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An efficient mechanism for the sequence‐specific inhibition of gene expression is RNA interference. In this process, double‐stranded RNA molecules induce cleavage of a selected target RNA (see picture). This technique has in recent years developed into a standard method of molecular biology. Successful applications in animal models have already led to the initiation of RNAi‐based clinical trials as a new therapeutic option.

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RNA interference (RNAi) mediated by small interfering RNA (siRNA) duplexes is a powerful therapeutic modality, but the translation of siRNAs from the bench into clinical application has been hampered by inefficient delivery in vivo. An innovative delivery strategy involves fusing siRNAs to a three-way junction (3WJ) motif derived from the phi29 bacteriophage prohead RNA (pRNA). Chimeric siRNA-3WJ molecules are presumed to enter the RNAi pathway through Dicer cleavage. Here, we fused siRNAs to the phi29 3WJ and two phylogenetically related 3WJs. We confirmed that the siRNA-3WJs are substrates for Dicer in vitro. However, our results reveal that siRNA-3WJs transfected into Dicer-deficient cell lines trigger potent gene silencing. Interestingly, siRNA-3WJs transfected into an Argonaute 2-deficient cell line also retain some gene silencing activity. siRNA-3WJs are most efficient when the antisense strand of the siRNA duplex is positioned 5′ of the 3WJ (5′-siRNA-3WJ) relative to 3′ of the 3WJ (3′-siRNA-3WJ). This work sheds light on the functional properties of siRNA-3WJs and offers a design rule for maximizing their potency in the human RNAi pathway.  相似文献   

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The analysis of small interfering RNA (siRNA) is important for gene function studies and drug developments. We employed CE to study the separation of siRNA ladder marker, which were ten double‐stranded RNA (dsRNA) fragments ranged from 20 to 1000 bp, in solutions of hydroxyethylcellulose (HEC) polymer with different concentrations and molecular weights (Mws). Migration mechanism of dsRNA during CE was studied by the mobility and resolution length (RL) plots. We found that the RL depended on not only the concentration of HEC, but also the Mw of HEC. For instance, RL of small dsRNA fragment was more influenced by concentration of high Mw HEC than large dsRNA fragment and RL of large dsRNA fragment was more influenced by concentration of low Mw HEC than small dsRNA fragment. In addition, we found electrophoretic evidence that the structure of dsRNA was more compact than dsDNA with the same length. In practice, we succeeded to separate the glyceraldehyde 3‐phosphate dehydrogenase siRNA in the mixture of the siRNA ladder marker within 4 min.  相似文献   

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As a new treatment technique,photothermal therapy(PTT) has aroused worldwide attention in cancer treatment,mainly due to its excellent absorption ability,easy regulation,and biodegradability.Photothermal conversion materials with enhanced permeability and retention effect can be targeted easily to tumor tissue.They can accumulate efficiently to tumor tissues and allow normal tissues and organs not to be affected by temperature,thus significantly helping to reduce the systemic toxicity and improve the antitumor effect.However,PTT alo ne often suffers from the rapeutic resistance and reduced therapeutic efficacy,due to photothermal nanomaterial-mediated fundamental cellular defense mechanism of heat shock response,which could be inhibited by small interfering RNA(siRNA).Nevertheless,photothermal conversion materials as an excellent siRNA delivery carrier may conside rably enhance the delivery efficiency of siRNA.Therefore,photothermal and RNA interfering(RNAi) synergistic therapy has recently aroused extensive attention in tumor treatment.In this review,we mainly summarize the recent advances of photothermal and RNAi synergistic therapy,including some synergistic therapeutic nanoplatforms of inorganic and organic photothermal materials and other combined therapies such as combining with small molecular antitumor agents or PDT/imaging.The combination of various treatment techniques may considerably improve the synergistic therapeutic effect of PTT and RNAi in the treatment of cancers.  相似文献   

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用实时荧光定量PCR方法检测56对人卵巢癌组织及对应的癌旁组织中iASPP(Inhibitor of ASPP fami-ly)mRNA表达水平,应用受试者工作特征曲线(Receiver operating characteristic curve,ROC曲线)分析癌组织与癌旁组织中iASPP mRNA表达水平,探索iASPP在卵巢癌发生中的作用。从细胞水平进一步研究其作用机制,用siRNA干扰的方法使iASPP表达降低后用Hoechst 33342和CCK-8分别检测iASPP沉默后对人类卵巢癌细胞株OVCA420的影响。结果显示,卵巢癌组织中iASPP表达较癌旁组织明显增高;iASPP沉默后,细胞凋亡增多,细胞增殖水平降低。据此推断,iASPP mRNA水平对卵巢癌的临床诊断具有一定价值,可作为卵巢癌治疗的一个重要靶点。  相似文献   

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RNAs are a promising class of therapeutics given their ability to regulate protein concentrations at the cellular level. Developing safe and effective strategies to deliver RNAs remains important for realizing their full clinical potential. Here, we develop lipid nanoparticle formulations that can deliver short interfering RNAs (for gene silencing) or messenger RNAs (for gene upregulation). Specifically, we study how the tail length, tail geometry, and linker spacing in diketopiperazine lipid materials influences LNP potency with siRNAs and mRNAs. Eight lipid materials are synthesized, and 16 total formulations are screened for activity in vitro; the lead material is evaluated with mRNA for in vivo use and demonstrates luciferase protein expression in the spleen. In undertaking this approach, not only do we develop synthetic routes to delivery materials, but we also reveal structural criteria that could be useful for developing next‐generation delivery materials for RNA therapeutics.  相似文献   

