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 要比那些医疗器械行业分析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（背景图片动漫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。 |
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以下为一些文献中提出的原则补充，很不错。
General Guidelines
1. siRNA targeted quence is usually 21 nt in length.
2. Avoid regions within 50-100 bp of the start codon and the termination codon
3. Avoid intron regions
4. Avoid stretches of 4 or more bas such as AAAA, CCCC
5. Avoid regions with GC content <30% or > 60%.
6. Avoid repeats and low complex quence
7. Avoid single nucleotide polymorphism (SNP) sites
8. Perform BLAST homology arch to avoid off-target effects on other genes or quences
9. Always design negative controls by scrambling targeted siRNA quence. The control RNA should have the same length and nucleotide composition as the siRNA but have at least 4-5 bas mismatched to the siRNA. Make sure the scrambling will not create new homology to other genes.
Tom Tuschl's rules
1. Select targeted region from a given cDNA quence beginning 50-100 nt downstream of start condon
2. First arch for 23-nt quence motif AA(N19). If no suitable quence is found, then,
3. Search for 23-nt quence motif NA(N21) and convert the 3' end of the n siRNA to TT
4. Or arch for NAR(N17)YNN
5. Target quence 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 quence features of individual siRNAs, Reynolds et al at Dharmacon, Inc identified eight characteristics associated with siRNA functionality. The characteristics are ud by rational siRNA design algorithm to evaluate potential targeted quences and assign scores to them. Sequences with higher scores will have higher chance of success in RNAi. The table below lists the 8 criteria and the methods of score assignment.
Criteria | Description | Score |
Yes | No |
1 | Moderate to low (30%-52%) GC Content | 1 point | 梅花的花语是什么 |
2 | At least 3 A/Us at positions 15-19 (n) | 1 point /per A or U | |
3 | Lack of internal repeats (Tm*<20¡ãC) | 1 point | |
4 | A at position 19 (n)保密审查 | 1 point | |
5 | A at position 3 (n) | 1 point | |
6 | U at position 10 (n) | 1 point | |
7 | No G/C at position 19 (n) | | -1 point | 严峻的近义词
8 | No G at position 13 (n) qq网页登陆 | | -1 point |
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A sum score of 6 defines the cutoff for lecting siRNAs. All siRNAs scoring higher than 6 are acceptable candidates.
*Tm = 79.8 + 18.5*log10([Na+]) + (58.4 * GC%/100) + (11.8 * (GC%/100)2) - (820/Length)
For example, the Tm can be calculated as follows for the siRNA UUCUCCAGCUUCUAAAAUA
关于分享的作文Tm = 79.8 + 18.5*log10(0.05) + (58.4 * 31.6/100) + (11.8 * (31.6/100)2) - (820/19)
Tm = 32.19
There are two siRNA design tools which implement this siRNA design algorithm: one is offered by Dharmacon, Inc; the other is a downloadable Excel template, written by Maurice Ho at boz094.ust.hk/RNAi/siRNA.
References
1. Elbashir SM et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411:494-498.
2. Elbahir SM et al. (2001). Functional anatomy of 辛苦的近义词是什么siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20:6877-6888.
3. Elbashir SM et al. (2002). Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods. 26:199-213.
4. Reynolds A, Leake D, Boe Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004 Mar;22(3):326-30.
5. hwestern.edu/biotools/oligocalc.html
6. Maurice Ho, Rational siRNA Design
RNAi target lection rules:
1. Targeted regions on the cDNA quence of a targeted gene should be located 50-
100 nt downstream of the start codon (ATG).
2. Search for quence motif AA(N19)TT or NA(N21), or NAR(N17)YNN, where N is any nucleotide, R is purine (A, G) and Y is pyrimidine (C, U).
3. Avoid targeting introns, since RNAi only works in the cytoplasm and not within the nucleus.
4. Avoid quences with > 50% G+C content.
5. Avoid stretches of 4 or more nucleotide repeats.
6. Avoid 5URT and 3UTR, although siRNAs targeting UTRs have successfully induced gene inhibition.
