Spatially Localized Entropy-Driven Evolution of Nucleic Acid-Based Constitutional Dynamic Networks for Intracellular Imaging and Spatiotemporal Programmable Gene Therapy

The primer-guided entropy-driven high-throughput evolution of the DNA-based constitutional dynamic network, CDN, is introduced. The entropy gain associated with the process provides a catalytic principle for the amplified emergence of the CDN. The concept is applied to develop a programmable, spatially localized DNA circuit for effective in vitro and in vivo theranostic, gene-regulated treatment of cancer cells. The localized circuit consists of a DNA tetrahedron core modified at its corners with four tethers that include encoded base sequences exhibiting the capacity to emerge and assemble into a 2 × 2 CDN. Two of the tethers are caged by a pair of siRNA subunits, blocking the circuit into a mute, dynamically inactive configuration. In the presence of miRNA-21 as primer, the siRNA subunits are displaced, resulting in amplified release of the siRNAs silencing the HIF-1α mRNA and fast dynamic reconfiguration of the tethers into a CDN. The resulting CDN is, however, engineered to be dynamically reconfigured by miRNA-155 into an equilibrated mixture enriched with a DNAzyme component, catalyzing the cleavage of EGR-1 mRNA. The DNA tetrahedron nanostructure stimulates enhanced permeation into cancer cells. The miRNA-triggered entropy-driven reconfiguration of the spatially localized circuit leads to the programmable, cooperative bis-gene-silencing of HIF-1α and EGR-1 mRNAs, resulting in the effective and selective apoptosis of breast cancer cells and effective inhibition of tumors in tumor bearing mice.