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: 3.Designappropriatecontrols. AcompletesiRNAexperimentshouldincludeanumberofcontrolstoensurethevalidityofthedata.TheeditorsofNatureCellBiologyhaverecommendedseveralcontrols(2).Twoofthesecontrolsare: 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). Figure1.SchematicofaTypicalHairpinsiRNAProducedbyansiRNAExpressionVectororansiRNAExpressionCassetteandItsRelationshiptotheRNATargetSequence. 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). Figure2.InsertDesignforpSilencer1.0-U6.ThisinsertisspecificforthepSilencer1.0-U6Vectorandcontainstheappropriate3"overhangsfordirectionalcloningintothisvector.Theloopsequenceandlengthcanbevariedasdesired. Figure3.InsertDesignforpSilencer2.0-U6andpSilencer3.0-H1.TheinsertdesignisspecificforthepSilencer2.0-U6,2.1-U6,3.0-H1and3.1-H1ExpressionVectorsandcontainstheappropriateoverhanging5"endsfordirectionalcloningintotheseplasmids.AswithpSilencer1.0-U6showninFigure2,earlyindicationssuggestthatagreatdealoflatitudeispossibleinthedesignoftheloop;hereweprovideoneloopsequencethatwefindworkswell. 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). Figure4.InsertDesignforpSilencer™adeno1.0-CMVVector.ThisinsertdesignisspecificforthepSilenceradeno1.0-CMVvectorandcontainstheappropriateoverhangsfordirectionalcloningintothisvector.Theloopsequenceandlengthcanbevariedasdesired. Figure5.InsertDesignforpSilencer™4.1-CMVVector.ThisinsertdesignisspecificforthepSilencer4.1-CMVvectorandcontainstheappropriateoverhangsfordirectionalcloningintothisvector.Theloopsequenceandlengthcanbevariedasdesired. 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: 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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