Anders H. Okholm

Construction of a 4 Zeptoliters Switchable 3D DNA Box Origami

The DNA origami technique is a recently developed self-assembly method that allows construction of 3D objects at the nanoscale for various applications. In the current study we report the production of a 18 × 18 × 24 nm(3) hollow DNA box origami structure with a switchable lid. The structure was efficiently produced and characterized by atomic force microscopy, transmission electron microscopy, and Förster resonance energy transfer spectroscopy. The DNA box has a unique reclosing mechanism, which enables it to repeatedly open and close in response to a unique set of DNA keys. This DNA device can potentially be used for a broad range of applications such as controlling the function of single molecules, controlled drug delivery, and molecular computing.

Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins

DNA-protein conjugates are important in bioanalytical chemistry, molecular diagnostics and bionanotechnology, as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, preparing such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and technically challenging approach. Here we demonstrate a simpler method to create site-selective DNA-protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the production of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the constant domain.

Quantification of cellular uptake of DNA nanostructures by qPCR

DNA nanostructures facilitating drug delivery are likely soon to be realized. In the past few decades programmed self-assembly of DNA building blocks have successfully been employed to construct sophisticated nanoscale objects. By conjugating functionalities to DNA, other molecules such as peptides, proteins and polymers can be precisely positioned on DNA nanostructures. This exceptional ability to produce modular nanoscale devices with tunable and controlled behavior has initiated an interest in employing DNA nanostructures for drug delivery. However, to obtain this the relationship between cellular interactions and structural and functional features of the DNA delivery device must be thoroughly investigated. Here, we present a rapid and robust method for the precise quantification of the component materials of DNA origami structures capable of entering cells in vitro. The quantification is performed by quantitative polymerase chain reaction, allowing a linear dynamic range of detection of five orders of magnitude. We demonstrate the use of this method for high-throughput screening, which could prove efficient to identify key features of DNA nanostructures enabling cell penetration. The method described here is suitable for quantification of in vitro uptake studies but should easily be extended to quantify DNA nanostructures in blood or tissue samples.

Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway

DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site-selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22-fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface.

A quencher-free molecular beacon design based on pyrene excimer fluorescence using pyrene-labeled UNA (unlocked nucleic acid)

A quencher-free molecular beacon capable of generating pyrene excimer fluorescence has been constructed using strategically positioned pyrene-UNA monomers. Hybridization of a fully complementary RNA target was accompanied by a pyrene excimer emission increase of more than 900%, and detection of RNA in living cells was demonstrated.

DNA nanovehicles and the biological barriers

DNA is emerging as a smart material to construct nanovehicles for targeted drug delivery. The programmability of Watson-Crick base paring enables construction of defined and dynamic DNA nanostructures of almost arbitrary shape and DNA can readily be functionalized with a variety of molecular modules. The applications of DNA nanostructures are still in its infancy, but one of the high expectations are to deliver solutions for targeted therapy. Nucleic acids, however, do not easily enter cells unassisted and biological barriers and harsh nucleolytic conditions in the human body must also be overcome. Here, we highlight recent strategies for DNA nanostructures in drug delivery, DNA nanovehicles, to facilitate targeting and crossing of the biological barriers. In light of this, we discuss future solutions and challenges for DNA nanovehicles to unravel their great potential to facilitate targeted drug delivery.

A DNA-programmed liposome fusion cascade

Chemically engineered and functionalized nanoscale compartments are used in bottom-up synthetic biology to construct compartmentalized chemical processes. Progressively more complex designs demand spatial and temporal control over entrapped species. Here, we address this demand with a DNA-encoded design for the successive fusion of multiple liposome populations. Three individual stages of fusion are induced by orthogonally hybridizing sets of membrane-anchored oligonucleotides. Each fusion event leads to efficient content mixing and transfer of the recognition unit for the subsequent stage. In contrast to fusion-protein-dependent eukaryotic vesicle processing, this artificial fusion cascade exploits the versatile encoding potential of DNA hybridization and is generally applicable to small and giant unilamellar vesicles. This platform could thus enable numerous applications in artificial cellular systems and liposome-based synthetic pathways.

Fluorescence detection of natural RNA using rationally designed “clickable” oligonucleotide probes

Herein a reliable approach to the design of effective fluorescent probes for RNA detection is described. The fluorescence signalling of hybridization by internally positioned polyaromatic hydrocarbons and rhodamine dyes was achieved with a low fluorescence background signal, high fluorescence quantum yields at ambient and elevated temperature, high selectivity and signal specificity of the probes when binding to miR-7 and circRNA targets.

The utility of DNA nanostructures for drug delivery in vivo

DNA nanotechnology has moved from a structure-centric field into more focus on functionalities and applications. The delivery of drugs is among the most intensely studied applications for DNA nanotechnology and for obvious reasons. Methods such as Rothemund’s DNA origami, single-stranded DNA tiles, and the more recent polygonal DNA origami have enabled formation of structurally defined nanoparticles of various shapes and sizes, which appear to be crucial parameters for the fate of systemic nanoparticles. The inherent structural principles of DNA nanostructures, the programmability of the individual building blocks, allow for attachment of molecules in rationally designed numbers and patterns. This can, for example, affect their interaction with living cells by tuning either the position, or stoichiometry of the functional molecules. In addition, DNA nanostructures have the unique potential to control large dynamic changes induced by strand displacement reactions. Although clinical applications are still far from realization, several in vitro experiments have been conducted throughout the past decade. This has yielded new insights into the behavior of DNA nanostructure stability toward DNases and serum proteins, uptake into cultured cells, and the delivery of drugs. The results and conclusions from these studies have already been reviewed in great detail [1,2]. The full complexity and multifaceted challenges related to in vivo experiments, however, cannot be truly mimicked in vitro and results can rarely be extrapolated directly to in vivo experiments. Therefore, in vivo experiments have emerged during the past 5 years (Figure 1 and Table 1) where DNA nanostructures are injected into animals and the pharmacodynamics and pharmacokinetics are studied. Here, we review the methods and results from the 12 papers published so far on this topic.

Complexes of DNA with fluorescent dyes are effective reagents for detection of autoimmune antibodies

To date, there are multiple assays developed that detect and quantify antibodies in biofluids. Nevertheless, there is still a lack of simple approaches that specifically detect autoimmune antibodies to double-stranded DNA. Herein we investigate the potential of novel nucleic acid complexes as targets for these antibodies. This is done in a simple, rapid and specific immunofluorescence assay. Specifically, employing 3D nanostructures (DNA origami), we present a new approach in the detection and study of human antibodies to DNA. We demonstrate the detection of anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We tested the most potent non-covalent pairs of DNA and fluorescent dyes. Several complexes showed specific recognition of autoimmune antibodies in human samples of lupus patients using a simple one-step immunofluorescence method. This makes the novel assay developed herein a promising tool for research and point-of-care monitoring of anti-DNA antibodies. Using this method, we for the first time experimentally confirm that the disease-specific autoimmune antibodies are sensitive to the 3D structure of nucleic acids and not only to the nucleotide sequence, as was previously thought.