Simple chemical modifications to oligonucleotide ends with hexaethylene glycol and hexanediol are shown to significantly increase nuclease resistance under serum conditions. The modified oligonucleotides were used to construct DNA prismatic cages in a single step and in quantitative yield. These cages further stabilize their strands towards nucleases, with lifetimes of 62 hours in serum. The cages contain a large number of single-stranded regions for functionalization, illustrating their versatility for biological applications.

Long-range ordering of DNA crossover tiles with blunt ends on lipid bilayers is investigated using atomic force microscopy. "Blunt-ended" tiles do not have single-stranded complementary ends, and thus instead of assembling via base-pairing, they can interact by π-stacking of their duplex ends. This work demonstrates that the balance of base π-stacking interactions between the ends of DNA duplexes, cholesterol-mediated DNA anchoring, and electrostatic DNA binding to supported lipid bilayers (SLBs) presents an opportunity to build dynamic materials with long-range order on a soft support. The tiles are shown to organize into novel tunable surface packing morphologies on the micrometer scale. This work focuses on three-point star (3PS) tiles that are either unmodified or modified with a cholesterol unit and investigates their interactions on supported lipid bilayers. On fluid bilayers, the cholesterol tiles form extended hexagonal arrays with few defects, while the unmodified tiles do not bind. In contrast, both modified and unmodified tiles bind to gel-phase bilayers and produce arrays of new organized morphologies. With increasing tile concentration, we observe a range of motifs, that progressively favor tile-tile packing over duplex-end π-stacking. These structures can selectively pattern domains of phase-separated lipid bilayers, and the patterning is also observed for four-arm cross-tiles. Dynamic blunt end contacts promote error correction and network reconfiguration to maximize favorable interactions with the substrate and are required for the observed tile organization. These results suggest that small blunt-ended tiles can be used as a platform to organize oligonucleotides, nanoparticles, and proteins into extensive networks at the interface with biologically relevant membrane systems or other soft surface materials for applications in cellular recognition, plasmonics, light harvesting, model systems for membrane protein assemblies, or analytical devices.

RNA interference (RNAi) is a powerful therapeutic strategy that induces gene silencing by targeting disease-causing mRNA and can lead to their removal through degradation pathways. The potential of RNAi is especially relevant in cancer therapy, as it can be designed to regulate the expression of genes involved in all stages of tumor development (initiation, growth, and metastasis). We have generated gene silencing 3D DNA prisms that integrate antisense oligonucleotide therapeutics at 1, 2, 4, and 6 positions. Synthesis of these structures is readily achieved and leads to the assembly of highly monodisperse and well-characterized structures. We have shown that antisense strands scaffolded on DNA cages can readily induce gene silencing in mammalian cells and maintain gene knockdown levels more effectively than single and double stranded controls through increased stability of bound antisense units.

We report the anchoring of 3D-DNA-cholesterol labeled cages on spherically supported lipid bilayer membranes (SSLBM) formed on silica beads, and their addressability through strand displacement reactions, controlled membrane orientation and templated dimerization. The bilayer-anchored cages can load three different DNA-fluorophores by hybridization to their "top" face (furthest from bilayer) and unload each of them selectively upon addition of a specific input displacement strand. We introduce a method to control strand displacement from their less accessible "bottom" face (closest to the bilayer), by adding cholesterol-substituted displacing strands that insert into the bilayer themselves in order to access the toehold region. The orientation of DNA cages within the bilayer is tunable by positioning multiple cholesterol anchoring units on the opposing two faces of the cage, thereby controlling their accessibility to proteins and enzymes. A population of two distinct DNA cages anchored to the SSLBMs exhibited significant membrane fluidity and have been directed into dimer assemblies on bilayer via input of a complementary linking strand. Displacement experiments performed on these anchored dimers indicate that removal of only one prism's anchoring cholesterol strand was not sufficient to release the dimers from the bilayer; however, removal of both cholesterol anchors from the dimerized prisms via two displacement strands cleanly released the dimers from the bilayer. This methodology allows for the anchoring of DNA cages on supported lipid bilayers, the control of their orientation and accessibility within the bilayer, and the programmable dimerization and selective removal of any of their components. The facile coupling of DNA to other functional materials makes this an attractive method for developing stimuli-responsive protein or nanoparticle arrays, drug releasing biomedical device surfaces and self-healing materials for light harvesting applications, using a highly modular, DNA-economic scaffold.

Here, we introduce a 3D-DNA construction method that assembles a minimum number of DNA strands in quantitative yield, to give a scaffold with a large number of single-stranded arms. This DNA frame is used as a core structure to organize other functional materials in 3D as the shell. We use the ring-opening metathesis polymerization (ROMP) to generate block copolymers that are covalently attached to DNA strands. Site-specific hybridization of these DNA-polymer chains on the single-stranded arms of the 3D-DNA scaffold gives efficient access to DNA-block copolymer cages. These biohybrid cages possess polymer chains that are programmably positioned in three dimensions on a DNA core and display increased nuclease resistance as compared to unfunctionalized DNA cages.

Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into 'gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.

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