The functionality of DNA biomacromolecules has been widely excavated,as therapeutic drugs,carriers,and functionalized modification derivatives.In this study,we developed a series of DNA tetrahedron nanocages(Td),via s...The functionality of DNA biomacromolecules has been widely excavated,as therapeutic drugs,carriers,and functionalized modification derivatives.In this study,we developed a series of DNA tetrahedron nanocages(Td),via synchronous conjugating different numbers of i-(X)and therapeutic siRNA on four vertexes of tetrahedral DNA nanocage(aX-Td@bsiRNA,a+b=4).This i-motif-conjugated Td exhibited good endosomal escape behaviours in A549 tumor cells,and the escape efficiency was affected by the number of i-motif.Furthermore,the downregulating mRNA and protein expression level of epidermal growth factor receptor(EGFR)caused by this siRNA embedded Td were verified in A549 cells.The tumor growth inhibition efficiency of the 2X-Td@2siRNA treated group in tumorbearing mice was significantly higher than that of non-i-motif-conjugated Td@2siRNA(3.14-fold)and free siRNA(3.63-fold).These results demonstrate a general strategy for endowing DNA nanostructures with endosomal escape behaviours to achieve effective in vivo gene delivery and therapy.展开更多
DNA methylation plays an important role in gene expression in eukaryote. But DNA methylation of transgene usually leads to target gene silencing in plant genetic engineering. In this research, reporter gene β-glu-cur...DNA methylation plays an important role in gene expression in eukaryote. But DNA methylation of transgene usually leads to target gene silencing in plant genetic engineering. In this research, reporter gene β-glu-curonidase (GUS) gene (uidA) was introduced into tobaccos via Agrobacterium-mediated transformation method, and the foreign uidA gene became inactive in some transgenic tobaccos. No mRNA of uidA was detected in these plants by Northern blotting analysis, and DNA methylation of promoter region was found. The results indicated that gene silencing might be caused by DNA methylation of promoter.展开更多
Although DNA methylation is known to play an important role in the silencing of transposable elements （TEs） and introduced transgenes, the mechanisms that generate DNA methylation-independent transcrip- tional silen...Although DNA methylation is known to play an important role in the silencing of transposable elements （TEs） and introduced transgenes, the mechanisms that generate DNA methylation-independent transcrip- tional silencing are poorly understood. Previous studies suggest that RNA-directed DNA methylation （RdDM） is required for the silencing of the RD29A-LUC transgene in the Arabidopsis rosl mutant back- ground with defective DNA demethylase. Loss of function of ARGONAUTE 4 （AGO4） gene, which encodes a core RdDM component, partially released the silencing of RD29A-LUC in the rosl/ago4 double mutant plants. A forward genetic screen was performed to identify the mutants with elevated RD29A-LUC trans- gene expression in the rosl/ago4 mutant background. We identified a mutation in the homologous gene of PRP31, which encodes a conserved pre-mRNA splicing factor that regulates the formation of the U4/ U6.U5 snRNP complex in fungi and animals. We previously demonstrated that the splicing factors ZOP1 and STA1 contribute to transcriptional gene silencing. Here, we reveal that Arabidopsis PRP31 associates with ZOP1, STA1, and several other splicing-related proteins, suggesting that these splicing factors are both physically and functionally connected. We show that Arabidopsis PRP31 participates in transcrip- tional gene silencing. Moreover, we report that PRP31, STA1, and ZOP1 are required for development and stress response. Under cold stress, PRP31 is not only necessary for pre-mRNA splicing but also for regulation of cold-responsive gene expression. Our results suggest that the splicing machinery has multiple functions including pre-mRNA splicing, gene regulation, transcriptional gene silencing, and stress response.展开更多
Effective and low toxicity delivery of siRNA is of great importance for clinical gene therapy. Herein, self-assembled DNA nanoparticles (NPs) based on rolling circle amplification (RCA) with a small interfering RNA (s...Effective and low toxicity delivery of siRNA is of great importance for clinical gene therapy. Herein, self-assembled DNA nanoparticles (NPs) based on rolling circle amplification (RCA) with a small interfering RNA (siRNA) payload were successfully developed as a facile and efficient siRNA delivery strategy. This intracellular gene silencing strategy exhibits various advantages including low toxicity, high efficiency, and good stability. The synthesized DNA NPs serve as siRNA carriers, protecting the siRNA against nuclease degradation. We demonstrate that the obtained self-assembled siRNA/NP/PEI system can successfully deliver enhanced green fluorescent protein (EGFP)-siRNA into HeLa cells, realizing the same EGFP knockdown efficiency with less toxicity as that of commercial Lipofectamine 2000.展开更多
基金supported by Double First-Class Innovation Team of China Pharmaceutical University(CPU2018GY40,China)National Mega-project for Innovative Drugs(2019ZX09721001,China)。
文摘The functionality of DNA biomacromolecules has been widely excavated,as therapeutic drugs,carriers,and functionalized modification derivatives.In this study,we developed a series of DNA tetrahedron nanocages(Td),via synchronous conjugating different numbers of i-(X)and therapeutic siRNA on four vertexes of tetrahedral DNA nanocage(aX-Td@bsiRNA,a+b=4).This i-motif-conjugated Td exhibited good endosomal escape behaviours in A549 tumor cells,and the escape efficiency was affected by the number of i-motif.Furthermore,the downregulating mRNA and protein expression level of epidermal growth factor receptor(EGFR)caused by this siRNA embedded Td were verified in A549 cells.The tumor growth inhibition efficiency of the 2X-Td@2siRNA treated group in tumorbearing mice was significantly higher than that of non-i-motif-conjugated Td@2siRNA(3.14-fold)and free siRNA(3.63-fold).These results demonstrate a general strategy for endowing DNA nanostructures with endosomal escape behaviours to achieve effective in vivo gene delivery and therapy.
