siRNA特异性及脱靶效应的研究
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摘要
RNA干扰(RNA interfering, RNAi)是指进化保守的小干扰RNA (small interfering RNA, siRNA)介导的抑制靶基因表达的现象。主要通过降解靶标mRNA从而抑制目标蛋白合成的转录后调控,有效抑制基因表达。参与这一过程的作用因子是siRNA(约21-23nt),作为关键的媒介与RISC蛋白在细胞质中结合,进而特异性的降解靶标mRNA。由于siRNA的这种特性可以抑制致病基因的突变位点,因此siRNA被广泛希望于作为新一代的生物临床治疗药物。
RNA干扰过程中,siRNA和mRNA特异结合能够使得靶基因沉默。但研究证实,siRNA还可以与非靶基因结合而导致非靶基因沉默,这种现象称为siRNA的脱靶效应。
siQuant系统以双荧光报告检测技术为基础,是一种检测siRNA抑制活性的高通量检测工具。在siQuant中引入siRNA的靶位点,siRNA的沉默效率可以通过荧光报告基团的活性直接反映出来。
siRNA的沉默特异性和脱靶效应是其作为临床应用,特别是在选择性沉默SNP突变基因过程中遇到的关键问题。然而,目前尚没有设计等位基因特异性siRNA的可行性指导原则。本论文以基于siQuant的双荧光报告系统为主要检测手段,使用20条siRNAs和相应的240条错配靶序列,以siRNA的4、12和17三个位点为例,发现siRNA的错配容忍模式是由其错配位点决定的,在不同位置具有不同的错配容忍模式,并且不受周围序列的影响。本论文进一步使用20条siRNAs和相应的260条错配靶序列对siRNA的错配模式进行了全面研究,建立了siRNA各碱基在各位点的错配容忍模式图谱。
根据上述得到的siRNA各碱基各位点错配容忍规律,将错配位点放置在siRNA高敏感区域,从而实现将完全匹配与单碱基错配选择性地沉默,即通过人为设计等位基因特异性siRNA来有效抑制突变治病的等位基因而对正常基因的影响达到最小。本论文设计抑制PIK3CA基因的两个致病突变位点的特异性siRNA,成功地对PIK3CA基因的两个致病突变位点进行了选择性沉默。
本论文建立的siRNA错配容忍模式可以为等位基因特异性siRNA的设计提供可行的规律,选择性的基因沉默对治疗因SNP导致的疾病具有重大价值,为siRNA作为临床治疗药物研究提供有力的技术支持。
RNA interfering (RNAi) is an evolutionarily conserved process where small interfering RNA (siRNA) specifically represses the expression of target genes. The effectors of this process are siRNA duplexes (approximately 21-23nt) that are key intermediaries in the specific degradation of target mRNA following incorporation into the RNA-induced silencing complex (RISC) in the cytoplasma
siRNAs are widely expected to become next generation of biological therapeutics, and they are initially anticipated to play a major role in treatment of diseases involving single nucleotide polymorphisms (SNPs) where discrimination against single nucleotide variation between wild-type and mutant alleles is demanded.
In RNA interference, siRNAs can silence target genes specifically by binding corresponding mRNA. But recent researches confirm that siRNAs are likely to act on non-target genes and degrade them. This is so-called "off target effects".
siQuant is a reporter-based siRNA validation system that provides a rapid and precise approach to evaluate individual siRNA in high-throughput manner. The efficacy of a siRNA could be read out by measuring the activity of a reporter enzyme, of which the mRNA was tagged by a short sequence representing the target site of tested siRNA.
Silencing specificity and off target effects are critical issues in the therapeutic applications of siRNA, particularly in the treatment of single nucleotide polymorphism (SNP) diseases. However, no generally applicable guidelines are available for the design of such allele-specific siRNAs. In this paper, the issue was approached by using a reporter-based assay. With a panel of 20 siRNAs and 240 variously mismatched target reporters, we first demonstrated that the mismatches were discriminated in a position-dependent order, which was however independent of their sequence contexts using position 4th,12th and 17th as examples. A general model was further built for mismatch discrimination at all positions using 260 additional reporter constructs specifically designed to contain mismatches distributed evenly along the target regions of different siRNAs. This model was successfully employed to design allele-specific siRNAs targeting disease-causing mutations of PIK3CA gene at two SNP sites. Furthermore, conformational distortion of siRNA-target duplex was observed to correlate with the compromise of gene silencing. In summary, these findings could dramatically simplify the design of allele-specific siRNAs and might also provide guide to increase the specificity of therapeutic siRNAs.
