Hybrid DNA-protein nanogels represent potential protein vectors and enzymatic nanoreactors for modern biotechnology. Here we describe new, easy and robust method for preparation of tunable DNA-protein nanogels with controllable size and density. For this purpose, polymerase chain reaction (PCR) is used to prepare highly biotinylated DNA as soft biopolymeric backbone which can be efficiently cross-linked via streptavidin-biotin binding. This approach enables to control both density and size of the resulting nanogels not only by adjusting the amount of the cross-linking streptavidin, but also by using different rates of DNA biotinylation, to give DNA-streptavidin nanogels with size ranging from 80 nm, for the most compact state, to up to 200 nm. Furthermore, using streptavidin-enzyme conjugates allows the straightforward one-pot incorporation of enzymes during the preparation of the nanogels. Monoenzymatic and multienzymatic nanogels have been obtained in this manner and their catalytic activities characterized. All tested enzymes (alkaline phosphatase (AP), horseradish peroxidase (HRP), βgalactosidase (βGal), incorporated individually or in a coupled manner (glucose oxidase (GOx)-HRP cascade), were shown to remain functional. The activities of AP and βGal were unchanged, while that of the HRP was slightly improved inside the nanogels. We demonstrate that for the HRP it is not the DNA-to-enzyme ratio, but the physical density of the functionalized DNA nanogels which is responsible of the improvement of its enzymatic activity.

We report that user‐defined DNA nanostructures, such as two‐dimensional (2D) origamis and nanogrids, undergo a rapid higher‐order folding transition, referred to as supra‐folding, into three‐dimensional (3D) compact structures (origamis) or well‐defined μm‐long ribbons (nanogrids), when they adsorb on a soft cationic substrate prepared by layer‐by‐layer deposition of polyelectrolytes. Once supra‐folded, origamis can be switched back on the surface into their 2D original shape through addition of heparin, a highly charged anionic polyelectrolyte known as an efficient competitor of DNA‐polyelectrolyte complexation. Orthogonal to DNA base‐pairing principles, this reversible structural reconfiguration is also versatile; we show in particular that 1) it is compatible with various origami shapes, 2) it perfectly preserves fine structural details as well as site‐specific functionality, and 3) it can be applied to dynamically address the spatial distribution of origami‐tethered proteins.

We report that user‐defined DNA nanostructures, such as two‐dimensional (2D) origamis and nanogrids, undergo a rapid higher‐order folding transition, referred to as supra‐folding, into three‐dimensional (3D) compact structures (origamis) or well‐defined μm‐long ribbons (nanogrids), when they adsorb on a soft cationic substrate prepared by layer‐by‐layer deposition of polyelectrolytes. Once supra‐folded, origamis can be switched back on the surface into their 2D original shape through addition of heparin, a highly charged anionic polyelectrolyte known as an efficient competitor of DNA‐polyelectrolyte complexation. Orthogonal to DNA base‐pairing principles, this reversible structural reconfiguration is also versatile; we show in particular that 1) it is compatible with various origami shapes, 2) it perfectly preserves fine structural details as well as site‐specific functionality, and 3) it can be applied to dynamically address the spatial distribution of origami‐tethered proteins.

Using functional proteins for therapeutic purposes due to their high selectivity and/or catalytic properties can enable the control of various cellular processes, however, the transport of active proteins inside living cells remains a major challenge. In contrast, intracellular delivery of nucleic acids has become a routine method for a number of applications in gene therapy, genome-editing or immunization. Here we report a functionalizable platform constituting of DNA-protein nanogel carriers crosslinked through streptavidin-biotin interactions and demonstrate its applicability for intracellular delivery of active proteins. We demonstrate that the nanogels can be loaded with proteins bearing either biotin or streptavidin tags, and can be transfected into living cells after complexation with cationic lipid vectors. In particular we use this approach for transfection of alkaline phosphatase enzyme which is shown to keep its catalytic activity after internalization by mouse melanoma B16 cells, as demonstrated by DDAO-phosphate assay. The resulting functionalized nanogels have dimensions of the order of 100 nm, contain around 100 enzyme molecules and are shown to be transfectable at low lipid concentrations (charge ratio R+/- = 0.75). Low lipid concentrations requested for transfection of the 3-dimensional DNA nanogels ensure low toxicity of our system, which in combination with high local enzyme concentration (~ 100 µM) underlines potential interest of this nanoplatform for biomedical applications.