4/30/2023 0 Comments Sirna probe design beacon designerThe complexity in the therapeutic delivery of nucleic acids and the heterogeneity of side effects make the interpretation of the therapeutic outcome difficult. The purpose of this review is to reflect on the complexity in the therapeutic delivery of RNA interference-based drugs emerging from the recent clinical experiences and report the actual technological and analytical advances introduced to solve it. These properties enable for the enhancement of the nano-formulation's therapeutic efficacy, but on the other hand, the nanomatters interactions in biological fluids are also responsible for adverse effects. Nano-sized matters own specific features responsible for inducing characteristic interactions with biological molecules and tissues. However, a more wide-spread development of therapeutics based on nucleic acids is restricted by their poor chemical and enzymatic stability in vivo, lack of cellular uptake and insufficient capability to reach intracellular targets.Īdvanced formulation of nucleic acids in nano-sized carriers may help unlocking their potential as therapeutic agents. Nucleic acids have witnessed a dramatic acceleration in their therapeutic exploitation and currently represent a growing number of applications in drug development pipelines. Delivery of siRNA via antibody-directed endosomolytic nanoparticles may be a promising strategy for cancer therapy. Detection of the expected mRNA cleavage product by 5' RLM-RACE assay confirmed that suppression occurs via the expected RNAi pathway. Mice bearing intraperitoneal human ovarian tumor xenografts demonstrated increased tumor accumulation of Cy5.5 fluorescently labeled siRNA and 70% target gene suppression after treatment with HER2 antibody-directed siRNA nanocarriers. RNAi-mediated site specific cleavage of target mRNA was demonstrated using the 5' RLM-RACE (RNA ligase mediated-rapid amplification of cDNA ends) assay. We investigated the utility of linking an internalizing streptavidin-conjugated HER2 antibody to an endosome-disruptive biotinylated polymeric nanocarrier to improve the functional cytoplasmic delivery of siRNA in breast and ovarian cancer cells in vitro and in an intraperitoneal ovarian cancer xenograft model in vivo, yielding an 80% reduction of target mRNA and protein levels with sustained repression for at least 96 hours. Tumor-specific recognition can be effectively achieved by antibodies directed against highly expressed cancer cell surface receptors. The therapeutic potential of RNA interference (RNAi) has been limited by inefficient delivery of short interfering RNA (siRNA). All MBRACE experiments were performed in duplicate, but for clarity only a single, representative replicate is shown. (E) Amplification curves from MBRACE reactions using the template from siRNA-treated cells as in (C) filled symbols represent samples transfected with ApoB-1 siRNA, and open symbols are samples transfected with mismatch control siRNA at 10nM (open square) or 1nM (open triangle). The correct product indicated by the arrow has a size of 290 bp. (D) The same samples were used as template in standard 5′-RLM-RACE analysis, and analyzed by electrophoresis. (C) RT–qPCR analysis of ApoB mRNA in Hepa1-6 cells transfected with either 1 (a) or 10nM (b) ApoB-1 siRNA, or 1 (c) or 10 nM (d) mismatch control siRNA. (B) Amplification curves showing fluorescent signal generated from positive (filled square) and negative (open square) cDNA clones. (A) Schematic representation of sequences of: (1) the RNA linker (blue) (2) ApoB cleavage site (3) positive (RNA linker ligated to cleavage product) and (4) negative (4 bp insertion between linker and mRNA fragment underlined) clones and (5) the molecular beacon probe used to detect linker ligation to the cleaved mRNA fragment (red text represents the 7-bp stem). Specific detection of mRNA cleavage products with MBRACE in vitro. Prove a useful tool to detect mRNA cleavage and corroborate knockdown studies following siRNA use in vivo. With its sensitivity and specificity, this variation on the 5′RACE method should Was also observed when RNA from mouse liver following administration of ApoB-specific siRNA was analysed, even in cases where ApoB knockdown measured by real-time PCR was <10%. In subsequent real-time PCR analysis, the specific mRNA cleavage product was detected. When RNA from siRNA-transfected cells was used for 5′-RLM-RACE and a cleavage site-specific molecular beacon probe was included We have combined 5′-RNA-linker-mediated RACE (5′-RLM-RACE) with real-time PCR using a molecular beacon to developĪ rapid and specific method termed MBRACE, which we have used to detect small-interfering RNA (siRNA)-induced cleavage ofĪpoB, RRM1 and YBX1 transcripts in vitro, and ApoB in vivo. Specific detection of mRNA cleavage by 5′RACE is the only method to confirm the knockdown of mRNA by RNA interference, but
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