RNA silencing nanozymes
View Patent ↗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.
1. 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. The nanozyme of claim 1 , further comprising a second RNA recognition moiety comprising a nucleic acid sequence complementary to a second region of the RNA target, wherein the second RNA recognition moiety is found on a separate DNA oligonucleotide independently attached to the gold nanoparticle or is attached to the first RNA recognition moiety by a brancher molecule.
3. The nanozyme of claim 1 , wherein the DNA oligonucleotide is thiol-modified and is directly attached to the gold nanoparticle by a gold-sulfur bond.
4. The nanozyme of claim 2 , wherein the second RNA recognition moiety is located on a second thiol-modified DNA oligonucleotide attached to the nanoparticle by a gold-sulfur bond.
5. The nanozyme of claim 2 , wherein the brancher molecule comprises a 5-Me-dC brancher.
6. The nanozyme of claim 1 , wherein the DNA oligonucleotide further comprises a blocker moiety comprising a nucleic acid sequence complementary to a blocker oligonucleotide, wherein binding of the blocker oligonucleotide to the blocker moiety sterically inhibits the RNA target from binding to the first RNA recognition site.
7. The nanozyme of claim 1 , wherein in the nanozyme, a unibody molecule is attached to the gold nanoparticle by a gold-sulfur bond, wherein the unibody molecule comprises (i) the recombinant RNase-A enzyme, and (ii) the first RNA recognition moiety and/or a second RNA recognition moiety.
8. A method for silencing RNA in a cell, comprising contacting the cell with the nanozyme of claim 1 .