate that the enzymes can operate on RNA or RNA-containing substrates and thus might possess an intrinsic RNA topoisomerase activity, as has previously been demonstrated for Escherichia coli topoisomerase III.

Eukaryotic topoisomerase I catalysis was characterized by the use of a DNA substrate system, which allows uncoupling of cleavage and ligation half-reactions. Covalent topoisomerase I-DNA intermediates formed by cleavage without concomitant ligation were able to catalyze hydrolysis of the 3'-phosphotyrosyl bond in the pH range 7.5-10, with a broad pH optimum between pH 8.5 and 9.5. In comparison, the DNA cleavage and ligation activity of topoisomerase I were found to be independent of pH in the pH range 7-10 and strongly impaired at higher pH values. Moreover, different polyhydric alcohol compounds were found to function as nucleophiles at pH 9 to facilitate the release of topoisomerase I. The hydrolysis and alcoholysis activities of topoisomerase I were specific for the 3'-phosphotyrosyl bond and blocked by enzyme denaturation or proteolysis. Taken together the data suggest that site-specific hydrolysis or alcoholysis mediated by topoisomerase I-DNA complexes reflects the ability of the enzyme to direct the activation of the 3'-phosphotyrosyl bond or the incoming nucleophile. Analysis of enzyme-directed coupling of non-DNA nucleophiles to the cleaved DNA strand may provide a useful tool for elucidation of the biochemical mechanism of type I DNA topoisomerases.