High-Affinity Host-Guest Recognition for Efficient Assembly and Enzymatic Responsiveness of DNA Nanostructures.

The combination of multiple orthogonal interactions enables hierarchical complexity in self-assembled nanoscale materials. Here, efficient supramolecular polymerization of DNA origami nanostructures is demonstrated using a multivalent display of small molecule host-guest interactions. Modification of DNA strands with cucurbit[7]uril (CB[7]) and its adamantane guest, yielding a supramolecular complex with an affinity of order 1010 m-1 , directs hierarchical assembly of origami monomers into 1D nanofibers. This affinity regime enables efficient polymerization; a lower-affinity ß-cyclodextrin-adamantane complex does not promote extended structures at a similar valency. Finally, the utility of the high-affinity CB[7]-adamantane interactions is exploited to enable responsive enzymatic actuation of origami nanofibers assembled using peptide linkers. This work demonstrates the power of high-affinity CB[7]-guest recognition as an orthogonal axis to drive self-assembly in DNA nanotechnology.

Site-Specific Arrangement and Structure Determination of Minor Groove Binding Molecules in Self-Assembled Three-Dimensional DNA Crystals.

The structural analysis of guest molecules in rationally designed and self-assembling DNA crystals has proven an elusive goal since its conception. Oligonucleotide frameworks provide an especially attractive route toward studying DNA-binding molecules by using three-dimensional lattices with defined sequence and structure. In this work, we site-specifically position a suite of minor groove binding molecules, and solve their structures via X-ray crystallography as a proof-of-principle toward scaffolding larger guest species. Two crystal motifs were used to precisely immobilize the molecules DAPI, Hoechst, and netropsin at defined positions in the lattice, allowing us to control occupancy within the crystal. We also solved the structure of a three-ring imidazole-pyrrole-pyrrole polyamide molecule, which sequence-specifically packs in an antiparallel dimeric arrangement within the minor groove. Finally, we engineered a crystal designed to position both netropsin and the polyamide at two distinct locations within the same lattice. Our work elucidates the design principles for the spatial arrangement of functional guests within lattices and opens new potential opportunities for the use of DNA crystals to display and structurally characterize small molecules, peptides, and ultimately proteins of unknown structure.

A Self-Assembled Rhombohedral DNA Crystal Scaffold with Tunable Cavity Sizes and High-Resolution Structural Detail.

DNA is an ideal molecule for the construction of 3D crystals with tunable properties owing to its high programmability based on canonical Watson-Crick base pairing, with crystal assembly in all three dimensions facilitated by immobile Holliday junctions and sticky end cohesion. Despite the promise of these systems, only a handful of unique crystal scaffolds have been reported. Herein, we describe a new crystal system with a repeating sequence that mediates the assembly of a 3D scaffold via a series of Holliday junctions linked together with complementary sticky ends. By using an optimized junction sequence, we could determine a high-resolution (2.7 Å) structure containing R3 crystal symmetry, with a slight subsequent improvement (2.6 Å) using a modified sticky-end sequence. The immobile Holliday junction sequence allowed us to produce crystals that provided unprecedented atomic detail. In addition, we expanded the crystal cavities by 50 % by adding an additional helical turn between junctions, and we solved the structure to 4.5 Šresolution by molecular replacement.

Emerging applications of peptide-oligonucleotide conjugates: bioactive scaffolds, self-assembling systems, and hybrid nanomaterials.

Peptide-oligonucleotide conjugates (POCs) are covalent constructs that link a molecule like DNA to a synthetic peptide sequences. These materials merge the programmable self-assembly of oligonucleotides with the bioactivity and chemical diversity of polypeptides. Recent years have seen the widespread use of POCs in a range of fields, driven the by relative advantages of each molecular type. In this review, we will present an overview of the synthesis and application of POCs, with an emphasis on emerging areas where these molecules will have a unique impact. We first discuss two main strategies for synthesizing POCs from synthetic monomers such as phosphoramidites and functionalized amino acids. We then describe four key fields of research in POCs: (1) biomaterials for interfacing with, and controlling the behavior of cells; (2) hybrid self-assembling systems that balance peptide and oligonucleotide intermolecular forces; (3) template-enhanced coupling of POCs into larger molecules; and (4) display of peptides on self-assembled oligonucleotide scaffolds. We also highlight several promising areas for future applications in each of these four directions, and anticipate ever increasing uses of POCs in interdisciplinary research.

