Molecular DNA dendron vaccines.

A foundational principle of rational vaccinology is that vaccine structure plays a critical role in determining therapeutic efficacy, but in order to establish fundamental, effective, and translatable vaccine design parameters, a highly modular and well-defined platform is required. Herein, we report a DNA dendron vaccine, a molecular nanostructure that consists of an adjuvant DNA strand that splits into multiple DNA branches with a varied number of conjugated peptide antigens that is capable of dendritic cell uptake, immune activation, and potent cancer killing. We leveraged the well-defined architecture and chemical modularity of the DNA dendron to study structure-function relationships that dictate molecular vaccine efficacy, particularly regarding the delivery of immune-activating DNA sequences and antigenic peptides on a single chemical construct. We investigated how adjuvant and antigen placement and number impact dendron cellular uptake and immune activation, in vitro. These parameters also played a significant role in raising a potent and specific immune response against target cancer cells. By gaining this structural understanding of molecular vaccines, DNA dendrons successfully treated a mouse cervical human papillomavirus TC-1 cancer model, in vivo, where the vaccine structure defined its efficacy; the top-performing design effectively reduced tumor burden (<150 mm3 through day 30) and maintained 100% survival through 44 d after tumor inoculation.

Symmetry-Breaking Dendrimer Synthons in Colloidal Crystal Engineering with DNA.

Breaking symmetry in colloidal crystals is challenging due to the inherent chemical and structural isotropy of many nanoscale building blocks. If a non-particle component could be used to anisotropically encode such building blocks with orthogonal recognition properties, one could expand the scope of structural and compositional possibilities of colloidal crystals beyond what is thus far possible with purely particle-based systems. Herein, we report the synthesis and characterization of novel DNA dendrimers that function as symmetry-breaking synthons, capable of programming anisotropic and orthogonal interactions within colloidal crystals. When the DNA dendrimers have identical sticky ends, they hybridize with DNA-functionalized nanoparticles to yield three distinct colloidal crystals, dictated by dendrimer size, including a structure not previously reported in the field of colloidal crystal engineering, Si2Sr. When used as symmetry-breaking synthons (when the sticky ends deliberately consist of orthogonal sequences), the synthesis of binary and ternary colloidal alloys with structures that can only be realized through directional interactions is possible. Furthermore, by modulating the extent of shape anisotropy within the DNA dendrimers, the local distribution of the nanoparticles within the crystals can be directed.

Transferrin Aptamers Increase the In Vivo Blood-Brain Barrier Targeting of Protein Spherical Nucleic Acids.

The systemic delivery of exogenous proteins to cells within the brain and central nervous system (CNS) is challenging due to the selective impermeability of the blood-brain barrier (BBB). Herein, we hypothesized that protein delivery to the brain could be improved via functionalization with DNA aptamers designed to bind transferrin (TfR) receptors present on the endothelial cells that line the BBB. Using ß-galactosidase (ß-Gal) as a model protein, we synthesized protein spherical nucleic acids (ProSNAs) comprised of ß-Gal decorated with TfR aptamers (Transferrin-ProSNAs). The TfR aptamer motif significantly increases the accumulation of ß-Gal in brain tissue in vivo following intravenous injection over both the native protein and ProSNAs containing nontargeting DNA sequences. Furthermore, the widespread distribution of ß-Gal throughout the brain is only observed for Transferrin-ProSNAs. Together, this work shows that the SNA architecture can be used to selectively deliver protein cargo to the brain and CNS if the appropriate aptamer sequence is employed as the DNA shell. Moreover, this highlights the importance of DNA sequence design and provides a potential new avenue for designing highly targeted protein delivery systems by combining the power of DNA aptamers together with the SNA platform.

Spherical nucleic acids as an infectious disease vaccine platform.

