Spatially-interactive biomolecular networks organized by nucleic acid nanostructures

Abstract

Living systems have evolved a variety of nanostructures to control the molecular interactions that mediate many functions including the recognition of targets by receptors, the binding of enzymes to substrates, and the regulation of enzymatic activity. Mimicking these structures outside of the cell requires methods that offer nanoscale control over the organization of individual network components. Advances in DNA nanotechnology have enabled the design and fabrication of sophisticated one-, two- and three-dimensional (1D, 2D, and 3D) nanostructures that utilize spontaneous and sequence-specific DNA hybridization. Compared with other self-assembling biopolymers, DNA nanostructures offer predictable and programmable interactions and surface features to which other nanoparticles and biomolecules can be precisely positioned. The ability to control the spatial arrangement of the components while constructing highly organized networks will lead to various applications of these systems. For example, DNA nanoarrays with surface displays of molecular probes can sense noncovalent hybridization interactions with DNA, RNA, and proteins and covalent chemical reactions. DNA nanostructures can also align external molecules into well-defined arrays, which may improve the resolution of many structural determination methods, such as X-ray diffraction, cryo-EM, NMR, and super-resolution fluorescence. Moreover, by constraint of target entities to specific conformations, self-assembled DNA nanostructures can serve as molecular rulers to evaluate conformation-dependent activities. This Account describes the most recent advances in the DNA nanostructure directed assembly of biomolecular networks and explores the possibility of applying this technology to other fields of study. Recently, several reports have demonstrated the DNA nanostructure directed assembly of spatially interactive biomolecular networks. For example, researchers have constructed synthetic multienzyme cascades by organizing the position of the components using DNA nanoscaffolds in vitro or by utilizing RNA matrices in vivo. These structures display enhanced efficiency compared with the corresponding unstructured enzyme mixtures. Such systems are designed to mimic cellular function, where substrate diffusion between enzymes is facilitated and reactions are catalyzed with high efficiency and specificity. In addition, researchers have assembled multiple choromophores into arrays using a DNA nanoscaffold that optimizes the relative distance between the dyes and their spatial organization. The resulting artificial light-harvesting system exhibits efficient cascading energy transfers. Finally, DNA nanostructures have been used as assembly templates to construct nanodevices that execute rationally designed behaviors, including cargo loading, transportation, and route control.

Figure 1

Introduction to structural DNA nanotechnology. (A) Self-assembly of nanostructures based on complementary DNA base pairing. (B) DNA helix bundles (left),^– 2-D arrays (middle),^– and 3-D objects (right).^– (C) DNA origami for constructing 2-D nanostructures and (D) 3-D architectures - hollow box (left), multi-layer monolith and a square-toothed gear(middle),^, and semi-sphere and a nanoflask (right). Figures are reproduced with permissions from ACS, AAAS and NPG.

Figure 2

DNA-directed assembly. (A) Seeman’s proposal to organize macromolecules within a DNA nanoscaffold. (B) Patterning macromolecules on DNA origami: streptavidin (top), virus capsid (middle) and orthogonal protein decoration (bottom). Reproduce from ref (C) Site-specific protein-oligo conjugation using His-tag, ybbR-tag, STV-tag, HALO-tag, SNAP-tag and Intein-tag (from top to bottom). Figures are reproduced with permissions from IOP Publishing, ACS, and Wiley.

Figure 3

DNA nanostructures as a template for label-free detection of bimolecular interactions: (A) RNA hybridization assay; (B) SNP detection; (C) Spatially-dependent multivalent ligand-protein binding; (D) Chemical bond formation and (E) bond cleavage. Figures are reproduced with permissions from AAAS, ACS, NPG and Wiley.

Figure 4

DNA nanostructures as biophysical study tools: (A) X-ray diffraction, (B) cyro-EM and (C) super-resolution imaging. Conformational studies of (D) G-quadruplex formation, (E) DNA methylation and (F) constrained intermolecular forces. Figures are reproduced with permissions from NPG, ACS and Wiley.

Figure 5

DNA/RNA nanostructures for engineering multi-enzyme systems. A linear, double-stranded DNA scaffold for (A) assembling an enzyme cascade: NAD(P)H:FMN (NFOR) oxidoreductase and luciferase (Luc); and (B) evaluating the distance-dependent activity of Cytochrome P450 BM3 by varying the spacing between the BMR reductase domain and the BMP porphyrin domain. (C) 2D DNA strip for organizing GOx/HRP cascades. (D) In vivo assembly of RNA nanostructures to organize the [FeFe]-hydrogenase and ferredoxin enzyme pathway for improved hydrogen production. (E) Organization of a GOx/HRP cascade on DNA origami tiles with controlled spatial positions (top), and a protein bridge for facilitating surface-limited intermediate diffusion between enzymes (bottom). Figures are reproduced with permissions from Wiley, ACS, NPG and AAAS.

Figure 6

Energy-transfer within DNA nanostructures. (A) Four-color FRET and (B) artificial light-harvesting network. Figures are reproduced with permissions from ACS.

Figure 7

Responsive DNA nanodevices (A) a cargo transportation system consisting of an assembly template, cargo loading apparatus and DNA walker, (B) walker movement along a 2-D deoxyribonucleotide substrate surface and (C) forceps for sensing various noncovalent interactions. Figures are reproduced with permissions from NPG.

Figure 8

(A) Engineering enzyme pathways to achieve directional substrate diffusion (top), constrained substrate tunneling (middle) and split enzyme pathways as feedback mechanisms (bottom). (B) Schematic illustration of an artificial photosynthesis system that couples light harvesting and charge separation components within a multi-layer DNA nanostructure. (C) DNA nanocontainer for target-specific drug delivery and in vivo regulation of cellular activities.