Tuning the Cavity Size and Chirality of Self-Assembling 3D DNA Crystals.

The foundational goal of structural DNA nanotechnology-the field that uses oligonucleotides as a molecular building block for the programmable self-assembly of nanostructured systems-was to use DNA to construct three-dimensional (3D) lattices for solving macromolecular structures. The programmable nature of DNA makes it an ideal system for rationally constructing self-assembled crystals and immobilizing guest molecules in a repeating 3D array through their specific stereospatial interactions with the scaffold. In this work, we have extended a previously described motif (4 × 5) by expanding the structure to a system that links four double-helical layers; we use a central weaving oligonucleotide containing a sequence of four six-base repeats (4 × 6), forming a matrix of layers that are organized and dictated by a series of Holliday junctions. In addition, we have assembled mirror image crystals (l-DNA) with the identical sequence that are completely resistant to nucleases. Bromine and selenium derivatives were obtained for the l- and d-DNA forms, respectively, allowing phase determination for both forms and solution of the resulting structures to 3.0 and 3.05 Å resolution. Both right- and left-handed forms crystallized in the trigonal space groups with mirror image 3-fold helical screw axes P32 and P31 for each motif, respectively. The structures reveal a highly organized array of discrete and well-defined cavities that are suitable for hosting guest molecules and allow us to dictate a priori the assembly of guest-DNA conjugates with a specified crystalline hand.

Using dynamic biomaterials to study the temporal role of bioactive peptides during osteogenesis.

The extracellular matrix is a highly dynamic environment, and the precise temporal presentation of biochemical signals is critical for regulating cell behavior during development, healing, and disease progression. To mimic this behavior, we developed a modular DNA-based hydrogel platform to enable independent and reversible control over the immobilization of multiple biomolecules during in vitro cell culture. We combined reversible DNA handles with a norbornene-modified hyaluronic acid hydrogel to orthogonally add and remove multiple biomolecule-DNA conjugates at user-defined timepoints. We demonstrated that the persistent presentation of the cell adhesion peptide RGD was required to maintain cell spreading on hyaluronic acid hydrogels. Further, we discovered the delayed presentation of osteogenic growth peptide (OGP) increased alkaline phosphatase activity compared to other temporal variations. This finding is critically important when considering the design of OGP delivery approaches for bone repair. More broadly, this platform provides a unique approach to tease apart the temporal role of multiple biomolecules during development, regeneration, and disease progression.

Using dynamic biomaterials to study the temporal role of osteogenic growth peptide during osteogenesis.

The extracellular matrix is a highly dynamic environment, and the precise temporal presentation of biochemical signals is critical for regulating cell behavior during development, healing, and disease progression. To mimic this behavior, we developed a modular DNA-based hydrogel platform to enable independent and reversible control over the immobilization of multiple biomolecules during in vitro cell culture. We combined reversible DNA handles with a norbornene-modified hyaluronic acid hydrogel to orthogonally add and remove multiple biomolecule-DNA conjugates at user-defined timepoints. We demonstrated that the persistent presentation of the cell adhesion peptide RGD was required to maintain cell spreading on hyaluronic acid hydrogels. Further, we discovered the delayed presentation of osteogenic growth peptide (OGP) increased alkaline phosphatase activity compared to other temporal variations. This finding is critically important when considering the design of OGP delivery approaches for bone repair. More broadly, this platform provides a unique approach to tease apart the temporal role of multiple biomolecules during development, regeneration, and disease progression.

Site-specific arrangement and structure determination of minor groove binding molecules in self-assembled three-dimensional DNA crystals.