RNA干扰过程中,siRNA和mRNA特异结合能够使得靶基因沉默。但研究证实,siRNA还可以与非靶基因结合而导致非靶基因沉默,这种现象称为siRNA的脱靶效应。
siQuant系统以双荧光报告检测技术为基础,是一种检测siRNA抑制活性的高通量检测工具。在siQuant中引入siRNA的靶位点,siRNA的沉默效率可以通过荧光报告基团的活性直接反映出来。
siRNA的沉默特异性和脱靶效应是其作为临床应用,特别是在选择性沉默SNP突变基因过程中遇到的关键问题。然而,目前尚没有设计等位基因特异性siRNA的可行性指导原则。本论文以基于siQuant的双荧光报告系统为主要检测手段,使用20条siRNAs和相应的240条错配靶序列,以siRNA的4、12和17三个位点为例,发现siRNA的错配容忍模式是由其错配位点决定的,在不同位置具有不同的错配容忍模式,并且不受周围序列的影响。本论文进一步使用20条siRNAs和相应的260条错配靶序列对siRNA的错配模式进行了全面研究,建立了siRNA各碱基在各位点的错配容忍模式图谱。
根据上述得到的siRNA各碱基各位点错配容忍规律,将错配位点放置在siRNA高敏感区域,从而实现将完全匹配与单碱基错配选择性地沉默,即通过人为设计等位基因特异性siRNA来有效抑制突变治病的等位基因而对正常基因的影响达到最小。本论文设计抑制PIK3CA基因的两个致病突变位点的特异性siRNA,成功地对PIK3CA基因的两个致病突变位点进行了选择性沉默。
本论文建立的siRNA错配容忍模式可以为等位基因特异性siRNA的设计提供可行的规律,选择性的基因沉默对治疗因SNP导致的疾病具有重大价值,为siRNA作为临床治疗药物研究提供有力的技术支持。
RNA interfering (RNAi) is an evolutionarily conserved process where small interfering RNA (siRNA) specifically represses the expression of target genes. The effectors of this process are siRNA duplexes (approximately 21-23nt) that are key intermediaries in the specific degradation of target mRNA following incorporation into the RNA-induced silencing complex (RISC) in the cytoplasma
siRNAs are widely expected to become next generation of biological therapeutics, and they are initially anticipated to play a major role in treatment of diseases involving single nucleotide polymorphisms (SNPs) where discrimination against single nucleotide variation between wild-type and mutant alleles is demanded.
In RNA interference, siRNAs can silence target genes specifically by binding corresponding mRNA. But recent researches confirm that siRNAs are likely to act on non-target genes and degrade them. This is so-called "off target effects".
siQuant is a reporter-based siRNA validation system that provides a rapid and precise approach to evaluate individual siRNA in high-throughput manner. The efficacy of a siRNA could be read out by measuring the activity of a reporter enzyme, of which the mRNA was tagged by a short sequence representing the target site of tested siRNA.
Silencing specificity and off target effects are critical issues in the therapeutic applications of siRNA, particularly in the treatment of single nucleotide polymorphism (SNP) diseases. However, no generally applicable guidelines are available for the design of such allele-specific siRNAs. In this paper, the issue was approached by using a reporter-based assay. With a panel of 20 siRNAs and 240 variously mismatched target reporters, we first demonstrated that the mismatches were discriminated in a position-dependent order, which was however independent of their sequence contexts using position 4th,12th and 17th as examples. A general model was further built for mismatch discrimination at all positions using 260 additional reporter constructs specifically designed to contain mismatches distributed evenly along the target regions of different siRNAs. This model was successfully employed to design allele-specific siRNAs targeting disease-causing mutations of PIK3CA gene at two SNP sites. Furthermore, conformational distortion of siRNA-target duplex was observed to correlate with the compromise of gene silencing. In summary, these findings could dramatically simplify the design of allele-specific siRNAs and might also provide guide to increase the specificity of therapeutic siRNAs.
引文
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