RNAi片段siRNA設(shè)計原則RNAi目標(biāo)序列選取原則應(yīng)遵循以下幾個

RNAi片段siRNA設(shè)計原則RNAi目標(biāo)序列的采用原則應(yīng)依據(jù)以下幾個RNAi片段siRNA設(shè)計原則RNAi目標(biāo)序列的采用原則應(yīng)依據(jù)以下幾個RNAi片段siRNA設(shè)計原則RNAi目標(biāo)序列的采用原則應(yīng)依據(jù)以下幾個RNAi片段siRNA設(shè)計原則RNAi目標(biāo)序列的采用原則應(yīng)依據(jù)以下幾個方面的原則：(1)從轉(zhuǎn)錄本（mRNA）的AUG初步密碼開始，搜尋“AA”二連序列，并記下其3'端的19個堿基序列，作為潛藏的siRNA靶位點。有研究結(jié)果顯示GC含量在45%-55%左右的siRNA要比那些GC含量偏高的更為有效。Tuschl等建議在設(shè)計siRNA時不要針對5'和3'端的非編碼區(qū)（untranslatedregions，UTRs），原因是這些地方有豐富的調(diào)控蛋白結(jié)合地域，而這些UTR結(jié)合蛋白也許翻譯初步復(fù)合物可能會影響siRNP核酸內(nèi)切酶復(fù)合物結(jié)合mRNA從而影響siRNA的收效。(2)將潛藏的序列和相應(yīng)的基因組數(shù)據(jù)庫（人，也許小鼠，大鼠等等）進(jìn)行比較，消除那些和其他編碼序列/EST同源的序列。比方使用BLAST(3)選出合適的目標(biāo)序列進(jìn)行合成。平時一個基因需要設(shè)計多個靶序列的siRNA，以找到最有效的siRNA序列。(4)一個目標(biāo)基因最少設(shè)計3-5個以上的siRNA，平行實驗以期提高成功率。據(jù)評估，隨機設(shè)計的siRNA有25%的機遇有效默然基因表達(dá)（減少75%-95%以上的mRNA），一半以上的幾率能達(dá)到50%的默然收效。siRNA的反義鏈3’端最好以UU結(jié)尾，這被公認(rèn)是最有效的siRNA結(jié)構(gòu)。現(xiàn)在以其他堿基結(jié)尾的siRNA也有報道能成功惹起RNAi。以下為一些文件中提出的原則補充，很不錯。GeneralGuidelinessiRNAtargetedsequenceisusually21ntinlength.Avoidregionswithin50-100bpofthestartcodonandtheterminationcodonAvoidintronregionsAvoidstretchesof4ormorebasessuchasAAAA,CCCCAvoidregionswithGCcontent<30%or>60%.AvoidrepeatsandlowcomplexsequenceAvoidsinglenucleotidepolymorphism(SNP)sitesPerformBLASThomologysearchtoavoidoff-targeteffectsonothergenesorsequencesAlwaysdesignnegativecontrolsbyscramblingtargetedsiRNAsequence.ThecontrolRNAshouldhavethesamelengthandnucleotidecompositionasthesiRNAbuthaveatleast4-5basesmismatchedtothesiRNA.Makesurethescramblingwillnotcreatenewhomologytoothergenes.TomTuschl'srulesSelecttargetedregionfromagivencDNAsequencebeginning50-100ntdownstreamofstartcondonFirstsearchfor23-ntsequencemotifAA(N19).Ifnosuitablesequenceisfound,then,Searchfor23-ntsequencemotifNA(N21)andconvertthe3'endofthesensesiRNAtoTTOrsearchforNAR(N17)YNNTargetsequenceshouldhaveaGCcontentofaround50%A=Adenine;T=Thymine;R=AdenineorGuanine(Purines);Y=ThymineorCytosine(Pyrimidines);N=Any.Byexperimentallyanalyzingthesilencingefficiencyof180siRNAstargetingthemRNAoftwogenesandcorrelatingitwithvarioussequencefeaturesofindividualsiRNAs,ReynoldsetalatDharmacon,IncidentifiedeightcharacteristicsassociatedwithsiRNAfunctionality.ThesecharacteristicsareusedbyrationalsiRNAdesignalgorithmtoevaluatepotentialtargetedsequencesandassignscorestothem.SequenceswithhigherscoreswillhavehigherchanceofsuccessinRNAi.Thetablebelowliststhe8criteriaandthemethodsofscoreassignment.CriteriaDescriptionScoreYesNo1Moderatetolow(30%-52%)GC1pointContent2Atleast3A/Usatpositions15-191point/perAor(sense)U3Lackofinternalrepeats1point(Tm*<20??C)4Aatposition19(sense)1point5Aatposition3(sense)1point6Uatposition10(sense)1point7NoG/Catposition19(sense)-1point8NoGatposition13(sense)-1pointAsumscoreof6definesthecutoffforselectingsiRNAs.AllsiRNAsscoringhigherthan6areacceptablecandidates.