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siRNA specificity and off-target effects

Author: ZhaoDeYao
Tutor: GuoJiangFeng；DingXianFeng
School: Zhejiang University of Technology
Course: Biochemistry and Molecular Biology
Keywords: siRNA Specificity Off-target effects
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 103
Quote: 1
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Abstract


RNA interference (RNA interfering RNAi) is evolutionarily conserved small interfering RNA (small interfering RNA, siRNA)-mediated inhibition of target gene expression phenomenon. Mainly by degradation of the target mRNA, thereby inhibiting the post-transcriptional regulation of the target protein synthesis, effectively inhibit gene expression. Acting factors are involved in this process siRNA (21-23nt), combined, and then the specific degradation of target mRNA as a key media and RISC protein in the cytoplasm. This characteristic of the siRNA can inhibit pathogenic gene becomes mutated sites, siRNA as a new generation of biological clinical therapy is widely hoped. The process of RNA interference, siRNA and mRNA-specific binding can make the target gene silencing. However, the study confirmed that siRNA can also be combined with non-target genes and lead to non-target gene silencing, a phenomenon known as off-target effects of siRNA. siQuant the report of the dual fluorescence detection technology based on a detection siRNA inhibits the activity of high-throughput detection tool. Of siRNA target sites introduced in siQuant siRNA silencing efficiency by fluorescent reporter activity directly reflected. The siRNA silencing specificity and off-target effects as clinical applications, especially in the process of selective silent SNP mutation gene key problems. However, there is currently no design feasibility of the guiding principles of the allele-specific siRNA. In this thesis, dual fluorescent reporter system based on siQuant as the main means of detection, the use of 20 siRNAs and the corresponding mismatch target sequence 240, siRNA 4,12 and 17 three-point, for example, found that siRNA mismatch tolerance the pattern is determined by its point of mismatch in different positions, with the type of mismatch tolerance mode, and is insensitive to the influence of the surrounding sequence. The paper further 20 siRNAs and the corresponding 260 mismatch target sequences conducted a comprehensive study on the the siRNA mismatch of mode, the siRNA base with tolerance mode you point the wrong map. According to the siRNA base you Point wrong with tolerance law, the the mismatch point in the siRNA highly sensitive area, in order to achieve an exact match with the single-base mismatch selectively silence, by artificially designed allele specific siRNA to inhibit the cure mutation allele and with minimal impact on normal gene. This paper is designed to inhibit the sites of two pathogenic mutations of the PIK3CA gene-specific siRNA, two pathogenic mutations of the PIK3CA gene sites selective silence. This thesis is to establish the the siRNA mismatch tolerance mode can provide a viable law of allele-specific siRNA design, selective gene silencing of great value to the treatment of diseases caused by SNP, siRNA as a clinical drug research provide strong technical support.

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