UsingsiRNAforgenesilencingisarapidlyevolvingtoolinmolecularBIOLOGy.ThereareseveralmethodsforpreparingsiRNA,suchaschemicalsynthesis,invitrotranscription,siRNAexpressionvectors,andPCRexpressioncassettes.Irrespectiveofwhichmethodoneuses,thefirststepindesigningasiRNAistochoosethesiRNAtargetsite.TheguidelinesbelowforchoosingsiRNAtargetsitesarebasedonboththecurrentliterature,andonempiricalobservationsbyscientistsatAmbion.Usingtheseguidelines,approximatelyhalfofallsiRNAsyield>50%reductionintargetmRNAlevels.

FortheBestResults,LetUsDesignYoursiRNAs

AmbionhasrecentlypartneredwithCenixBioScience,aleaderinthefieldofRNAi.CenixhasdevelopedaproprietarysiRNAdesignalgorithmthatyieldsamuchhigherpercentageofeffectivesiRNAswhencomparedtosiRNAsdesignedusingtherulesoutlinedbelow.Forinformationonthatalgorithm,seeDesigningaBettersiRNA.YoucanorderchemicallysynthesizedsiRNAspre-designedusingtheCenixalgorithmfromAmbion.Designsarecurrentlyavailablefor>98%ofthehuman,mouse,andratgenesintheRefSeqdatabase.SeethePre-designedsiRNACatalogPageformoreinformation.Inaddition,AmbionoffersSilencerValidatedsiRNAstoanumberofimportanthumangenes.ThesesiRNAshaveactuallybeentestedandverifiedtoreducetargetmRNAlevels>70%.

GeneralDesignGuidelines

IfyouprefertodesignyourownsiRNAs,youcanchoosesiRNAtargetsitesinavarietyofdifferentorganismsbasedonthefollowingguidelines.CorrespondingsiRNAscanthenbechemicallysynthesized,createdbyinvitrotranscription,orexpressedfromavectororPCRproduct.

1.Find21ntsequencesinthetargetmRNAthatbeginwithanAAdinucleotide.

BeginningwiththeAUGstartcodonofyourtranscript,scanforAAdinucleotidesequences.RecordeachAAandthe3"adjacent19nucleotidesaspotentialsiRNAtargetsites.ThisstrategyforchoosingsiRNAtargetsitesisbasedontheobservationbyElbashiretal.(1)thatsiRNAswith3"overhangingUUdinucleotidesarethemosteffective.ThisisalsocompatIBLewithusingRNApolIIItotranscribehairpinsiRNAsbecauseRNApolIIIterminatestranscriptionat4-6nucleotidepoly(T)tractscreatingRNAmoleculeswithashortpoly(U)tail.InElbashir"sandsubsequentpublications,siRNAswithother3"terminaldinucleotideoverhangshavebeenshowntoeffectivelyinduceRNAi.Ifdesired,youmaymodifythistargetsiteselectionstrategytodesignsiRNAswithotherdinucleotideoverhangs,butitisrecommendedthatyouavoidGresiduesintheoverhangbecauseofthepotentialforthesiRNAtobecleavedbyRNaseatsingle-strandedGresidues.

2.Select2-4targetsequences.

ResearchatAmbionhasfoundthattypicallymorethanhalfofrandomlydesignedsiRNAsprovideatleasta50%reductionintargetmRNAlevelsandapproximately1of4siRNAsprovidea75-95%reduction.ChoosetargetsitesfromamongthesequencesidentifiedinStep1basedonthefollowingguidelines:

• AmbionresearchersfindthatsiRNAswith30-50%GCcontentaremoreactivethanthosewithahigherG/Ccontent.
• Sincea4-6nucleotidepoly(T)tractactsasaterminationsignalforRNApolIII,avoidstretchesof>4T"sorA"sinthetargetsequencewhendesigningsequencestobeexpressedfromanRNApolIIIpromoter.
• SincesomeregionsofmRNAmaybeeitherhighlystructuredorboundbyregulatoryproteins,wegenerallyselectsiRNAtargetsitesatdifferentpositionsalongthelengthofthegenesequence.WehavenotseenanycorrelationbetweenthepositionoftargetsitesonthemRNAandsiRNApotency.
• Comparethepotentialtargetsitestotheappropriategenomedatabase(human,mouse,rat,etc.)andeliminatefromconsiderationanytargetsequenceswithmorethan16-17contiguousbasepairsofhomologytoothercodingsequences.WesuggestusingBLAST,whichcanbefoundontheNCBIserverat:www.ncbi.nlm.nih.gov/BLAST.

