Enzymatically Active DNA-Protein Nanogels with Tunable Cross-Linking Density

Abstract

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.

Computational DNA Droplets Recognizing miRNA Sequence Inputs Based on Liquid–Liquid Phase Separation

“…DNA nanotechnology is categorized into structural DNA nanotechnology [11] and DNA computing. [12] In structural DNA nanotechnology, sophisticated nanostructures such as DNA polyhedra, [13] DNA origami, [14,15] and DNA hydrogels [16][17][18][19][20] with varying sizes, functions, and complexities have been produced. In addition, by designing and regulating interactions between DNA and other molecules, DNA nanostructures have shown critical potential applications in molecule detection, [21] sensing capability, [22] drug delivery, [23] and disease treatment.…”

Biopolymer-Based Nanogel Approach in Drug Delivery: Basic Concept and Current Developments

“…A further example involves the introduction of a novel, simple, and reliable technique for making tunable DNAprotein nanogels with adjustable size and density. Highly biotinylated DNA was manufactured for this use using polymerase chain reaction as a soft biopolymeric backbone that can be effectively cross-linked using streptavidin-biotin binding [45].…”

Multiscale Biofabrication: Integrating Additive Manufacturing with DNA‐Programmable Self‐Assembly

“…[51] Also hybrid DNA-protein nanogels were developed using biotin-streptavidin interactions for crosslinking, where the gel properties could be controlled via the protein crosslinks rather than via DNA hybridization. [52] A large variety of methods have been employed to make DNA-based gels switchable and shape-changing, including DNA strand displacement, [53,54] pH, [55] ion responsive DNA motifs, [56] aptamers, [57] enzyme action, [58,59] or temperature. [60] For reviews of responsive DNA gels see also.…”