*Tm=79.8+18.5*log10([Na+])+(58.4*GC%/100)+(11.8*(GC%/100)2)-(820/Length)Forexample,theTmcanbecalculatedasfollowsforthesiRNAUUCUCCAGCUUCUAAAAUATm=79.8+18.5*log10(0.05)+(58.4*31.6/100)+(11.8*(31.6/100)2)-(820/19)ReferencesElbashirSMetal.(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.MauriceHo,RationalsiRNADesignTargetedregionsonthecDNAsequenceofatargetedgeneshouldbelocated50-100ntdownstreamofthestartcodon(ATG).SearchforsequencemotifAA(N19)TTorNA(N21),orNAR(N17)YNN,whereNisanynucleotide,Rispurine(A,G)andYispyrimidine(C,U).Avoidtargetingintrons,sinceRNAionlyworksinthecytoplasmandnotwithinthenucleus.Avoidsequenceswith>50%G+Ccontent.Avoidstretchesof4ormorenucleotiderepeats.Avoid5URTand3UTR,althoughsiRNAstargetingUTRshavesuccessfullyinducedgeneinhibition.Avoidsequencesthatshareacertaindegreeofhomologywithotherrelatedorunrelatedgenes.HowtoobtainacDNAsequencefortargetselectionBeforefindingaRNAitargetonthegeneofyourinterest,firstyouhavetogetitsmRNAsequenceorsequenceaccessionnumberassomesiRNAdesigntoolscantakeaccessionnumberasinput.Itisrecommendedtousethegene'sRefSeqfromNCBI,sincetheRefSeqrepresentsnon-redundant,curatedandvalidatedsequences.RefSeqmRNAsequenceshaveuniqueaccessionnumberswhichstartwithNMorXM,followedby6digits.Forexample,NM_123456(curatedmRNAsequence)orXM_0123456(modelmRNAsequencepredictedbygenomesequenceanalysis).ThereareseveralwaysofqueryingRefSeq.geneisfound,scrolldowntothe"NCBIReferenceSequence(RefSeq)"sectionandlookformRNA.SearchEntrezGeneat/entrez/query.fcgi?db=gene,andselecttherightgeneofdesiredorganism.Oncethepageforthegeneisshown,scrolldowntothe"NCBIReferenceSequence(RefSeq)"andlookformRNA.SearchNucleotidedatabaseusingEntrezquerytoolatanduseEntrezLimitssettingstorestrictyourquerytotheRefSeqdatabaseonlyoselect"RefSeq"fromthe"Onlyfrom"menu,thisrestrictsthequerytotheRefSeqcollectionoselect"mRNA"fromthe"Molecule"menu,thisrestrictsthequerytomRNARefSeqrecordsHomologysearchTheRNAitargetedregiononthemRNAsequenceofageneshouldnotsharesignificanthomologywithothergenesorsequencesinthegenome,therefore,homologysearchisessentialtominimizeoff-targeteffects.AlthoughmostsiRNAdesigntoolsprovideBLASToption,somesimplyuseNCBIBLASTtoolswhichsometimesarequiteslow.HerearesomeBLASTtoolsforhomologysearch.NCBIBlasttool:Nucleotide-nucleotideBLAST(blastn)orSearchforshort,nearlyexactmatchesBlattoolonUCSCGenomeWebsiteReferenc