3.Designappropriatecontrols.

AcompletesiRNAexperimentshouldincludeanumberofcontrolstoensurethevalidityofthedata.TheeditorsofNatureCellBiologyhaverecommendedseveralcontrols(2).Twoofthesecontrolsare:

• AnegativecontrolsiRNAwiththesamenucleotidecompositionasyoursiRNAbutwhichlackssignificantsequencehomologytothegenome.TodesignanegativecontrolsiRNA,scramblethenucleotidesequenceofthegene-specificsiRNAandconductasearchtomakesureitlackshomologytoanyothergene.
• AdditionalsiRNAsequencestargetingthesamemRNA.PerhapsthebestwaytoensureconfidenceinRNAidataistoperformexperiments,usingasinglesiRNAatatime,withtwoormoredifferentsiRNAstargetingthesamegene.Priortotheseexperiments,eachsiRNAshouldbetestedtoensurethatitreducestargetgeneexpressionbycomparablelevels.

Ambion"ssiRNATargetFinder

Useouronlinetargetfindertofindpotentialsequencesbasedonthedesignguidelinesdescribedabove.SimplypasteyourmRNAsequenceintothewindowandthisprogramwillscanyoursequenceforAAdinucleotides.AreportisgeneratedindicatingthepositionoftheAAdinucleotide,the21basetargetandthecorrespondingsenseandantisensesiRNAoligonucleotides.siRNAtargetscanthenbesentdirectlytooneofourkit-specificdesigntoolsorsubjectedtoaBLASTsearchbyclickingontheappropriatelinkbelowthetargetofinterest.

Alternatively,theWhiteheadInstituteofBiomedicalResearchatMIThasapubliclyavailablesiRNAdesigntoolthatincorporatesadditionalselectionparametersandintegratesBLASTsearchesofthehumanandmousegenomedatabases.Seehttp://jura.wi.mit.edu/pubint/http://iona.wi.mit.edu/siRNAext/(registrationrequired).

SpecificGuidelinesforDesigningsiRNAHairpinsEncodedbysiRNAExpressionVectorsandsiRNAExpressionCassettes

ResearcherswhoinitiallyreportedtheuseofsiRNAexpressionvectorstoinduceRNAihaddifferentdesigncriteriafortheirinsertsencodingtheexpressedsiRNA.MostofthedesignshadtwoinvertedrepeatsseparatedbyashortspacersequenceandendedwithastringofT"sthatservedasatranscriptionterminationsite.ThesedesignsproduceanRNAtranscriptthatispredictedtofoldintoashorthairpinsiRNAasshowninFigure1.TheselectionofsiRNAtargetsequence,thelengthoftheinvertedrepeatsthatencodethestemofaputativehairpin,theorderoftheinvertedrepeats,thelengthandcompositionofthespacersequencethatencodestheloopofthehairpin,andthepresenceorabsenceof5"-overhangs,varyamongdifferentreports(3-11).

Ambion"sRecommendedProcedureforsiRNAHairpinDesign

ThefollowingrecommendationsforsiRNAhairpindesignandcloningstrategyaremadebasedonresearchbyAmbionscientists.ThefirststepindesigninganappropriateinsertistochoosethesiRNAtargetsitebyfollowingthestepsdescribedunder"GeneralDesignGuidelines"above.

