Programming Structured DNA Assemblies to Probe Biophysical Processes

Abstract

Structural DNA nanotechnology is beginning to emerge as a widely accessible research tool to mechanistically study diverse biophysical processes. Enabled by scaffolded DNA origami in which a long single strand of DNA is weaved throughout an entire target nucleic acid assembly to ensure its proper folding, assemblies of nearly any geometric shape can now be programmed in a fully automatic manner to interface with biology on the 1-100-nm scale. Here, we review the major design and synthesis principles that have enabled the fabrication of a specific subclass of scaffolded DNA origami objects called wireframe assemblies. These objects offer unprecedented control over the nanoscale organization of biomolecules, including biomolecular copy numbers, presentation on convex or concave geometries, and internal versus external functionalization, in addition to stability in physiological buffer. To highlight the power and versatility of this synthetic structural biology approach to probing molecular and cellular biophysics, we feature its application to three leading areas of investigation: light harvesting and nanoscale energy transport, RNA structural biology, and immune receptor signaling, with an outlook toward unique mechanistic insight that may be gained in these areas in the coming decade.

Keywords: DNA origami; RNA structural biology; computational design; immunology; light harvesting; nanoscale energy transport; nanotechnology; synthetic structural biology.

Figure 1

Design and synthesis of wireframe scaffolded DNA origami nanostructures for biophysical research. (a) This panel shows the workflow for designing DNA origami objects using the software DAEDALUS (186). First, a target polyhedron is used as input to the algorithm. DAEDALUS then automatically routes the scaffold through every edge and assigns the staples needed to fold the target object. An atomic model is also generated to allow researchers to visualize the object and design molecular functionalization patterns. Using the computationally designed set of oligonucleotide sequences, the DNA nanoparticle is self-assembled via thermal annealing with an excess of staples over scaffold, which is typically based on the M13 phage or a synthetic sequence. Folding is characterized using agarose gel electrophoresis and structural analysis using atomic force microscopy (AFM), transmission electron microscopy (TEM), or 3D cryo-electron microscopy (cryo-EM). Adapted with permission from Reference . (b) A vast number of different wireframe architectures of arbitrary shape can now be designed from the top down using algorithms such as DAEDALUS (; http://daedalus-dna-origami.org/) and PERDIX (; http://perdix-dna-origami.org/). Adapted with permission from References and . (c) Biomolecular functionalization on wireframe objects can be controlled with single-molecule precision. Strategies for the functionalization of staples at 3′ or 5′ locations include hybridization of single-stranded DNA overhangs with complementary locked nucleic acid or peptide nucleic acid sequences and direct, covalent chemical modifications.

Figure 2

Computational design strategies for scaffolded DNA origami nanostructures. Following the early development of the bottom-up sequence design program caDNAno for rectilinear, brick-like scaffolded DNA origami assemblies, several approaches for the top-down, fully automated design of wireframe scaffolded DNA origami objects were developed, including PERDIX, METIS, DAEDALUS, TALOS, and vHelix-BSCOR. (a) Sequence designs from caDNAno can be imported into the online software CanDo to predict three-dimensional (3D) solution structures. Adapted with permission from References , , , and . (b) Both PERDIX and METIS enable the design of any free-form 2D geometry using either DX- or honeycomb-based edges, respectively. Adapted with permission from Reference . (c) DAEDALUS and TALOS program arbitrary 3D polyhedral geometries using DX- or honeycomb-based edges, respectively. Adapted with permission from References and . (d) vHelix-BSCOR enables the semiautomated top-down design of both 2D and 3D wireframe objects using predominantly single-helix edge architectures (right). Adapted with permission from References and . Structural characterization of the target objects is typically achieved using atomic force microscopy for 2D assemblies (a, bottom left, b, bottom left, and d, bottom left, where the white arrow indicates blunt-end stacking of Rothemund rectangles in panel a), cryo-electron microscopy with 3D reconstruction (c, bottom left and right) or transmission electron microscopy (a, bottom right; b, bottom right; d, bottom right)

