Research On DNAzyme-based Circular Oligonucleotide Drugs For High-efficiency In Vivo Gene Silencing

Oligonucleotide drugs have been used widely as therapeutic agents for gene therapy.Multiple types of oligonucleotide drugs have been utilized for the treatment of diseases ranging from viral infections to hereditary disorders and cancers.However,their instability in biological media and inefficiency for intracellular delivery remain major hurdles for practical in vivo applications.Herein,we report a circular Y-shaped aptamer-DNAzyme conjugate(cYAD)for highly efficient in vivo gene silencing via RNA cleavage,which can be employed in malignant tumors gene therapy.Based on this concept,this project uses the Sgc8aptamer that specifically binds to the PTK 7 protein and the widely used Na~+-dependent Na A43 DNAzyme to construct cYAD.Through the programmed annealing process with precise temperature control,under the action of T4 DNA Ligase,the Sgc8 aptamer and Na A43 DNAzyme are connected at a molar ratio of 2:1to form cYAD.Systemically studies have shown that the cyclization of Sgc8 aptamer and Na A43 DNAzyme DNA structure not only retains its function,but also improves its stability in physiological environment.In addition,the bivalent aptamer motif provides specific and enhanced target cell targeting capabilities,which can accurately identify targets in complex biological environments to achieve specific targeting of tumor cells by nanocarriers,for accumulation and retention of the oligonucleotide drugs at the tumor site.In the actual delivery process,cYAD enters the cell through PTK 7-mediated endocytosis and is stored stably in the cytoplasm.A large amount of Na~+in the cytoplasm will activate the catalytic activity of DNAzyme and quickly cleavge the target m RNA(MET),thereby inhibiting the expression of MET protein,inhibiting cell proliferation,and inducing cell apoptosis.Moreover,different cell models have proved that cYAD is based on the specificity of gene silencing of cell receptor binding.The programmability of the cYAD sequence,that is,between different nucleic acid aptamers and DNAzymes,or even the combination of nucleic acid aptamers and DNAzymes in different proportions,greatly expands its potential to be applied to different diseases.We have confirmed in cell experiments and animal experiments that compared with single-stranded aptamers,single-stranded DNAzymes,the circular Y-shaped aptamer-DNAzyme conjugate exhibits efficient gene silencing performance in vivo.The proposal of this strategy provides new ideas and methods for oligonucleotide drugs to achieve precise and efficient gene therapy in vivo.