Transmembrane nanostructures like ion channels and transporters perform key biological functions by controlling flow of molecules across lipid bilayers. Much work has gone into engineering artificial nanopores and applications in selective gating of molecules, label-free detection/sensing of biomolecules and DNA sequencing have shown promise. Here, we use DNA origami to create a synthetic 9 nm wide DNA nanopore, controlled by programmable, lipidated flaps and equipped with a size-selective gating system for the translocation of macromolecules. Successful assembly and insertion of the nanopore into lipid bilayers are validated by transmission electron microscopy (TEM), while selective translocation of cargo and the pore mechanosensitivity are studied using optical methods, including single-molecule, total internal reflection fluorescence (TIRF) microscopy. Size-specific cargo translocation and oligonucleotide-triggered opening of the pore are demonstrated showing that the DNA nanopore can function as a real-time detection system for external signals, offering potential for a variety of highly parallelized sensing applications.

Developing reliable methods of constructing cell-free multienzyme biocatalytic systems is a milestone goal of synthetic biology. It would enable overcoming the limitations of current cell-based systems, which suffer from the presence of competing pathways, toxicity, and inefficient access to extracellular reactants and removal of products. DNA nanostructures have been suggested as ideal scaffolds for assembling sequential enzymatic cascades in close enough proximity to potentially allow for exploiting of channeling effects; however, initial demonstrations have provided somewhat contradictory results toward confirming this phenomenon. In this work, a three-enzyme sequential cascade was realized by site-specifically immobilizing DNA-conjugated amylase, maltase, and glucokinase on a self-assembled DNA origami triangle. The kinetics of seven different enzyme configurations were evaluated experimentally and compared to simulations of optimized activity. A 30-fold increase in the pathway’s kinetic activity was observed for enzymes assembled to the DNA. Detailed kinetic analysis suggests that this catalytic enhancement originated from increased enzyme stability and a localized DNA surface affinity or hydration layer effect and not from a directed enzyme-to-enzyme channeling mechanism. Nevertheless, the approach used to construct this pathway still shows promise toward improving other more elaborate multienzymatic cascades and could potentially allow for the custom synthesis of complex (bio)­molecules that cannot be realized with conventional organic chemistry approaches.

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.

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide–oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.

DNA nanostructures of different sizes and forms are internalized in cells through the LOX-1 receptor with different intracellular fate and lifetime.

Green luminescent carbon dots with a high quantum yield and superior stability over a range of pH are synthesised for the first time via a new heating method. The carbon dots can be assembled into defined nanocomplexes as theranostic carriers for doxorubicin delivery.

The development of methods for conjugation of DNAt op roteins is of high relevance for the integration of protein function and DNAs tructures.H ere,w ed emonstrate that protein-binding peptides can direct aD NA-templated reaction, selectively furnishing DNA-protein conjugates with one DNAlabel. Quantitative conversion of oligonucleotides is achieved at lows toichiometries and the reaction can be performed in complex biological matrixes,such as cell lysates. Further,w eh ave used as tar-like pentameric DNAn anostructure to assemble five DNA-Rituximab conjugates,m ade by our reported method, into apseudo-IgM antibody structure that was subsequently characterized by negative-stain transmission electron microscopy( nsTEM) analysis.

A variety of different tiles for the construction of DNA lattices have been developed since the structural DNA nanotechnology field was born. The majority of these are designed for the realization of close-packed structures, where DNA helices are arranged in parallel and tiles are connected through sticky ends. Assembly of such structures requires the use of cation-rich buffers to minimize repulsion between parallel helices, which poses limits to the application of DNA nanostructures. Wireframe structures, on the other hand, are less susceptible to salt concentration, but the assembly of wireframe lattices is limited by the availability of tiles and motifs. Herein, we report the construction of a polyhedral 12-arm junction for the self-assembly of wireframe DNA lattices. Our approach differs from traditional assembly of DNA tiles through hybridization of sticky ends. Instead, the assembly approach presented here uses small polyhedral shapes as connecting points and branch points of wires in a lattice structure. Using this design principle and characterization techniques, such as transmission electron microscopy, single-particle reconstruction, patterning of gold nanoparticles, dynamic light scattering, UV melting analyses, and small-angle X-ray scattering among others, we demonstrated formation of finite 12-way junction structures, as well as 1D and 2D short assemblies, demonstrating an alternative way of designing polyhedral structures and lattices.