Tunable Nanoscale Cages from Self-Assembling DNA and Protein Building Blocks

Abstract

cages are one of the most important targets for nanotechnology. Both proteins and DNA have been used as building blocks to create tunable nanoscale cages for a wide range of applications, but each molecular type has its own limitations. Here, we report a cage constructed from both protein and DNA building blocks through the use of covalent protein−DNA conjugates. We modified a homotrimeric protein (KDPG aldolase) with three identical single-stranded DNA handles by functionalizing a reactive cysteine residue introduced via site-directed mutagenesis. This protein−DNA building block was coassembled with a triangular DNA structure bearing three complementary arms to the handles, resulting in tetrahedral cages comprising six DNA sides capped by the protein trimer. The dimensions of the cage could be tuned through the number of turns per DNA arm (3 turns ∼ 10 nm, 4 turns ∼ 14 nm), and the hybrid structures were purified and characterized to confirm the three-dimensional structure. Cages were also modified with DNA using click chemistry and using aldolase trimers bearing the noncanonical amino acid 4-azidophenylalanine, demonstrating the generality of the method. Our approach will allow for the construction of nanomaterials that possess the advantages of both protein and DNA nanotechnology and find applications in fields such as targeted delivery, structural biology, biomedicine, and catalytic materials.

DNAzyme-Functionalized R-Phycoerythrin as a Cost-Effective and Environment-Friendly Fluorescent Biosensor for Aqueous Pb2+ Detection

“…An obvious increase in the size of R-PE was observed, suggesting the successful conjugation of the enzyme-substrate complex onto R-PE [30]. Meanwhile, the native PAGE (8%) experiment indicated the coupling of the enzyme-substrate complex to R-PE, which led to a clear difference in the band shift between R-PE and DNAzyme-R-PE (Figure S2) [31]. This further confirmed that the enzyme-substrate complex was successfully immobilized on R-PE.…”

Hierarchical Assembly of DNA Origami Nanostructures Using Coiled-coil Peptides

“…For example, a homotrimeric parallel coiled-coil could serve as a C3-symmetric "cap" for a DNA structure, similar to a recent report from our lab using homotrimeric proteins. 10 Different coiled-coil systems have a wide range of stabilities, so it may be possible to sequentially form different domains during the annealing process, and effectively "fold" a DNA structure using orthogonal peptide interactions, akin to intramolecular coiled-coil folded cages. 22 Using coils that are closer to charge-neutral as described by Woolfson and Turberfield (DOI: 10.1021/acsnano.9b04251) could help avoid some of the challenges with DNA conjugation and nonspecific aggregation.…”

“…coiled-coils, collagen triple helices) that impart unique functional and mechanical properties. While several examples exist of multivalent recombinant proteins covalently modified with DNA to form cages, 10 nanofibers, 11,12 and three-dimensional crystals, 13 the chemical coupling of synthetic self-assembled peptides with DNA has only recently been demonstrated. The Ke and Conticello labs demonstrated the first example of linking DNA origami nanostructures with a synthetic peptide self-assembly motif, using collagen-mimetic triple helices fused with a cationic domain to electrostatically associate with the negatively charged oligonucleotide structures.…”

Bioengineered Polyhydroxyalkanoates as Immobilized Enzyme Scaffolds for Industrial Applications

“…Although these scaffolds have been manufactured in numerous recombinant expression systems (e.g., various prokaryotic and eukaryotic organisms), they have been mainly assembled in Escherichia coli strains (Diaz et al, 2018). Advances in protein engineering in recent years harnessed the unique structural assembly of EZPs to enable the de novo and in silico design of novel EZPs (King et al, 2014;Bale et al, 2016;Hsia et al, 2016;Jakobson et al, 2017;Xu et al, 2019).…”