基金This work was supported by the State "863" High-Tech Project (Grant Nos. Z-17-01-01 and BH-02-02-02) the National Natural Science Foundation of China (Grant No. 39989001) and Chinese Department of Science and Technology (Grant No. ZJY-B-01).
文摘DNA methylation plays an important role in gene expression in eukaryote. But DNA methylation of transgene usually leads to target gene silencing in plant genetic engineering. In this research, reporter gene β-glu-curonidase (GUS) gene (uidA) was introduced into tobaccos via Agrobacterium-mediated transformation method, and the foreign uidA gene became inactive in some transgenic tobaccos. No mRNA of uidA was detected in these plants by Northern blotting analysis, and DNA methylation of promoter region was found. The results indicated that gene silencing might be caused by DNA methylation of promoter.
文摘Although DNA methylation is known to play an important role in the silencing of transposable elements （TEs） and introduced transgenes, the mechanisms that generate DNA methylation-independent transcrip- tional silencing are poorly understood. Previous studies suggest that RNA-directed DNA methylation （RdDM） is required for the silencing of the RD29A-LUC transgene in the Arabidopsis rosl mutant back- ground with defective DNA demethylase. Loss of function of ARGONAUTE 4 （AGO4） gene, which encodes a core RdDM component, partially released the silencing of RD29A-LUC in the rosl/ago4 double mutant plants. A forward genetic screen was performed to identify the mutants with elevated RD29A-LUC trans- gene expression in the rosl/ago4 mutant background. We identified a mutation in the homologous gene of PRP31, which encodes a conserved pre-mRNA splicing factor that regulates the formation of the U4/ U6.U5 snRNP complex in fungi and animals. We previously demonstrated that the splicing factors ZOP1 and STA1 contribute to transcriptional gene silencing. Here, we reveal that Arabidopsis PRP31 associates with ZOP1, STA1, and several other splicing-related proteins, suggesting that these splicing factors are both physically and functionally connected. We show that Arabidopsis PRP31 participates in transcrip- tional gene silencing. Moreover, we report that PRP31, STA1, and ZOP1 are required for development and stress response. Under cold stress, PRP31 is not only necessary for pre-mRNA splicing but also for regulation of cold-responsive gene expression. Our results suggest that the splicing machinery has multiple functions including pre-mRNA splicing, gene regulation, transcriptional gene silencing, and stress response.
基金the National Natural Science Foundation of China (Nos.21432008,21721005 and 91753201)Major new drug innovation in the Ministry of Science and Technology (No.2017ZX09303013).
文摘Effective and low toxicity delivery of siRNA is of great importance for clinical gene therapy. Herein, self-assembled DNA nanoparticles (NPs) based on rolling circle amplification (RCA) with a small interfering RNA (siRNA) payload were successfully developed as a facile and efficient siRNA delivery strategy. This intracellular gene silencing strategy exhibits various advantages including low toxicity, high efficiency, and good stability. The synthesized DNA NPs serve as siRNA carriers, protecting the siRNA against nuclease degradation. We demonstrate that the obtained self-assembled siRNA/NP/PEI system can successfully deliver enhanced green fluorescent protein (EGFP)-siRNA into HeLa cells, realizing the same EGFP knockdown efficiency with less toxicity as that of commercial Lipofectamine 2000.