SignificanceUsing SARS-CoV-2 as a relevant case study for infectious disease, we investigate the structure-function relationships that dictate antiviral spherical nucleic acid (SNA) vaccine efficacy. We show that the SNA architecture can be rapidly employed to target COVID-19 through incorporation of the receptor-binding domain, and that the resulting vaccine potently activates human cells in vitro and mice in vivo. Furthermore, when challenged with a lethal viral infection, only mice treated with the SNA vaccine survived. Taken together, this work underscores the importance of rational vaccine design for infectious disease to yield vaccines that elicit more potent immune responses to effectively fight disease.

Nanoparticle Superlattices through Template-Encoded DNA Dendrimers.

The chemical interactions that lead to the emergence of hierarchical structures are often highly complex and difficult to program. Herein, the synthesis of a series of superlattices based upon 30 different structurally reconfigurable DNA dendrimers is reported, each of which presents a well-defined number of single-stranded oligonucleotides (i.e., sticky ends) on its surface. Such building blocks assemble with complementary DNA-functionalized gold nanoparticles (AuNPs) to yield five distinct crystal structures, depending upon choice of dendrimer and defined by phase symmetry. These DNA dendrimers can associate to form micelle-dendrimers, whereby the extent of association can be modulated based upon surfactant concentration and dendrimer length to produce a low-symmetry Ti5Ga4-type phase that has yet to be reported in the field of colloidal crystal engineering. Taken together, colloidal crystals that feature three different types of particle bonding interactions-template-dendron, dendrimer-dendrimer, and DNA-modified AuNP-dendrimer-are reported, illustrating how sequence-defined recognition and dynamic association can be combined to yield complex hierarchical materials.

A General DNA-Gated Hydrogel Strategy for Selective Transport of Chemical and Biological Cargos.

The selective transport of molecular cargo is critical in many biological and chemical/materials processes and applications. Although nature has evolved highly efficient in vivo biological transport systems, synthetic transport systems are often limited by the challenges associated with fine-tuning interactions between cargo and synthetic or natural transport barriers. Herein, deliberately designed DNA-DNA interactions are explored as a new modality for selective DNA-modified cargo transport through DNA-grafted hydrogel supports. The chemical and physical characteristics of the cargo and hydrogel barrier, including the number of nucleic acid strands on the cargo (i.e., the cargo valency) and DNA-DNA binding strength, can be used to regulate the efficiency of cargo transport. Regimes exist where a cargo-barrier interaction is attractive enough to yield high selectivity yet high mobility, while there are others where the attractive interactions are too strong to allow mobility. These observations led to the design of a DNA-dendron transport tag, which can be used to universally modify macromolecular cargo so that the barrier can differentiate specific species to be transported. These novel transport systems that leverage DNA-DNA interactions provide new chemical insights into the factors that control selective cargo mobility in hydrogels and open the door to designing a wide variety of drug/probe-delivery systems.

DNA Dendrons as Agents for Intracellular Delivery.

Herein, a method for synthesizing and utilizing DNA dendrons to deliver biomolecules to living cells is reported. Inspired by high-density nucleic acid nanostructures, such as spherical nucleic acids, we hypothesized that small clusters of nucleic acids, in the form of DNA dendrons, could be conjugated to biomolecules and facilitate their cellular uptake. We show that DNA dendrons are internalized by 90% of dendritic cells after just 1 h of treatment, with a >20-fold increase in DNA delivery per cell compared with their linear counterparts. This effect is due to the interaction of the DNA dendrons with scavenger receptor-A on cell surfaces, which results in their rapid endocytosis. Moreover, when conjugated to peptides at a single attachment site, dendrons enhance the cellular delivery and activity of both the model ovalbumin 1 peptide and the therapeutically relevant thymosin alpha 1 peptide. These findings show that high-density, multivalent DNA ligands play a significant role in dictating cellular uptake of biomolecules and consequently will expand the scope of deliverable biomolecules to cells. Indeed, DNA dendrons are poised to become agents for the cellular delivery of many molecular and nanoscale materials.