Figure 3

Natural photosynthetic light-harvesting systems. (a) Schematic representation of a light-harvesting complex in the green sulfur bacterium. Chlorosomes have high-absorption cross sections that harvest light and transfer energy to the Fenna-Matthews-Olson (FMO) complex through the baseplate. Absorbed energy is then transferred to reaction centers. (b) Illustrative representation of the phycobilisome light-harvesting antennae. Bilin-containing proteins, phycoerythrin, phycocyanin, and allophycocyanin, absorb light in the green, orange, and red regions of the visible light spectrum. The energy-transfer cascade from the phycoerythrin to allophycocyanin via phycocyanin funnels the energy to the photosystem I and II reaction centers (59, 60, 119). (c) Protein scaffolds in the FMO control the spatial organization and influence the site energies of bacteriochlorophyll pigments (184) [Protein Data Bank identification (PDB ID) 3EOJ]. (d) The light-harvesting complex (LH2) in purple bacteria has a characteristic circular structure, which contains the B800 and B850 rings (PDB ID 2FKW) (31). Proteins control the spatial organization of bacteriochlorophyll pigments in the B800 and B850 rings. These different organizations lead to exciton delocalization (118) and fast energy transfer (105).

Figure 4

Biologically inspired artificial light-harvesting systems. (a) Three-dimensional energy transfer on DNA duplex bundles. DNA nanostructures enable the controlled variation of the distances and numbers of dyes scaffolded to investigate directed energy transfer. Adapted with permission from Reference . (b) The energy-transfer efficiency of DNA-templated PIC J-aggregates to Alexa Fluor® 647 decreases with the length of the DNA template due to static disorder. Adapted with permission from Reference . (c) The sequence-selectivity of PIC aggregation presents an opportunity to create higher-order excitonic circuits to understand the dynamics of interaggregate energy transfer. Adapted with permission from Reference . (d) DNA nanostructures can be leveraged as designer nanoscale scaffolds to understand energy funneling and directed energy transport mechanisms that are typically found in natural light-harvesting systems. The ability to program light-harvesting structures across different length scales using DNA, from the nanoscale distance of dyes to the microscale organization of light-harvesting DNA constructs (102), provides a path toward mimicking photosynthesis. Adapted with permission from Reference . Abbreviations: AF, Alexa Fluor® AF647, Alexa Fluor® 647; bp, base pair; Cy3, C3-indocyanine; PIC, pseudoisocyanine; hv, energy of an incoming photon; hv′, energy of an outgoing photon; J-bit, specific, noncovalent complex of aggregated PIC monomers templated by an A-tract of duplex DNA.

Figure 5

RNA tertiary structure and function in natural and synthetic systems. (a) The structure of the ribosome has been solved to atomic resolution (top) [Protein Data Bank identification (PDB ID) 1GIY and 1JGO; References 198, 199], which has yielded biological insight into ribosome activity (bottom; adapted with permission from Reference 7), including coordination of tRNAs in the peptidyl-transferase center (PTC). (b) Folding intermediates of the ribosome show late assembly of an active PTC (adapted with permission from Reference 124), with the active conformation in the bottom panel (PDB ID 4V6F; Reference 82). (c) DNA origami objects of different sizes and shapes may be used to coordinate RNA structures, modeled for size comparison here with a tRNA (PDB ID 1WZ2), RNase P (PDB ID 3Q1R; Reference 140), group II intron (PDB ID 4DS6; Reference 29), and the 50S subunit of a ribosome (PDB ID 2WWQ; Reference 155), for applications in RNA structural biology (top) and synthetic catalysts (bottom) with a hypothetical minimized PTC, shown in blue (modified from PDB ID 1GIY; Reference 198), modeled into an octahedral origami. The P-site tRNA is shown in red, the A-site tRNA in green, and the E-site tRNA in purple.

Figure 6

Templated antigen and ligand systems to probe immune cell surface receptor signaling. (a) Schematic of TCR and BCR activation showing the importance of the nanoscale organization of receptors on triggering immune responses. (b) Effects of TCR ligand density and cluster size on TCR activation (left). Adapted with permission from Reference . A minimum number of TCR ligands is needed to induce T cell synapse formation (middle). Adapted with permission from Reference . DNA origami has been used to present ligands at specific distances to study receptor activation (right). Adapted with permission from Reference . (c) DNA origami nanoparticles can be designed to investigate the effects of antigen distance, stoichiometry, dimensionality, and multiplexing through site-specific control of functionalization. Additionally, DNA origami can be used for surface patterning to study the effect of antigen precluster distance on immune receptor activation. Atomic force microscopy data adapted with permission from Reference . Abbreviations: APC, antigen-presenting cell; BCR, B cell receptor; CD4/8, cluster of differentiation 4/8; Igα/β, immunoglobulin-α/β; MHC, major histocompatibility complex; RICM, reflection interference contrast microscopy; TCR, T cell receptor; Syk, spleen tyrosine kinase; Lyn, Lck/Yes novel tyrosine kinase.