Advanced Nanozyme Designs for Developing High Selectivity RNA Silencing Therapeutics

Increases Target Selectivity and Enzymatic Activity of Nanozymes and Improves their Synthesis Process

These nanozyme designs use a recombinant RNase A loading mechanism strong enough to support high-density surface packing of DNA oligonucleotides, facilitating stable and reproducible synthesis of nanozymes with high target selectivity and enzymatic activity for use as RNA silencing therapeutic agents. The market for RNA-based therapeutics and vaccines is projected to exceed $630 million by 2026. RNA-based therapies, such as RNA interference or antisense therapy, treat certain cancers and viral infections by preventing harmful protein translation through delivery of predesigned synthetic nucleic acids to targeted tissues and cells in order to neutralize messenger RNA. However, these therapies rely on cellular machineries such as RNA-induced silencing complex (RISC) or RNase H, which can disrupt natural gene regulation pathways, potentially causing toxicity and side effects. Nanozymes are engineered cellular machines that mimic the RNA silencing function of RISC without disrupting natural processes. For effective RNA silencing, a nanozyme must exhibit high target selectivity, which requires a high surface density of targeting oligonucleotides. Available designs for nanozymes with dense oligonucleotide packing have an unstable synthesis process that results in inconsistent synthetic yields of nanozymes with easily-diminishing enzymatic activity, which hinders their development as RNA-based therapeutic agents.

Researchers at the University of Florida have developed RNA silencing nanozymes that employ an RNase A loading mechanism to enable high-density loading of targeting oligonucleotides for increased RNA target selectivity. Preparation of these nanozymes is reproducible and achieves consistently higher synthetic yields with better, more stable enzymatic activity, expanding the application of nanozymes in RNA-based therapies for viral infections and cancers.

Application

RNA silencing nanozymes that exhibit better target selectivity and enzymatic activity and also have a stable, high-yield synthesis process

Advantages

• Supports high-density loading of DNA oligonucleotides, producing nanozymes highly selective to specific RNA targets, with low off-target effects
• Employs double capturer strands and combined DNA-enzyme unibodies, further increasing enzymatic activity and site selectivity, as well as minimized off-target effects
• Augments the bond strength of RNase A, producing nanozymes that maintain enzymatic activity and target selectivity over relatively-long term storage
• Stabilizes the nanozyme synthesis process, facilitating their clinical development into antiviral and anticancer therapeutics
• Incorporates nanozyme activity on-off switching controls, enabling specific targeting of cell types with certain environmental triggers
• Exists also in an inorganic-core-free hollow format, eliminating potential long-term toxicity associated with inorganic nanoparticles and creating an organic cavity to hold small molecule drugs for delivery

Technology

These designs enable controllable preparation of RNA silencing nanozymes with high target selectivity for use as a therapeutic for viral infections and cancers. Recombinant RNase A enzymes with cysteine-substituted mutations link with multi-thiol tethers. These multi-thiol functionalized enzymes then load onto gold nanoparticles through multiple gold-sulfur bonds. These bonds are strong enough to support a densely-packed arrangement of alkylthiol-modified DNA oligonucleotides around the enzyme, which provides the high level of RNA target selectivity necessary for efficient RNA silencing. The RNase A loading mechanism further supports optimizing the activity of the nanozyme through double capturer DNA strands, DNA-RNase-A unibodies, and multi-branched targeting oligonucleotides.