rnai片段sirna设计原则rnai目标序列的选取原则应遵循以下几个.doc

RNAi片段siRNA设计原则 RNAi 目标序列的选取原则应遵循以下几个方面的原则 (1)从转录本（mRNA）的AUG起始密码开始，寻找“AA”二连序列，并记下其3端的19个碱基序列，作为潜在的siRNA靶位点。有研究结果显示GC 含量在45%-55%左右的siRNA 要比那些GC 含量偏高的更为有效。Tuschl 等建议在设计siRNA 时不要针对5和3端的非编码区（untranslated regions，UTRs），原因是这些地方有丰富的调控蛋白结合区域，而这些UTR结合蛋白或者翻译起始复合物可能会影响siRNP 核酸内切酶复合物结合mRNA从而影响siRNA 的效果。(2)将潜在的序列和相应的基因组数据库（人，或者小鼠，大鼠等等）进行比较，排除那些和其他编码序列/EST 同源的序列。例如使用BLAST(3)选出合适的目标序列进行合成。通常一个基因需要设计多个靶序列的siRNA，以找到最有效的siRNA序列。(4)一个目标基因至少设计3-5个以上的siRNA，平行实验以期提高成功率。据评估，随机设计的siRNA有25%的机会有效沉默基因表达（减少75%-95%以上的mRNA），一半以上的几率能达到50%的沉默效果。(5) siRNA的反义链3’端最好以UU结尾，这被公认是最有效的siRNA结构。现在以其他碱基结尾的siRNA也有报道能成功引发RNAi。 General Guidelines siRNA targeted sequence is usually 21 nt in length. Avoid regions within 50-100 bp of the start codon and the termination codon Avoid intron regions Avoid stretches of 4 or more bases such as AAAA, CCCC Avoid regions with GC content 30% or 60%. Avoid repeats and low complex sequence Avoid single nucleotide polymorphism (SNP) sites Perform BLAST homology search to avoid off-target effects on other genes or sequences Always design negative controls by scrambling targeted siRNA sequence. The control RNA should have the same length and nucleotide composition as the siRNA but have at least 4-5 bases mismatched to the siRNA. Make sure the scrambling will not create new homology to other genes.?? Tom Tuschls rules Select targeted region from a given cDNA sequence beginning 50-100 nt downstream of start condon First search for 23-nt sequence motif AA(N19). If no suitable sequence is found, then, Search for 23-nt sequence motif NA(N21) and convert the 3 end of the sense siRNA to TT Or search for NAR(N17)YNN Target sequence should have a GC content of around 50% A = Adenine; T = Thymine; R = Adenine or Guanine (Purines); Y = Thymine or Cytosine (Pyrimidines); N = Any. Rational siRNA design ?By experimentally analyzing the silencing efficiency of 180 siRNAs targeting the mRNA of two genes and correlating it with various sequence features of individual siRNAs,