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1、PredictionforTargetSitesofSmallInterferingRNADuplexesinSARSCoronavirusThisinformationhasnotbeenpeer-reviewed.Responsibilityforthefindingsrestssolelywiththeauthor(三).DepositedresearcharticlePredictionforTargetSitesofSmallInterferingRNDuplexesinSRSCoronavirusFengminJi1and1.iaofu1.uo23Addresses:1Instit
2、uteforBiologicalScienceandTechnology,FacultyofSciences,NorthJiaotongUniversity,Beijing,100044China.21.aboratoryofTheoreticalBiophysics,FacultyofSciencesandTechnology,InnerMongoliaUniversity,Hohhot,010021China.3CenterforTheoreticalBiology,PekingUniversity,Beijing,100871China.Correspondence:1.iaofu1.u
3、o.E-mail:lfluomail.imu.eduPosted:16January2004Received:14January2001GenomeBiology2001,5:P6Theelectronicversionofthisarticleisthecompleteoneandcanbefoundonlineat200452P6Thisisthefirstversionofthisarticletobemadeavailableublicly.2004BioMedCentral1.td.depositedresearchASASERVICETOTHERESEARCHCOMMUNlTY,G
4、EN0MEBlO1.OGYPROVIDESAPREPRINTDEPOSITORYT0WHICHANYORIGINA1.RESEARCHCANBESUBMITTEDANDWHlCHA1.1.INDIVIDUA1.SCANACCESSFREEOFCHARGE.ANYARTIC1.ECANBESUBMITTEDBYAUT1IORS,WH0HAVESO1.ERESPONSTBI1.ITYFORTHEARTIC1.E,SCONTENT.THEON1.YSCREENINGISTOENSURERE1.EVNCEOFTHEPREPRINTTOGENOMEBIO1.OGY,SSCOPEANDTOAVOIDABU
5、SIVE,1.IBE1.1.OUSORINDECENTARTIC1.ES.ARTIC1.ESINTHISSECTIONOFTHEJOURNA1.HAVENOTBEENPEER-REVIEWED.EACHPREPRINTHASPERMNENTUR1.,BYWHICHITCANBECITED.RESEARCHSUBMITTEDTOTHEPREPRINTDEPOSITORYMAYBESIMU1.TNEOUS1.YORSUBSEQUENT1.YSUBMITTEDTOGENOMEBIO1.OGYORANYOTHERPUB1.ICATIONFORPEERREVIEW;THEON1.YREQUIREMENT
6、ISANEXP1.ICITCITTIONOF,AND1.INKTO,THEPREPRINTINANYVERSIONOFTHEARTIC1.ETHATISEVENTUA1.1.YPUB1.ISHED.IFPOSSIB1.E,GENOMEBIO1.OGYWI1.1.PROVIDEARECIPROCA1.1.INKFROMTHEPREPRINTTOTHEPUB1.ISHEDARTIC1.E.GenomeBiology2004,5:P6PredictionforTargetSitesofSmallInterferingRNADuplexesinSARSCoronavirusFengminJi11.ia
7、ofu1.uo2,3*1InstituteforBiologicalScienceandTechnology,FacultyofSciences,NorthJiaotongUniversity,Beijing,100044China;21.aboratoryofTheoreticalBiophysics,FacultyofSciencesandTechnology,InnerMongoliaUniversity,Hohhot,010021China;3CenterforTheoreticalBiology,PekingCoronavirus,SmallInterferingRNADuplex,
8、TargetSite,Anti-virusdrugdesignRunningheadTargetSitesofsiRNAinSARS-CoV*correspondingauthor,towhomcorrespondenceshouldbeaddressed.Mailingaddress:1.iaofu1.uo1.aboratoryofTheoreticalBiophysics,FacultyofSciencesandTechnology,InnerMongoliaUniversity,Hohhot,010021ChinaEmai1address:IfIUoWmail.imu.eduFax:00
9、86-471-49517611AbstractRNinterferenceisusedforSARS-rcIatedpharmaceuticalresearchanddevelopment.Followingbioinformaticmethodtwentyseven2125base-longsequencesegmentsinSARS-CoVgenomearepredictedastheoptimaltargetsitesofsmallinterferingRNAduplexes.SARS(severeacuterespiratorysyndrome)wasfirstidentifiedin
10、GuangdongProvince,ChinaandrapidlyspreadtomanyregionsinChinaandaroundtheworld.Itcauseddeathanddisastertothousandsofhumanbeings.However,theactivedrugintreatingSARShasnotbeenfoundyet.Thegenomesequencesdeterminedbyseveralgroupsl-3showthatitisavariantofcoronaviruses,belongingtosingle-strandedplussenseRNv
11、iruses.Thegenomeisabout30kbinlength,anditsseveralencodedproteinshavebeenseparatedandpurified.ThisUniversity,Beijing,100871ChinaKeywordsSRSprovidesasoundbasisforSARSrelatedpharmaceuticalresearchanddevelopment.Theuseofdouble-strandedRNA(dsRN)tomanipulategeneexpression(RNAinterferenceorRNAi)hasbeenpiov
