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Source:ProtocolOnline

Abstract:SummarizebothTomTuschl"smotifsiRNAdesignrulesandsiRNArationaldesign.

GeneralGuidelines

siRNAtargetedsequenceisusually21ntinlength.
Avoidregionswithin50-100bpofthestartcodonandtheterminationcodon
Avoidintronregions
Avoidstretchesof4ormorebasessuchasAAAA,CCCC
AvoidregionswithGCcontent<30%or>60%.
Avoidrepeatsandlowcomplexsequence
Avoidsinglenucleotidepolymorphism(SNP)sites
PerformBLASThomologysearchtoavoidoff-targeteffectsonothergenesorsequences
AlwaysdesignnegativecontrolsbyscramblingtargetedsiRNAsequence.ThecontrolRNAshouldhavethesamelengthandnucleotidecompositionasthesiRNAbuthaveatleast4-5basesmismatchedtothesiRNA.Makesurethescramblingwillnotcreatenewhomologytoothergenes.

TomTuschl"srules

SelecttargetedregionfromagivenCDNAsequencebeginning50-100ntdownstreamofstartcondon
Firstsearchfor23-ntsequencemotifAA(N₁₉).Ifnosuitablesequenceisfound,then,
Searchfor23-ntsequencemotifNA(N₂₁)andconvertthe3"endofthesensesiRNAtoTT
OrsearchforNAR(N₁₇)YNN
TargetsequenceshouldhaveaGCcontentofaround50%

A=Adenine;T=Thymine;R=AdenineorGuanine(Purines);Y=ThymineorCytosine(Pyrimidines);N=Any.

RationalsiRNAdesign

Byexperimentallyanalyzingthesilencingefficiencyof180siRNAstargetingthemRNAoftwogenesandcorrelatingitwithvarioussequencefeaturesofindividualsiRNAs,ReynoldsetalatDharmacon,IncidentifiedeightcharacteristicsassociatedwithsiRNAfunctionality.ThesecharacteristicsareusedbyrationalsiRNAdesignalgorithmtoevaluatepotentialtargetedsequencesandassignscorestothem.SequenceswithhigherscoreswillhavehigherchanceofsuccessinRNAi.Thetablebelowliststhe8criteriaandthemethodsofscoreassignment.

Criteria	Description	Score
Criteria	Description	Yes	No
1	Moderatetolow(30%-52%)GCContent	1point
2	Atleast3A/Usatpositions15-19(sense)	1point/perAorU
3	Lackofinternalrepeats(Tm*<20¡ãC)	1point
4	Aatposition19(sense)	1point
5	Aatposition3(sense)	1point
6	Uatposition10(sense)	1point
7	NoG/Catposition19(sense)		-1point
8	NoGatposition13(sense)		-1point

Asumscoreof6definesthecutoffforselectingsiRNAs.AllsiRNAsscoringhigherthan6areacceptablecandidates.
*Tm=79.8+18.5*log₁₀([Na⁺])+(58.4*GC%/100)+(11.8*(GC%/100)²)-(820/Length)

Forexample,theTmcanbecalculatedasfollowsforthesiRNAUUCUCCAGCUUCUAAAAUA
Tm=79.8+18.5*log₁₀(0.05)+(58.4*31.6/100)+(11.8*(31.6/100)²)-(820/19)
Tm=32.19

TherearetwosiRNAdesigntoolswhichimplementthissiRNAdesignalgorithm:oneisofferedbyDharmacon,Inc;theotherisadownloadableExceltemplate,writtenbyMauriceHoathttp://boz094.ust.hk/RNAi/siRNA.References

ElbashirSMetal.(2001)Duplexesof21-nucleotideRNAsmediateRNAinterferenceinculturedmammaliancells.Nature.411:494-498.
ElbahirSMetal.(2001).FunctionalanatomyofsiRNAsformediatingefficientRNAiinDrosophilamelanogasterembryolysate.EMBOJ.20:6877-6888.
ElbashirSMetal.(2002).AnalysisofgenefunctioninsomaticmammaliancellsusingsmallinterferingRNAs.Methods.26:199-213.
ReynoldsA,LeakeD,BoeseQ,ScaringeS,MarshallWS,KhvorovaA.RationalsiRNAdesignforRNAinterference.NatBiotechnol.2004Mar;22(3):326-30.
http://www.basic.northwestern.edu/biotools/oligocalc.html
http://boz094.ust.hk/RNAi/siRNA