Break to Build: Isothermal Assembly of Nucleic Acid Nanoparticles (NANPs) via Enzymatic Degradation

Abstract

The intrinsic properties of RNA and DNA biopolymers emphasized by engineered nucleic acid nanoparticles (NANPs) offer accelerated development of next-generation therapies. The rational design of NANPs facilitates programmable architectures intended for regulated molecular and cellular interactions. The conventional bottom-up assembly of NANPs relies on the thermal annealing of individual strands. Here, we introduce a concept of nuclease-driven production of NANPs where selective digestion of functionally inert structures leads to isothermal selfassembly of liberated constituents. The working principles, morphological changes, assembly kinetics, and the retention of structural integrity for system components subjected to anhydrous processing and storage are assessed. We show that the assembly of precursors into a single structure improves stoichiometry and enhances the functionality of nuclease-driven products. Furthermore, the experiments with immune reporting cell lines show that the developed protocols retain the immunostimulatory functionality of tested NANPs. The presented approach enables exploitation of the advantages of conditionally produced NANPs and demonstrates that NANPs' stability, immunorecognition, and assembly can be regulated to allow for a more robust functional system.

Design and Characterization of Compact, Programmable, Multistranded Nonimmunostimulatory Nucleic Acid Nanoparticles Suitable for Biomedical Applications

“…In our study, we conduct a series of experiments to assess whether DNA triangles with various ssT regions would elicit an immune response or prove to be toxic to human cells. We utilize three different reporter cell lines (HEK-Blue hTLR7, HEK-Blue hTLR9, and HEK-Lucia RIG-I) engineered to turn up the activation of specific pathways related to the detection of exogenous nucleic acids. − While TLR9 is a well-known key receptor for the recognition of DNA, TLR7 primarily recognizes ssRNAs. However, it has been reported that TLR7 also has the ability to respond to deoxyguanosine, independent of RNA. , Furthermore, recent studies have indicated the possible recognition of DNA nanoparticles by TLR7 .…”

The motive forces in DNA-enabled nanomachinery

“…There have been several profound studies on bioproduction of ssDNA oligonucleotides. 119 Combining isothermal assembly assisted by enzymes 120 and kinetics control, it may be feasible to harvest designed DNA nanomachines with microbial production in a prescribed manner. With the fast developed computer-aided sequence design with cross-linked reactions and a sufficient supply of DNA materials, we believe DNA-enabled synthetic nanomachines with spatial and temporal controls can accomplish specified assignments in real-life biomedical and clinical applications.…”

A Photoresponsive Homing Endonuclease for Programmed DNA Cleavage

“…For gene-editing applications where cleavage precision is paramount, higher fidelity is first required before the photoresponsive I-TevI nuclease can be fully exploited. The modular I-TevI nuclease is however well suited for in vitro purposes and may have applications for controlling the top-down synthesis of DNA nanostructures for 2D and 3D DNA architectures and microarrays and the release of molecular payloads from DNA-based precursors. , As these applications typically do not require high fidelity, the light-responsive I-TevI nuclease developed here could be applied without further engineering for optical control. In summary, we have demonstrated that monomeric homing endonucleases can be reengineered for optical control with programmable specificity by fusion of independent functional domains to LOV domain photoreceptors with notable dynamic range for a first-generation platform.…”