【资源】siRNA 设计 英文介绍 挺详细
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1992
siRNA Design
1. Select siRNA 19-mers
First, look around in the literature if any siRNA for your gene has been already made or have a look at siRNA Database (small though)
There is a growing numbers of tools for selection of siRNA 19-mers based on a provided cDNA sequence. We use siRNA Selection Program devoloped at Whitehead Institute. You need to register (for free) in order to obtain username and password. I usually go for custom AAN19 (AA is present only in the target sequence and is not a part of your siRNA). Recently, a very nice software - iRNAi - was developed by MEK&Tosj from Amsterdam. iRNAi searches for oligos, does thermodynamic profile and outputs the oligo sequences in ready to order hairpin design (also fully customizable), and many more. It works great, it's free and only for MacOSX :).
It has been reported that effective RNAi depends on siRNA-specifc properties, rather then properties of target mRNA and the most potent siRNA are characterized by:
moderate to low G/C content 36-52%
low internal stability of the sense 3'end; at least one A/T duplex between position 15-19
lack of internal repeats; Tm < 60 °C
A at position 3, U at position 10, G at position 13, A at position 19 (sense strand) and absence of G at position 19
In order to minimize off-target effects use oligos with at least 3 mismatches to unrelated sequences. Please see Reynolds et al, Nat Biotech 22:326-30, 2004 for more details.
2. Fine tuning - thermodynamics
It has been reported that enhanced flexibility at 5' anti-sense terminal base pair play critical role in siRNA function. In another words, have e.g. G-C pair at pos. 1 (that is 5' sense end) and A-T or even a A-G mismatch at position 19 (that is 5' antisense) of your siRNA sequence. However, we have not tested this concept in our lab yet and (e.g. if that flexibility would not affect hairpin structure). But the data looks very nice and the strategy is worth to try. In case you did tested this strategy and have comments or suggetstions to share, please send me an email and I will post it here. Please see D. Schwarz et al, Cell 115:199, 2003 and A. Khvorova et al, ibid for details.
3. Design a hairpin
Avoid polyT (4 and more) stretches since it may lead to premature termination of shRNA transcription. Also, make sure that your N19 does not end with 2-3Ts; since the hairpin loop starts already with 2Ts. We order most of our oligos from Sigma at 0.05µmol range. Please consult our Cloning Strategies for details. More detailed informations can be found e.g. at T. Tuschl Website and Ambion, specially their siRNA design guidelines
4. Clone hairpin into a vector
Please consult our Cloning Strategies or recent publication for informations how to design expression vector with siRNA (shRNA).
1. Select siRNA 19-mers
First, look around in the literature if any siRNA for your gene has been already made or have a look at siRNA Database (small though)
There is a growing numbers of tools for selection of siRNA 19-mers based on a provided cDNA sequence. We use siRNA Selection Program devoloped at Whitehead Institute. You need to register (for free) in order to obtain username and password. I usually go for custom AAN19 (AA is present only in the target sequence and is not a part of your siRNA). Recently, a very nice software - iRNAi - was developed by MEK&Tosj from Amsterdam. iRNAi searches for oligos, does thermodynamic profile and outputs the oligo sequences in ready to order hairpin design (also fully customizable), and many more. It works great, it's free and only for MacOSX :).
It has been reported that effective RNAi depends on siRNA-specifc properties, rather then properties of target mRNA and the most potent siRNA are characterized by:
moderate to low G/C content 36-52%
low internal stability of the sense 3'end; at least one A/T duplex between position 15-19
lack of internal repeats; Tm < 60 °C
A at position 3, U at position 10, G at position 13, A at position 19 (sense strand) and absence of G at position 19
In order to minimize off-target effects use oligos with at least 3 mismatches to unrelated sequences. Please see Reynolds et al, Nat Biotech 22:326-30, 2004 for more details.
2. Fine tuning - thermodynamics
It has been reported that enhanced flexibility at 5' anti-sense terminal base pair play critical role in siRNA function. In another words, have e.g. G-C pair at pos. 1 (that is 5' sense end) and A-T or even a A-G mismatch at position 19 (that is 5' antisense) of your siRNA sequence. However, we have not tested this concept in our lab yet and (e.g. if that flexibility would not affect hairpin structure). But the data looks very nice and the strategy is worth to try. In case you did tested this strategy and have comments or suggetstions to share, please send me an email and I will post it here. Please see D. Schwarz et al, Cell 115:199, 2003 and A. Khvorova et al, ibid for details.
3. Design a hairpin
Avoid polyT (4 and more) stretches since it may lead to premature termination of shRNA transcription. Also, make sure that your N19 does not end with 2-3Ts; since the hairpin loop starts already with 2Ts. We order most of our oligos from Sigma at 0.05µmol range. Please consult our Cloning Strategies for details. More detailed informations can be found e.g. at T. Tuschl Website and Ambion, specially their siRNA design guidelines
4. Clone hairpin into a vector
Please consult our Cloning Strategies or recent publication for informations how to design expression vector with siRNA (shRNA).