The structural analysis of guest molecules in rationally designed and self-assembling DNA crystals has proven elusive since its conception. Oligonucleotide frameworks provide an especially attractive route towards studying DNA-binding molecules by using three-dimensional lattices with defined sequence and structure. In this work, we site-specifically position a suite of minor groove binding molecules, and solve their structures via x-ray crystallography, as a proof-of-principle towards scaffolding larger guest species. Two crystal motifs were used to precisely immobilize the molecules DAPI, Hoechst, and netropsin at defined positions in the lattice, allowing us to control occupancy within the crystal. We also solved the structure of a three-ring imidazole-pyrrole-pyrrole polyamide molecule, which sequence-specifically packs in an anti-parallel dimeric arrangement within the minor groove. Finally, we engineered a crystal designed to position both netropsin and the polyamide at two distinct locations within the same lattice. Our work elucidates the design principles for the spatial arrangement of functional guests within lattices and opens new potential opportunities for the use of DNA crystals to display and structurally characterize small molecules, peptides, and ultimately proteins of unknown structure.

Proximity-enhanced synthesis of DNA-peptide-DNA triblock molecules.

We report a proximity-enhanced method to synthesize a peptide flanked by two different oligonucleotide handles. Our method relies on sequential bioorthogonal reactions, and partial hybridization of the second handle to the first. We demonstrate the synthesis of a protease-responsive DNA "latch" and a cyclic bioactive peptide using this method.

The influence of Holliday junction sequence and dynamics on DNA crystal self-assembly.

The programmable synthesis of rationally engineered crystal architectures for the precise arrangement of molecular species is a foundational goal in nanotechnology, and DNA has become one of the most prominent molecules for the construction of these materials. In particular, branched DNA junctions have been used as the central building block for the assembly of 3D lattices. Here, crystallography is used to probe the effect of all 36 immobile Holliday junction sequences on self-assembling DNA crystals. Contrary to the established paradigm in the field, most junctions yield crystals, with some enhancing the resolution or resulting in unique crystal symmetries. Unexpectedly, even the sequence adjacent to the junction has a significant effect on the crystal assemblies. Six of the immobile junction sequences are completely resistant to crystallization and thus deemed "fatal," and molecular dynamics simulations reveal that these junctions invariably lack two discrete ion binding sites that are pivotal for crystal formation. The structures and dynamics detailed here could be used to inform future designs of both crystals and DNA nanostructures more broadly, and have potential implications for the molecular engineering of applied nanoelectronics, nanophotonics, and catalysis within the crystalline context.

Uncovering temporospatial sensitive TBI targeting strategies via in vivo phage display.

The heterogeneous pathophysiology of traumatic brain injury (TBI) is a barrier to advancing diagnostics and therapeutics, including targeted drug delivery. We used a unique discovery pipeline to identify novel targeting motifs that recognize specific temporal phases of TBI pathology. This pipeline combined in vivo biopanning with domain antibody (dAb) phage display, next-generation sequencing analysis, and peptide synthesis. We identified targeting motifs based on the complementarity-determining region 3 structure of dAbs for acute (1 day post-injury) and subacute (7 days post-injury) post-injury time points in a preclinical TBI model (controlled cortical impact). Bioreactivity and temporal sensitivity of the targeting motifs were validated via immunohistochemistry. Immunoprecipitation-mass spectrometry indicated that the acute TBI targeting motif recognized targets associated with metabolic and mitochondrial dysfunction, whereas the subacute TBI motif was largely associated with neurodegenerative processes. This pipeline successfully discovered temporally specific TBI targeting motif/epitope pairs that will serve as the foundation for the next-generation targeted TBI therapeutics and diagnostics.

Protein Corona Inhibits Endosomal Escape of Functionalized DNA Nanostructures in Living Cells.

DNA nanostructures (DNs) can be designed in a controlled and programmable manner, and these structures are increasingly used in a variety of biomedical applications, such as the delivery of therapeutic agents. When exposed to biological liquids, most nanomaterials become covered by a protein corona, which in turn modulates their cellular uptake and the biological response they elicit. However, the interplay between living cells and designed DNs are still not well established. Namely, there are very limited studies that assess protein corona impact on DN biological activity. Here, we analyzed the uptake of functionalized DNs in three distinct hepatic cell lines. Our analysis indicates that cellular uptake is linearly dependent on the cell size. Further, we show that the protein corona determines the endolysosomal vesicle escape efficiency of DNs coated with an endosome escape peptide. Our study offers an important basis for future optimization of DNs as delivery systems for various biomedical applications.