Forscreening,wetypicallytestfoursiRNAsequencespertarget,spacingthesiRNAsequencesdownthelengthofthegenesequencetoreducethechancesoftargetingaregionofthemRNAthatiseitherhighlystructuredorboundbyregulatoryproteins.BecauseconstructingandtestingfoursiRNAexpressionplasmidspertargetistime-consuming,wefinditmucheasiertoscreenpotentialsiRNAsequencesusingPCR-derivedsiRNAexpressioncassettes(SECs).SECsarePCRproductsthatincludepromoterandterminatorsequencesflankingahairpinsiRNAtemplateandcanbepreparedwithAmbion"sSilencer™ExpressKits.ThisscreeningstrategyalsopermitstherapididentificationofthebestcombinationofpromoterandsiRNAsequenceintheexperimentalsystem.SECsfoundtoeffectivelyelicitgenesilencingcanbereADIlyclonedintoavectorforlongtermstudies.AmbionscientistshavedeterminedthatsequencesthatfunctionwellastransfectedsiRNAsalsofunctionwellassiRNAsthatareexpressedinvivo.TheonlyexceptionisthatsiRNAsequencestobeexpressedinvivoshouldnotcontainarunof4or5A"sorT"s,asthesecanactasterminationsitesforPolymeraseIII.

FortraditionalcloningintopSilencervectors,twoDNAoligonucleotidesthatencodethechosensiRNAsequencearedesignedforinsertionintothevector(Figures2and3).Ingeneral,theDNAoligonucleotidesconsistofa19-nucleotidesensesiRNAsequencelinkedtoitsreversecomplementaryantisensesiRNAsequencebyashortspacer.Ambionscientistshavesuccessfullyuseda9-nucleotidespacer(TTCAAGAGA),althoughotherspacerscanbedesigned.5-6T"sareaddedtothe3"endoftheoligonucleotide.Inaddition,forcloningintothepSilencer1.0-U6vector,nucleotideoverhangstotheEcoRIandApaIrestrictionsitesareaddedtothe5"and3"endoftheDNAoligonucleotides,respectively(Figure2).Incontrast,forcloningintothepSilencer2.0-U6,2.1-U6,3.0-H1,or3.1-H1vectors,nucleotideoverhangswithBamHIandHindIIIrestrictionsitesareaddedtothe5"and3"endoftheDNAoligonucleotides,respectively(Figure3).TheresultingRNAtranscriptisexpectedtofoldbackandformastem-loopstructurecomprisinga19bpstemand9ntloopwith2-3U"satthe3"end(Figure1).

ForcloningintothepSilenceradeno1.0-CMVvector,DNAoligonucleotideswithstem-loopstructuresarecreatedsimilartothoseofpSilencer2.0and3.0vectorsdescribedabove.However,onenotableexceptionistheabsenceof5-6T"sfromthe3"-endoftheoligonucleotidesfortheCMV-basedvectorsystemsincethetranscriptionterminationsignalfortheCMV-basedvectorsystemisprovidedbytheSV40polyAterminator.Inaddition,forcloningintothepSilenceradeno1.0-CMVvector,nucleotideoverhangscontainingtheXhoIandSpeIrestrictionsitesareaddedtothe5"and3"endoftheDNAoligonucleotides,respectively(Figure4).However,forcloningintothepSilencer4.1-CMVvector,nucleotideoverhangscontainingtheBamH1andHindIIIrestrictionsitesareaddedtothe5"and3"endoftheDNAoligonucleotides,respectively(Figure5).

ForpreparingSECscontaininganH1orU6promoterbyPCRusingtheSilencerExpressKits,(1)oneortwoDNAoligonucleotidesencodingthesiRNAsequencearedesignedandordered,(2)theoligonucleotidesareusedasprimersinoneormorePCRswiththepromoter-containingtemplateincludedinthekit,and(3)theresultingPCRproductiscolumnpurified.Ambionscientiststypicallyusealoopsequenceof5"-UUUGUGUAG-3"fortheirSECs,althoughotherloopsequencescanbedesigned.Aswithvectorinsertdesign,a5-6TterminationsequenceisaddedtoactasanRNApolIIIterminator.Forsubsequentcloningconvenience,EcoRIandHindIIIrestrictionsitesarealsoencodedbytheprimers.ThedetaileddesignparametersfortheoligonucleotideprimersusedwiththeSilencerExpressKitscanbefoundinthekits"InstructionManual.

