Control of the Assembly and Disassembly of Spherical Nucleic Acids Is Critical for Enhanced Gene Silencing.

Spherical nucleic acids─nanospheres with nucleic acids on their corona─have emerged as a promising class of nanocarriers, aiming to address the shortcomings of traditional nucleic therapeutics, namely, their poor stability, biodistribution, and cellular entry. By conjugating hydrophobic monomers to a growing nucleic acid strand in a sequence-defined manner, our group has developed self-assembled spherical nucleic acids (SaSNAs), for unaided, enhanced gene silencing. By virtue of their self-assembled nature, SaSNAs can disassemble under certain conditions in contrast to covalent or gold nanoparticle SNAs. Gene silencing involves multiple steps including cellular uptake, endosomal escape, and therapeutic cargo release. Whether assembly vs disassembly is advantageous to any of these steps has not been previously studied. In this work, we modify the DNA and hydrophobic portions of SaSNAs and examine their effects on stability, cellular uptake, and gene silencing. When the linkages between the hydrophobic units are changed from phosphate to phosphorothioate, we find that the SaSNAs disassemble better in endosomal conditions and exhibit more efficacious silencing, despite having cellular uptake similar to that of their phosphate counterparts. Thus, disassembly in the endolysosomal compartments is advantageous, facilitating the release of the nucleic acid cargo and the interactions between the hydrophobic units and endosomal lipids. We also find that SaSNAs partially disassemble in serum to bind albumin; the disassembled, albumin-bound strands are less efficient at cellular uptake and gene silencing than their assembled counterparts, which can engage scavenger receptors for internalization. When the DNA portion is cross-linked by G-quadruplex formation, disassembly decreases and cellular uptake significantly increases. However, this does not translate to greater gene silencing, again illustrating the need for disassembly of the SaSNAs when they are in the endosome. This work showcases the advantages of the dual nature of SaSNAs for gene silencing, requiring extracellular assembly and disassembly inside the cell compartments.

Minimalist Design of Wireframe DNA Nanotubes: Tunable Geometry, Size, Chirality, and Dynamics.

DNA nanotubes (NTs) have attracted extensive interest as artificial cytoskeletons for biomedical, synthetic biology, and materials applications. Here, we report the modular design and assembly of a minimalist yet robust DNA wireframe nanotube with tunable cross-sectional geometry, cavity size, chirality, and length, while using only four DNA strands. We introduce an h-motif structure incorporating double-crossover (DX) tile-like DNA edges to achieve structural rigidity and provide efficient self-assembly of h-motif-based DNA nanotube (H-NT) units, thus producing programmable, micrometer-long nanotubes. We demonstrate control of the H-NT nanotube length via short DNA modulators. Finally, we use an enzyme, RNase H, to take these structures out of equilibrium and trigger nanotube assembly at a physiologically relevant temperature, underlining future cellular applications. The minimalist H-NTs can assemble at near-physiological salt conditions and will serve as an easily synthesized, DNA-economical modular template for biosensors, plasmonics, or other functional materials and as cost-efficient drug-delivery vehicles for biomedical applications.