12、edhighlyeffective,atleast10timesmoreeffectivethaneitherusingsenseorantisenseRNAsalone4.TheRNAitriggeredbydsRNAisaphenomenonofhomo1ogy-dependentgenesilencing567.Itwasfoundthatthesmal1interferingRNA(SiRNA,21-25nt1ong)playsanimportantroleinRNAi-relatedgenesilencingpathways8.Progresshasalsobeenmadeinant
13、i-HIVandanti-HCVdrugdesignbyapplyingthemethodofRNinterference9-10.TodesignanIi-SARS-CoVdrug,onestrategyistosearchforsiRNAswhichspecificallyinterferethegeneexpressionandblockthegenomereplicationofSARS-associatedcoronavirus.Inthisnoteweshal1maketheoreticalpredictiononthepossibletargetsitesofsiRNAsinth
14、evirusgenome.2Uponinfectionofanappropriatehostcell,theviralenvelopeisfusedwithcel1membraneandtheviralplussenseRNAentersthehostcell.Thenthe5mostORFoftheviralgenomeistranslatedintoseveralnonstructuralproteinsincludinganRNA-dependentRNApolymeraseandanATPasehelicase.Theseproteinsinturnareresponsibleforr
15、eplicatingtheviralgenomeaswellasgeneratingnestedtranscriptsthatareusedinthesynthesisoftheviralproteins.Thetranscriptionallyactive,SUbgenomiC-SiZeminusstrandsarealsodiscovered2.ThesiRNA-mediatedRNAinterferencehasstrongspecificity,andmayplaycertainrolesinaffectingtheprocessofvirusexpressionandprolifer
16、ation.RNAsecondarystructureiscomposedofdouble-strandedregionofstackedbasepairs(stem)andsingle-strandedregion(loop).RNAstructurepredictionscomprisebase-pairedandnon-base-pairedregionsinvarioustypesofloopandjunctionarrangements(includinghairpinloop,bulgeloop,interior1oopandjunctionsormulti-loopsl1).On
17、ly2125ntlong(ormore)non-base-pairedregionscanbeservedasthetargetsitesofsiRNA.Theyarecalledfreesegments.Thelongnon-base-pairedregioncontainingoneorseveralshortstems(totallengthofstems3basepairs)isalsoconsideredinourstatistics.Thelatteriscalledquasi-freesegments.ByusingprogramRNAstructure(version3.7)1
18、2wefoldedtheRNsequenceofviralgenomeinawindowof3000nucleotides,andshiftedthewindow1500nucleotideseachstepalongthesequence,sothateachsiteinvirusgenomehasparticipatedindifferentfoldsmorethan10times.Weselected2125base-longfreeandquasi-freesegmentsasthecandidatesfortarget3sitesofRNinterferencewhenthesese
19、gmentsfrequentlyoccurredinnon-base-pairedregionsbasedontheabovecalculation.givenRNAsequencesegmentmayhavedifferentconfigurationsofsecondarystructurewithlowerfreeenergy,somecontainingshortstems(quasi-free)butsomenot(free).Thetotalfrequencyofasegmentoccurringinnon-base-pairedregionofdifferentfoldsisca
20、lledappearancerate.Ifeachquasi-freecaseismultipliedbyareducedfactorinnumeration,namely,by0.9for1basepair,0.8for2basepair,and0.7for3basepairs(basepairsmaybecontinuousinstructureordisconnected)thenthetotalnumberoffoldsiscalledreducedappearancerate.TheantisenseoligonucIeotide(AO)complementarytoaspecifi
21、csub-sequenceofanRNtargethasbeenextensivelyinvestigated.AOefficacyisaffectedbymanyfactors.Apartfromthebindingenergybetween0andRN,whichdescribesthe0accessibi1itytotheRNA1thesequencemotifisanotherimportantfactor.Thecorrelationof9sequencemotifswith0efficacywasdeducedempiricalIyin1314.Ifthetargetsequenc
22、econtainsCCAC,TCCC,CTC,GCCandCTCT,thenitwillmakeapositivescore.IfthetargetsequencecontainsGGGG,CTG,TandAA,thenitwillmakeanegativescore.Ontheotherhand,experimentshowsthat2nt3overhangsinsiRNAduplexhasplayedanimportantroleinitsstabiIization8.Thatmeansin5endofthesequencesegmentisfavorableforitstarget.In
