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 ∼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.

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 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. The structures of the cages were determined by small-angle X-ray scattering. With increasing linker length, there is a systematic increase of the cage size and decrease of the twist angle of the double helices with respect to the symmetry planes of the cage structure. In the present study, we demonstrate the length of the single-stranded linker regions, which impose a certain degree of flexibility to the structure, to be the important determinant for efficient assembly. The linker length can be decreased to three thymidines without affecting assembly yield or the overall structural characteristics of the DNA cages. A linker length of two thymidines represents a sharp cutoff abolishing cage assembly. This is supported by energy minimization calculations suggesting substantial hydrogen bond deformation in a cage with linkers of two thymidines.

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). An important inherent property of the presented setup is, at least in theory, the easy adaptability to multiplexed enzyme detection simply by using differently labeled probes for the detection of rolling circle products of different circularized substrates. In the present study we demonstrate the specific detection of three different enzyme activities, human topoisomerase I, and Flp and Cre recombinase in nuclear extracts from human cells one at a time or multiplexed using the rolling circle amplification based single-molecule detection system. Besides serving as a proof-of-principle for the feasibility of the presented assay for multiplexed enzyme detection in crude human cell extracts, the simultaneous detection of Flp and Cre activities in a single sample may find immediate practical use since these enzymes are often used in combination to control mammalian gene expression.

A DNA nanocage has been recently characterized by small-angle X-ray scattering (SAXS) and cryo-transmission electron microscopy as a DNA octahedron having a central cavity larger than the apertures in the surrounding DNA lattice. Starting from the SAXS data, a DNA nanocage has been modeled and simulated by classical molecular dynamics to evaluate in silico its structural properties and stability. Global properties, principal component analysis, and DNA geometrical parameters, calculated along the entire trajectory, indicate that the cage is stable and that the B-DNA conformation, also if slightly distorted, is maintained for all the simulation time. Starting from the initial model, the nanocage scaffold undergoes a contraction of the thymidine strands, connecting the DNA double helices, suggesting that the length of the thymidine strands is a crucial aspect in the modulation of the nanocage stability. A comparison of the average structure as obtained from the simulation shows good agreement with the SAXS experimental data.

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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. This setup allows the fast reliable analyses of enzyme activities in vast number of single cells thereby offering a valuable tool for basic research as well as theranostics.

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. Rolling Circle Amplification has been used to enhance signals of nucleic acid based sensors specific for a large variety of important biomarkers, including nucleotide sequences, small molecules, proteins and enzyme activities, allowing detection of biomarkers even at the aM concentration level. Moreover, Rolling Circle Products have been adapted to a broad variety of low technological and cost efficient readout formats making the technique appealing for future basic discovery research as well as for at-point-of-care diagnostic or prognostic tests.