The therapeutic promise of small interfering RNAs （siRNAs） for specific gene silencing is dependent on the successful delivery of functional siRNAs to the cytoplasm. Their conjugation to an established delivery plat...The therapeutic promise of small interfering RNAs （siRNAs） for specific gene silencing is dependent on the successful delivery of functional siRNAs to the cytoplasm. Their conjugation to an established delivery platform, such as gold nanoparticles, offers tremendous potential for treating diseases and advancing our understanding of cellular processes. Their success or failure is dependent on both the uptake of the nanoparticles into the cells and subsequent intracellular release of the functional siRNA. In this study, utilizing gold nanoparticle siRNA-mediated delivery against C-MYC, we aimed to determine if we could achieve knockdown in a cancer cell line with low levels of intracellular glutathione, and determine the influence, if any, of polyethylene glycol （PEG） ligand density on knockdown, with a view to determining the optimal nanoparticle design to achieve C-MYC knockdown. We demonstrate that, regardless of the PEG density, knockdown in cells with relatively low glutathione levels can be achieved, as well as the possible effect of steric hindrance of PEG on the availability of the siRNA for cleavage in the intracellular environment. Gold nanoparticle uptake was demonstrated via transmission electron microscopy and mass spectroscopy, while knockdown was determined at the protein and physiological levels （cells in S-phase） by in-cell westerns and BrdU incorporation, respectively.展开更多
Genetic transformation in some plant species, including cotton （Gossypium hirsutum）, is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing （VIGS） by double-stra...Genetic transformation in some plant species, including cotton （Gossypium hirsutum）, is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing （VIGS） by double-stranded RNAs can serve as a reverse-genetics tool to determine gene function. However, knockdown levels vary greatly when using a tobacco rattle virus-based vector that carries different cDNA fragments of a gene. How to choose the optional target fragment for high interference efficiency is very challenging. Addressing this challenge requires increasing the efficacy of small interference RNA （siRNA） in target fragment. Here, we describe a method to assess VIGS efficiency by comparing the following parameters of siRNA in target sequence： the disruptionenergy of the target （△Gdisruption）, the differential stability of siRNA duplex ends （DSSE）, and the internal stability at positions 9-14 of the siRNA antisense strand （AIS）, which are calculated by Sfold program （http：//sfold.wadsworth. org）. We find that the siRNAs with low mGdisruption, high DSSE and high AIS have high activity and easily result in high VIGS efficiency by experimentally testing the actual knockdown levels of the four target genes, GhPDS, GhCLA1, GhAOS1, and GhCXE1 via choosing different target sequences for each gene. Therefore, the Sfold pro- gram can be used to analyze target sequences when car- rying out VIGS design to increase gene-silencing effects in plants.展开更多
文摘The therapeutic promise of small interfering RNAs （siRNAs） for specific gene silencing is dependent on the successful delivery of functional siRNAs to the cytoplasm. Their conjugation to an established delivery platform, such as gold nanoparticles, offers tremendous potential for treating diseases and advancing our understanding of cellular processes. Their success or failure is dependent on both the uptake of the nanoparticles into the cells and subsequent intracellular release of the functional siRNA. In this study, utilizing gold nanoparticle siRNA-mediated delivery against C-MYC, we aimed to determine if we could achieve knockdown in a cancer cell line with low levels of intracellular glutathione, and determine the influence, if any, of polyethylene glycol （PEG） ligand density on knockdown, with a view to determining the optimal nanoparticle design to achieve C-MYC knockdown. We demonstrate that, regardless of the PEG density, knockdown in cells with relatively low glutathione levels can be achieved, as well as the possible effect of steric hindrance of PEG on the availability of the siRNA for cleavage in the intracellular environment. Gold nanoparticle uptake was demonstrated via transmission electron microscopy and mass spectroscopy, while knockdown was determined at the protein and physiological levels （cells in S-phase） by in-cell westerns and BrdU incorporation, respectively.
基金supported by Major Program of Joint Funds (Sinkiang) of the National Natural Science Foundation of China (No. U1303282)
文摘Genetic transformation in some plant species, including cotton （Gossypium hirsutum）, is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing （VIGS） by double-stranded RNAs can serve as a reverse-genetics tool to determine gene function. However, knockdown levels vary greatly when using a tobacco rattle virus-based vector that carries different cDNA fragments of a gene. How to choose the optional target fragment for high interference efficiency is very challenging. Addressing this challenge requires increasing the efficacy of small interference RNA （siRNA） in target fragment. Here, we describe a method to assess VIGS efficiency by comparing the following parameters of siRNA in target sequence： the disruptionenergy of the target （△Gdisruption）, the differential stability of siRNA duplex ends （DSSE）, and the internal stability at positions 9-14 of the siRNA antisense strand （AIS）, which are calculated by Sfold program （http：//sfold.wadsworth. org）. We find that the siRNAs with low mGdisruption, high DSSE and high AIS have high activity and easily result in high VIGS efficiency by experimentally testing the actual knockdown levels of the four target genes, GhPDS, GhCLA1, GhAOS1, and GhCXE1 via choosing different target sequences for each gene. Therefore, the Sfold pro- gram can be used to analyze target sequences when car- rying out VIGS design to increase gene-silencing effects in plants.