ForcloningoffunctionalSilencerExpressKit-derivedSECsintovectors,theSECanddestinationvectorshouldberestrictedwithEcoRIandHindIII.Linearizeddestinationvectorswithneomycin,hygromycinandpuromycinresistancegenes,calledpSECVectors,areavailable.

SelectionofsiRNATargets

InadditiontothealgorithmdevelopedbyCenixandoursuggestedprocedureforselectingsiRNAtargetsbyscanningamRNAsequenceforAAdinucleotidesandrecordingthe19nucleotidesimmediatelydownstreamoftheAA,twoothermethodshavebeenemployedbyotherresearchers.Inthefirstmethod,theselectionofthesiRNAtargetsequenceispurelyempiricallydetermined(4),aslongasthetargetsequencestartswithGGanddoesnotsharesignificantsequencehomologywithothergenesasanalyzedbyBLASTsearch.

Inthesecondreport,amoreelaboratemethodisemployedtoselectthesiRNAtargetsequences.ThisprocedureexploitsanobservationthatanyaccessiblesiteinendogenousmRNAcanbetargetedfordegradationbythesyntheticoligodeoxyribonucleotide/RNaseHmethod(5).AnyaccessiblesiteidentifiedinthisfashionisthenusedasinsertsequenceintheU6promoter-drivensiRNAconstructs.

OrderoftheSenseandAntisenseStrandswithintheHairpinsiRNAs

AhairpinsiRNAexpressioncassetteisusuallyconstructedtocontainthesensestrandofthetarget,followedbyashortspacer,thentheantisensestrandofthetarget,inthatorder.OnegroupofresearchershasfoundthatreversaloftheorderofsenseandantisensestrandswithinthesiRNAexpressionconstructsdidnotaffectthegenesilencingactivitiesofthehairpinsiRNA(6).Incontrast,anothergroupofresearchershasfoundthatsimilarreversaloforderinanothersiRNAexpressioncassettecausedpartialreductioninthegenesilencingactivitiesofthehairpinsiRNA(7).Itisnotclearwhatisresponsibleforthisdifferenceinobservation.Atthepresenttime,itisstilladvisabletoconstructthesiRNAexpressioncassetteintheorderofsensestrand,shortspacer,andantisensestrand.

LengthofthesiRNAStem

ThereappearstobesomedegreeofvariationinthelengthofnucleotidesequencebeingusedasthestemofsiRNAexpressioncassette.SeveralresearchgroupsincludingAmbionhaveused19nucleotides-longsequencesasthestemofsiRNAexpressioncassette(6-10).Incontrast,otherresearchgroupshaveusedsiRNAstemsrangingfrom21nucleotides-long(4-5)to25-29nucleotides-long(11).ItisfoundthathairpinsiRNAswiththesevariousstemlengthsallfunctionwellingenesilencingstudies.

LengthandSequenceoftheLoopLinkingSenseandAntisenseStrandsofHairpinsiRNA

VariousresearchgroupshavereportedsuccessfulgenesilencingresultsusinghairpinsiRNAswithloopsizerangingbetween3to23nucleotides(4,6-9,11).Thefollowingisasummaryofloopsizeandspecificloopsequencesusedbyvariousresearchgroups:

Presenceof5"OverhangsintheHairpinsiRNAs

Mostresearchgroupsdidnotusea5"overhangintheirhairpinsiRNAconstruct(4-8,10-11).However,oneresearchgroupincludeda6nucleotide5"overhanginthehairpinsiRNAconstructs(9).ThesehairpinsiRNAswith5"overhangswereshowntobefunctionalingenesilencing.

ChemicalSynthesisofsiRNA

AmbionsynthesizesbothcustomerdesignedsiRNAsandsiRNAspre-designedusingtheCenixalgorithm.

ToorderachemicallysynthesizedsiRNAforwhichyoualreadyhavethedesign,youcaneitherprovide:·the~21basemRNAsequence(startingwiththeAAdinucleotide)towhichthesiRNAwillbedirected

OR

·thesequenceofeachsiRNAstrand(ThisoptionisrecommendedifyouwishyoursiRNAtohave3"terminiotherthandTdTorUU.)

