RNAi片段siRNA设计原则.docxVIP

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(un tra nslated regio ns， 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。 ⑵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 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。 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 )，—半以上的几率能达到 50% 的沉默效果。 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。 以下为一些文献中提出的原则补充，很不错。 Gen eral Guideli nes siRNA targeted seque nee is usually 21 nt in len gth. Avoid regi ons withi n 50-100 bp of the start cod on and the term in atio n cod on Avoid intron regi ons Avoid stretches of 4 or more bases such as AAAA, CCCC Avoid regio ns with GC content 30% or 60%. Avoid repeats and low complex seque nee Avoid sin gle nu cleotide polymorphism (SNP) sites Perform BLAST homology search to avoid off-target effects on other genes or seque nces Always desig n n egative con trols by scrambli ng targeted siRNA seque nee. The con trol RNA should have the same len gth and nu cleotide compositi on 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 gen es. Tom Tuschls rules Select targeted regi on from a give n cDNA seque nee beg inning 50-100 nt dow nstream of start condon First search for 23-nt seque nee motif AA(N 19). If no suitable seque nee is found, the n, Search for 23-nt sequenee motif NA(N 21) and convert the 3 end of the sense siRNA to TT Or search for NAR(N 17 )YNN Target seque nee should have a GC content of around 50% A = Ade nine; T = Thymine; R = Ade nine or Guanine (Puri nes); Y 二 Thymine or Cytosine (Pyrimidines); N = Any. Ratio nal siRNA design By experime ntally an alyz ing the sile ncing efficie ncy of 180 siRNAs target ing the mRNA of two genes and correlat i