Intramolecularly Protein-Crosslinked DNA Gels: New Biohybrid Nanomaterials with Controllable Size and Catalytic Activity

Abstract

DNA micro- and nanogels-small-sized hydrogels made of a crosslinked DNA backbone-constitute new promising materials, but their functions have mainly been limited to those brought by DNA. Here a new way is described to prepare sub-micrometer-sized DNA gels of controllable crosslinking density that are able to embed novel functions, such as an enzymatic activity. It consists of using proteins, instead of traditional base-pairing assembly or covalent approaches, to form crosslinks inside individual DNA molecules, resulting in structures referred to as intramolecularly protein-crosslinked DNA gels (IPDGs). It is first shown that the addition of streptavidin to biotinylated T4DNA results in the successful formation of thermally stable IPDGs with a controllable crosslinking density, forming structures ranging from elongated to raspberry-shaped and pearl-necklace-like morphologies. Using reversible DNA condensation strategies, this paper shows that the gels can be reversibly actuated at a low crosslinking density, or further stabilized when they are highly crosslinked. Finally, by using streptavidin-protein conjugates, IPDGs with various enzymes are successfully functionalized. It is demonstrated that the enzymes keep their catalytic activity upon their incorporation into the gels, opening perspectives ranging from biotechnologies (e.g., enzyme manipulation) to nanomedicine (e.g., vectorization).

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

“…Indeed, the activity of free sHRP and sHRP in the presence of non-biotinylated 0%bt-DNA decreased down to 26 and 24%, respectively, indicating relatively rapid degradation of the enzyme even after incubation on ice, whereas when incorporated into the nanogels, sHRP demonstrated 73% of its initial activity. The effect of DNA on the activities of conjugated enzymes has been reported in the literature, and the enhancement of the activity of enzymes by DNA conjugation has been shown for several enzymes, , especially for HRP. ,− Recently, the enhancement of the EA for HRP anchored on DNA nanostructures was explained by the local decrease in pH in close proximity to DNA compared to that in the bulk solution. , In our experiments, EA was never observed to be over 100%, so we cannot conclude on the existence of such an enhancement, but the decrease in EA was much slower for HRP incorporated into the nanogels than the enzyme alone. This could be a consequence of two effects: (i) either a weak enhancement due to the favorable local ionic microenvironment inside the DNA nanogel or (ii) stabilization of the enzyme molecules against thermal degradation, aggregation, or adsorption by incorporating them into the nanogel structure.…”

“…Effect of DNA on the activities of conjugated enzymes has been reported in the literature, and enhancement of the activity of enzymes by DNA conjugation has been shown for several enzymes, 18,38 especially for HRP. 20,[39][40][41][42][43] Recently, enhancement of the EA for HRP anchored on DNA nanostructures was explained by the local decrease of pH in close proximity to DNA compared to that in the bulk solution. 44,45 In our experiments EA was never observed to be over 100%, so we cannot conclude on the existence of such an enhancement, but the decrease of EA was much slower for HRP incorporated into the nanogels compared to the enzyme alone.…”

“…Our strategy is based on the cross-linking of a multibiotinylated DNA backbone with streptavidin proteins. This approach has been recently explored to induce cross-linking within a giant DNA scaffold biotinylated by photochemical binding of apsoralene-biotin conjugate, but because of the low biotinylation efficiency, the resulting micrometer-size objects had a loose density . To obtain nanosized and dense DNA-streptavidin gels, the use of short and highly biotinylated DNA scaffolds was thus required.…”

Putting DNA to Work as Generic Polymeric Materials

“…In addition to biomedical applications,D NA gels have showed diverse uses in broader areas such as catalysis and self-assembly. [66,67] We describe here how DNAgels have been implemented for various applications in non-biomedical fields.…”

The Novel Nanomaterials Based Biosensors and Their Applications

“…In recent years, legion nucleic acid nanostructures have been applied to biological detection, including DNA tetrahedron, DNA gels, DNA dendrimers, and so on [73][74][75]. Y-shaped DNA (Y-DNA), as a constant nanostructure with high selectivity, provides an effective method for completely measuring target molecules [76].…”