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The ability of chemically modified siRNAs targeted to MDR1 mRNA to inhibit P-glycoprotein expression and to restore sensitivity of cancer cells to antibiotic vinblastine was investigated. The effects of chemical modifications on RNA stability in cell culture medium and inhibition of MDR1 gene expression were tested. We found that siRNAs containing 2′-O-methyl ribonucleotides within either sense or/and antisense strands display high stability in serum but exhibit a significant reduction in the biological activity. The protection of 3′-ends of siRNA by introduction of 3′-3′-inverted phosphodiester bonds and two 2′-O-methyl ribonucleotides in protruding 3′-ends considerably increase their biological activity, which allows a 30-fold decrease in the cytostatic agent concentration required for cancer cell death. The data obtained demonstrate that the chemically modified siRNAs can be considered as potential therapeutics, which enhances the efficiency of cancer chemotherapy. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1227–1235, July, 2006.  相似文献   

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Known molecular, “caged” siRNAs are activated by UV light. Since the light of this type is toxic to cells, the uncaging can cause undesired side effects. A modular, molecular system for designing siRNAs is reported, which can be activated by non‐toxic light in live cells. For example, siRNAs responsive to green and red light are described. The uncaging is mediated by 1O2 photogenerated on a photosensitizer, which is attached to the 3′‐terminus of the lagging strand. The 5′‐terminus of the guide strand is alkylated (“caged”) with a 9‐anthracenyl residue. The latter fragment reacts with the 1O2 with formation of the free (uncaged) 5′‐OH terminus. Simultaneously with the uncaging the photosensitizer is bleached and no more 1O2 is generated after this process is completed. The photoactivation of the siRNAs described here is not toxic to cells.  相似文献   

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We prepared reversed dye clusters by hybridizing two RNA oligomers, each of which tethered dyes (Methyl Red, 4′‐methylthioazobenzene, and thiazole orange) on D ‐threoninols (threoninol nucleotides) at the center of their strands. NMR spectroscopic analyses revealed that two dyes from each strand were axially stacked in an antiparallel manner to each other in the duplex, and were located adjacent to the 3′‐side of a natural nucleobase. Interestingly, this positional relationship of the dyes was completely the opposite of that assembled in DNA that we reported previously: dyes in DNA were located adjacent to the 5′‐side of a natural nucleobase. This observation was also consistent with the circular dichroism of dimerized dyes in which the Cotton effect of the dyes (i.e., the winding properties of two dyes) was inverted in RNA relative to that in DNA. Further spectroscopic analyses revealed that clustering of the dyes on RNA duplexes induced distinct hypsochromicity and narrowing of the band, thus demonstrating that the dyes were axially stacked (i.e., H‐aggregates) even on an A‐type helix. On the basis of these results, we also prepared heterodimers of a fluorophore (thiazole orange) and quencher (Methyl Red) in an RNA duplex. Fluorescence from thiazole orange was found to be strongly quenched by Methyl Red due to the excitonic interaction, so that the ratio of fluorescent intensities of the RNA–thiazole orange conjugate with and without its complementary strand carrying a quencher became as high as 27. We believe that these RNA–dye conjugates are potentially useful probes for real‐time monitoring of RNA interference (RNAi) mechanisms.  相似文献   

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The use of nanoparticle‐stabilized nanocapsules (NPSCs) for the direct cytosolic delivery of siRNA is reported. In this approach, siRNA is complexed with cationic arginine‐functionalized gold nanoparticles by electrostatic interactions, with the resulting ensemble self‐assembled onto the surface of fatty acid nanodroplets to form a NPSC/siRNA nanocomplex. The complex rapidly delivers siRNA into the cytosol through membrane fusion, a mechanism supported by cellular uptake studies. Using destabilized green fluorescent protein (deGFP) as a target, 90 % knockdown was observed in HEK293 cells. Moreover, the delivery of siRNA targeting polo‐like kinase 1 (siPLK1) efficiently silenced PLK1 expression in cancer cells with concomitant cytotoxicity.  相似文献   

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Research on RNA has led to many important biological discoveries and the improvement of therapeutic technologies. In particular, there is a great focus on small RNA and ribosomal RNA owing to their key functions in the cell, which make them excellent therapeutic targets. Although the study of these RNA classes is progressing, some limitations have been found regarding the use of suitable techniques that are able to produce and isolate biologically competent and chemically stable RNA. To address this, we have developed a novel histidine affinity chromatography-based isolation methodology for small and ribosomal RNA molecules. The new procedure involves three main steps: (1) cell lysis with guanidinium buffer, (2) RNA primary isolation with ammonium sulfate precipitation and (3) histidine affinity chromatography to specifically purify small RNA and ribosomal RNA from other Escherichia coli impurities (genomic DNA and proteins). The RNA quality assessment revealed that both RNA species were obtained with a high recovery, integrity and purity. The potential of this method to achieve a reproducible RNA isolation with appropriate quality has been demonstrated and it should have broad application in the structural, biophysical and biomedical investigation of systems involving RNA components.  相似文献   

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微小RNA(microRNA,miRNA)和短链干扰RNA (small interfering RNA,siRNA)是两类具有调节基因表达功能的内源性非编码性小RNA分子.它们已成为多种疾病的潜在治疗药物,逐渐被应用于基因治疗中,而将小RNA应用于基因治疗亟需一种安全高效的递送载体.壳聚糖及其衍生物作为一种可降解、低...  相似文献   

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