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

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。

以下为一些文献中提出的原则补充，很不错。

General Guidelines

1.

2.

siRNA targeted sequence is usually 21 nt in length.

Avoid regions within 50-100 bp of the start codon and the

termination codon

3.

4.

5.

6.

7.

8.

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

9. 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 Tuschl's rules

1. Select targeted region from a given cDNA sequence beginning

50-100 nt downstream of start condon

2. First search for 23-nt sequence motif AA(N

19

). If no suitable

sequence is found, then,

3. Search for 23-nt sequence motif NA(N

21

) and convert the 3' end

of the sense siRNA to TT

4.

5.

Or search for NAR(N

17

)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, Reynolds et al at Dharmacon,

Inc identified eight characteristics associated with siRNA

functionality. These characteristics are used by rational siRNA

design algorithm to evaluate potential targeted sequences 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

1 Moderate to low (30%-52%) GC

Content

2 At least 3 A/Us at positions 15-19 1 point /per A or

1 point

No

(sense)

3 Lack of internal repeats

(Tm*<20¡ãC)

4

5

6

7

8

A at position 19 (sense)

A at position 3 (sense)

U at position 10 (sense)

No G/C at position 19 (sense)

No G at position 13 (sense)

U

1 point

1 point

1 point

1 point

-1 point

-1 point

A sum score of 6 defines the cutoff for selecting siRNAs. All siRNAs

scoring higher than 6 are acceptable candidates.

*Tm = 79.8 + 18.5*log

10

([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

Tm = 79.8 + 18.5*log

10

(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

/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 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, Boese Q, Scaringe S, Marshall WS,

Khvorova A. Rational siRNA design for RNA interference. Nat

Biotechnol. 2004 Mar;22(3):326-30.

5.

6.

/biotools/

Maurice Ho, Rational siRNA Design

RNAi target selection rules:

1. Targeted regions on the cDNA sequence of a targeted gene

should be located 50-100 nt downstream of the start codon (ATG).

2. Search for sequence motif AA(N

19

)TT or NA(N

21

), or

NAR(N

17

)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.

5.

6.

Avoid sequences with > 50% G+C content.

Avoid stretches of 4 or more nucleotide repeats.

Avoid 5URT and 3UTR, although siRNAs targeting UTRs have

successfully induced gene inhibition.

7. Avoid sequences that share a certain degree of homology with

other related or unrelated genes.

How to obtain a cDNA sequence for target selection

Before finding a RNAi target on the gene of your interest, first you

have to get its mRNA sequence or sequence accession number as

some siRNA design tools can take accession number as input. It is

recommended to use the gene's RefSeq from NCBI, since the RefSeq

represents non-redundant, curated and validated sequences. RefSeq

mRNA sequences have unique accession numbers which start with

NM or XM, followed by 6 digits. For example, NM_123456 (curated

mRNA sequence) or XM_0123456 (model mRNA sequence predicted

by genome sequence analysis). There are several ways of querying

RefSeq.

1. Search LocusLink by gene name or symbol at

/LocusLink/. Once the locus of your

gene is found, scroll down to the "NCBI Reference Sequence

(RefSeq)" section and look for mRNA.

2. Search Entrez Gene at

/entrez/?db=gene, and select

the right gene of desired organism. Once the page for the gene is

shown, scroll down to the "NCBI Reference Sequence (RefSeq)"

and look for mRNA.

3. Search Nucleotide database using Entrez query tool

at /entrez/?db=Nucleotide

and use Entrez Limits settings to restrict your query to the RefSeq

database only

o

select "RefSeq" from the "Only from" menu, this

restricts the query to the RefSeq collection

o

select "mRNA" from the "Molecule" menu, this restricts

the query to mRNA RefSeq records

Homology search

The RNAi targeted region on the mRNA sequence of a gene should

not share significant homology with other genes or sequences in the

genome, therefore, homology search is essential to minimize

off-target effects. Although most siRNA design tools provide BLAST

option, some simply use NCBI BLAST tools which sometimes are

quite slow. Here are some BLAST tools for homology search.



NCBI Blast tool: Nucleotide-nucleotide BLAST (blastn) or

Search for short, nearly exact matches



Blat tool on UCSC Genome Website

/cgi-bin/hgBlat



Ensembl Blast /Multi/blastview

Examples of RNAi target selection

References

1. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K,

Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference

in cultured mammalian cells. Nature. 2001 May 24;411(6836):494-8.

2. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is

mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001 Jan

15;15(2):188-200.