US 12,116,603 B2 · RNA silencing nanozymes

Abstract

Disclosed herein are improved nanozymes for targeting RNA. The disclosed nanozymes are synthesized using recombinant RNase A with site-specific cysteine-substituted mutations that can be covalently functionalized with a length-tunable multi-thiol tether and then loaded onto gold particles through multiple gold-sulfur bonds. This new RNase A loading mechanism is site specific, and it allows high-density loading of alkylthiol modified DNA oligonucleotides. The disclosed nanozymes can also include additional capturer strands and/or involve DNA-recombinant-RNase-A unibodies to further increase the nanozyme's enzymatic activity and target selectivity. Also disclosed are functional on-off switchable nanozymes to control nanozyme activity. In some embodiments, the disclosed nanozyme are core-free hollow forms. The removal of the inorganic nanoparticle cores from nanozymes can effectively eliminate the potential long-term toxicity induced by the core, and also creates a cavity for loading and delivery of small molecule drugs.

Claims8

Dependent claims

A nanozyme comprising a gold nanoparticle, an engineered ribonuclease-A enzyme and a DNA oligonucleotide,

wherein the DNA oligonucleotide comprises a first RNA recognition moiety comprising a nucleic acid sequence complementary to a first region of an RNA target,
wherein the engineered ribonuclease-A enzyme comprises a cysteine substitution, wherein the cysteine is functionalized by the method of reacting the cysteine with propargyl-maleimide to introduce an alkynyl group;
synthesizing a PEG tether using 3′-dithiol serinol CPG, spacer phosphoramidite 18, and 5′-bromohexyl phosphoramidite, which introduces a 5′-bromide onto the PEG tether, converting the 5′-bromide into an azide using sodium azide, and using click chemistry to link the azide of the PEG tether to the alkynyl group on the cysteine,
wherein the DNA oligonucleotide is attached to the gold nanoparticle directly or indirectly by gold-sulfur bonds, and
wherein the RNA target is cleaved by the engineered ribonuclease-A enzyme when the first RNA recognition moiety binds to the RNA target.

2 more elements

Dependencies are inferred from the claim text.

RNA silencing nanozymes

Life of this patent

RNA silencing nanozymes

Life of this patent

Abstract

Claims8