Designer DNA–silica/carbon nanotube nanocomposites for traceable and targeted drug delivery

Due to their unique properties like porosity, high water content, softness and biocompatibility, hydrogels are of great interest for biomedical applications such as tissue engineering and drug delivery. We describe a programmable drug delivery system that is based on highly biocompatible SiNP/CNT–DANN nanocomposites, which can be synthesized in a highly modular fashion from DNA-functionalized carbon nanotubes and silica nanoparticles via enzymatic rolling circle amplification. Specific molecular recognition properties were implemented into the materials by DNA sequence design, as demonstrated by incorporation of GC/CG-rich stem loop and aptamer motifs that enable selective binding of intercalating drugs and cell surface receptors, respectively. In a proof-of-concept study we demonstrate the utility of this approach by targeting nanocomposites loaded with the anthracycline drug doxorubicin to HeLa cancer cells. Our observation that these designer materials work more efficiently than the pure drug alone suggests that further developments of the concept might be useful to selectively trigger more complex cellular pathways

Nucleic Acid‐based Enzyme Cascades—Current Trends and Future Perspectives

The natural micro- and nanoscale organization of biomacromolecules is a remarkable principle within living cells, allowing for the control of cellular functions by compartmentalization, dimensional diffusion and substrate channeling. In order to explore these biological mechanisms and harness their potential for applications such as sensing and catalysis, molecular scaffolding has emerged as a promising approach. In the case of synthetic enzyme cascades, developments in DNA nanotechnology have produced particularly powerful scaffolds whose addressability can be programmed with nanometer precision. In this minireview, we summarize recent developments in the field of biomimetic multicatalytic cascade reactions organized on DNA nanostructures. We emphasize the impact of the underlying design principles like DNA origami, efficient strategies for enzyme immobilization, as well as the importance of experimental design parameters and theoretical modeling. We show how DNA nanostructures have enabled a better understanding of diffusion and compartmentalization effects at the nanometer length scale, and discuss the challenges and future potential for commercial applications

Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin

Protein-DNA conjugates have found numerous applications in the field of diagnostics and nanobiotechnology, however, their intrinsic susceptibility to DNA degradation by nucleases represents a major obstacle for many applications. We here report the selective covalent conjugation of the protein streptavidin (STV) with phosphorothioate oligonucleotides (psDNA) containing a terminal alkylthiolgroup as the chemically addressable linking unit, using a heterobifunctional NHS-/maleimide crosslinker. The psDNA-STV conjugates were synthesized in about 10% isolated yields. We demonstrate that the terminal alkylthiol group selectively reacts with the maleimide while the backbone sulfur atoms are not engaged in chemical conjugation. The novel psDNA-STV conjugates retain their binding capabilities for both biotinylated macromolecules and the complementary nucleic acid. Moreover, the psDNA-STV conjugate retained its binding capacity for complementary oligomers even after a nuclease digestion step, which effectively degrades deoxyribonucleotide oligomers and thus the binding capability of regular DNA-STV conjugates. The psDNA-STV therefore hold particular promise for applications e.g. in proteome research and novel biosensing devices, where interfering endogenous nucleic acids need to be removed from analytes by nuclease digestion

Formulation and characterisation of enzyme-based biomaterials for μFluidic experiments

The fundamental principle of biological compartmentalisation of cellular life provides the basis for space-time resolved reaction processes. Based on this, intensive work is currently done on the use of interconnected, continuously flowing reaction chambers in order to improve the reaction control and efficiency of chemical syntheses, especially with the inclusion of biocatalysts (so called flow biocatalysis). Therefore, the Institute for Biological Interfaces IBG-1 aims at the formulation of enzyme-based biomaterials and the associated testing of novel gene-encoded coupling systems. The resulting enzyme fusions will be used as modular building blocks for the assembly of catalytically active materials, with different formulations (hydrogels or thin films) and characterized in terms of their immobilization and biocatalytic activity in miniaturized flow reactors. During the process of formulating an optimised biomaterial, we developed and established the self-assembling all-enzyme hydogels (AEHs). These protein materials consist of the two homotetrameric enzymes, (R)-selective alcohol dehydrogenase (ADH) and the cofactor regenerating glucose 1-dehydrogenase (GDH), that are genetically fused with either the SpyCatcher (SC) or the SpyTag (ST). The AEHs were characterised via dynamic light scattering (DLS) and scanning electron microscopy (SEM) in terms of physical properties. Moreover, the gels showed excellent stereoselectivity, stable conversion rates and high space-time yields (STY) for more than seven days in continuous flow experiments

An Orthogonal Covalent Connector System for the Efficient Assembly of Enzyme Cascades on DNA Nanostructures

Combining structural DNA nanotechnology with the virtually unlimited variety of enzymes offers unique opportunities for generating novel biocatalytic devices. However, the immobilization of enzymes is still restricted by a lack of efficient covalent coupling techniques. The rational re-engineering of the genetically fusible SNAP-tag linker is reported here. By replacing five amino acids that alter the electrostatic properties of the SNAP_R5 variant, up to 11-fold increased coupling efficiency with benzylguanine-modified oligonucleotides and DNA origami nanostructures (DON) was achieved, resulting in typical occupancy densities of 75%. The novel SNAP_R5 linker can be combined with the equally efficient Halo-based oligonucleotide binding tag (HOB). Since both linkers exhibit neither cross-reactivity nor non-specific binding, they allowed orthogonal assembly of an enzyme cascade consisting of the stereoselective ketoreductase Gre2p and the cofactor-regenerating isocitrate dehydrogenase on DON. The cascade showed approximately 1.6-fold higher activity in a stereoselective cascade reaction than the corresponding free solubilized enzymes. The connector system presented here and the methods used to validate it represent important tools for further development of DON-based multienzyme systems to investigate mechanistic effects of substrate channeling and compartmentalization relevant for exploitation in biosensing and catalysis

Postsynthetic Functionalization of DNA‐Nanocomposites with Proteins Yields Bioinstructive Matrices for Cell Culture Applications

We report on the directed postsynthetic functionalization of soft DNA nanocomposite materials with proteins. Using the example of the functionalization of silica nanoparticle‐modified DNA polymer materials with agonists or antagonists of the epidermal growth factor receptor EGFR cell membrane receptor, we demonstrate that hierarchically structured interfaces to living cells can be established. Owing to the modular design principle, even complex DNA nanostructures can be integrated into the materials, thereby enabling the high‐precision arrangement of ligands on the lower nanometer length scale. We believe that such complex biohybrid material systems can be used for new applications in biotechnology

Imine Reductase Based All-Enzyme Hydrogel with Intrinsic Cofactor Regeneration for Flow Biocatalysis

All-enzyme hydrogels are biocatalytic materials, with which various enzymes can be immobilized in microreactors in a simple, mild, and efficient manner to be used for continuous flow processes. Here we present the construction and application of a cofactor regenerating hydrogel based on the imine reductase GF3546 from Streptomyces sp. combined with the cofactor regenerating glucose-1-dehydrogenase from Bacillus subtilis. The resulting hydrogel materials were characterized in terms of binding kinetics and viscoelastic properties. The materials were formed by rapid covalent crosslinking in less than 5 min, and they showed a typical mesh size of 67 ± 2 nm. The gels were applied for continuous flow biocatalysis. In a microfluidic reactor setup, the hydrogels showed excellent conversions of imines to amines for up to 40 h in continuous flow mode. Variation of flow rates led to a process where the gels showed a maximum space-time-yield of 150 g·(L·day)−1 at 100 μL/mi