AmbionwillsynthesizeacomplementarypairofsiRNAoligonucleotidesaccordingtoyoursequence.Bydefault,siRNAsforwhichyouprovideonlythemRNAtargetsequencewillbesynthesizedwithdTdT3"overhangs.Ifyouwish,youcanchooseUUorotheroverhangs.OurscientistsobservenofunctionaldifferenceinthepotencyofsiRNAmadewithdTdTorUUoverhangs.(Note:the3"dTdTofthesensestranddoesnothavetobecomplementarytothetargetgene.)

Currently,Cenix-designedsiRNAsareavailablefor>98%ofallhuman,mouse,andratgenesintheRefSeqdatabasemaintainedbyNCBI.Toorderapre-designedsiRNA,searchoursiRNAdatabaseforyourgeneofinterest,choosethedesign(s)you"dliketopurchase,addthemtoyourcart,andtransfertherelevantinformationabouteachtoouronlineoligoorderform.SeeDesigningaBettersiRNAforinformationontheCenixdesignalgorithm.

OtherMethodsofsiRNAPreparation

TopreparesiRNAbyinvitrotranscription,siRNAexpressionvector,orPCR-generatedsiRNAexpressioncassette,appropriatetemplatesmustbeprepared.Web-basedtoolsfordesigningthesetemplatesareavailableforthefollowingAmbionkits/products:

• SilencersiRNAConstructionKit[ProductInfo][DesignTool]
• pSilencersiRNAExpressionVectors[ProductInfo][DesignTool]
• SilencerExpresssiRNAExpressionCassetteKits[ProductInfo][DesignTool]
• SilencersiRNACocktailKit(RnaseIII)[ProductInfo]

ThesetoolsarealsoaccessiblefromthesiRNATargetFinderdescribedabove.

References

1.Elbashir,etal.(2001)FunctionalanatomyofsiRNAformediatingefficientRNAiinDrosophilamelanogasterembryolysate.EMBOJ20:6877-6888.

2.EditorsofNatureCellBiology(2003)WhitherRNAi?NatCellBiol.5:489-490.

3.Brown,D.,Jarvis,R.,Pallotta,V.,Byrom,M.,andFord,L.(2002)RNAinterferenceinmammaliancellculture:design,execution,andanalysisofthesiRNAeffect.AmbionTechNotes9(1):3-5.

4.Sui,G.,Soohoo,C.,Affar,E.B.,Gay,F.,Shi,Y.,Forrester,W.C.,andShi,Y.(2002)ADNAvector-basedRNAitechnologytosuppressgeneexpressioninmammaliancells.Proc.Natl.Acad.Sci.USA99(8):5515-5520.

5.Lee,N.S.,Dohjima,T.,Bauer,G.,Li,H.,Li,M.-J.,Ehsani,A.,Salvaterra,P.,andRossi,J.(2002)ExpressionofsmallinterferingRNAstargetedagainstHIV-1revtranscriptsinhumancells.NatureBiotechnology20:500-505.

6.Yu,J.-Y.,DeRuiter,S.L.,andTurner,D.L.(2002)RNAinterferencebyexpressionofshort-interferingRNAsandhairpinRNAsinmammaliancells.Proc.Natl.Acad.Sci.USA99(9):6047-6052.

7.Paul,C.P.,Good,P.D.,Winer,I.,andEngelke,D.R.(2002)EffectiveexpressionofsmallinterferingRNAinhumancells.NatureBiotechnology20:505-508.

8.Brummelkamp,T.R.,Bernards,R.,andAgami,R.(2002)AsystemforstableexpressionofshortinterferingRNAsinmammaliancells.Science296:550-553.

9.Jacque,J.-M.,Triques,K.,andStevenson,M.(2002)ModulationofHIV-1replicationbyRNAinterference.Nature418:435-438.

10.Miyagishi,M.,andTaira,K.(2002)U6promoter-drivensiRNAswithfoururidine3"overhangseffectivelysuppresstargetedgeneexpressioninmammaliancells.NatureBiotechnology20:497-500.

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