23、SARS-CoVgenomewehavefoundseveraltenslongsegments(length20nt)matching4withthoseofhumanbeings.Toguaranteethesafetyofthedesigneddrug,wemakealignmentoffreeandquasi-freesegmentsofhighappearanceratewithhumangenomeanddeletethematchingones(morethan18exactlymatchingbases)insiRNAtargetcandidates.BytheuseofRNA
24、sequencedataofSARS-CoV,IsolateTor2,twentysevenoptimal2025base-longsiRNAtargetsareselectedfrom60000candidatesinbothstrands.Theyare1istedinTable1and2forminus-strandandplus-strandrespectively.Eachsegmentisscored.Themaintermofscoreisthevalueofreducedappearancerate(column5ofTable1and2).ThesumofAOefficaci
25、es(multipiicdby10)inasegmentisalsolistedforreference(column6).TheenhancingfactorofAAoccurredin5endisindicatedincolumn7bynotation+.Theresultsofmultiplesequencealignmentof19completeSARScoronavirusgenomegivethemutationalsitesbetweendifferentstrainsC15.Thelasttermofscoreisrelatedtomutationalsites.Eachpo
26、intmutationinsiRNAtargetsequencecontributes-1inscore(column8).Thoughtherelativeimportanceofthesetermscannotbequantitativelyestimatedatpresentweexpectthatthemaincontributiontothescorecomesfromthereducedappearancerate(column5).Generally,intheproliferationofplus-senseRNAvirusestheconcentrationofplus-st
27、randismuchhigherthanthatofminus-strand.Forexample,theymaydifferby100timesinTMV(tobaccomosaicvirus)16.Iftheconcentrationofminus-strandinSARS-CoVislower,thentheRNAinterferencetargetedatvirusminus-strandwillbemoreeffective.WesuggestthatthelatterpointShOUldbecheckedbyexperimentsimmediatelysinceitisimpor
28、tantfordesigninganeffectivesiRNAduplex.5Theaboveapproachisofbroadinteresttootheranti-virusdrugdesign.AcknowledgementAuthorsaregratefultoDr.J.C.1.uoforhissendingSARS-relatedmaterial.TheworkwassupportedbyNationalScienceFoundationofChina,Project90103030.References1.Rota,P.A.,Oberste,M.S.,Monroe,S.S.eta
29、l(2003)CharacterizationofaNovelCoronavirusAssociatedwithSevereAcuteRespiratorySyndrome,Science300(5624)1394-1399.2.Marra,M.A.,Jones,S.J.,stell,C.R.etal.(2003)TheGenomeSequenceoftheSARS-AssociatedCoronavirus.Science300(5624)1399-1404.3Qin,E.,Zhu,Q.Y.,Yu,M.etal.(2003)completesequenceandcomparativeanal
30、ysisofaSRS-associatedvirus(IsolateBJOl).ChineseScienceBulletin.48(10),941-948.4.Fire,A,Xu,S,Montgomery,M.K.,etal(1998)Potentandspecificgeneticinterencebydouble-strandedRNAinCaenorhabditiselcgans.Nature391(6669),806811.5.Bernstein,E.,Den1i,.M.,Hannon,G.J.(2001)Therestissilence.RNA7,1509-1521.6.Plaste
31、rk,R.H.A.RNsplicing:Thegenomesimmunesystem(2002).Science296,1263-1265.7.Zamore,P.D.(2002).AncientpathwaysprogrammedbysmallRNAs.Science296,1265-1269.68.Elbashir,S.M.,Harborth,J.,1.endeckel,W.etal.(2001)Duplexesof21-nucleotidcRNAsmediateRNAinterferenceinculturedmammaliancells.Nature411(6836),494.9.Jac
32、queJM.,TriquesK.,StevensonM.(2002)ModulationofHIV-IreplicationbyRNAinterference.Nature418(6896),435.10.Wi1sonJ,JayasenaS.,Khvorova.etal.(2003)RNinterferenceblocksgeneexpressionandRNAsynthesisfromhepatitisCrepliconspropagatedinhumanlivercells.Proc.Natl.Acad.Sci.USA100(5),2783.11.MountD.M.Bioinformati
33、cs.(2001)ColdSpringHarbor1.aboratoryPress.Ch.5.12.MathewsD.H.,SabinaJ.,ZukerM,TurnerD.H.(1999)ExpandedSequenceDependenceofThermodynamicParametersImprovesPredictionofRNASecondaryStructure.JMolBiol,288,911-940.13.Matveeva,0.V.,Tsodkov.D.,Giddings,M.etal.(2000)Identificationofsequencemotifsinoligonucle
34、otideswhosepresenceiscorrelatedwithantisenseactivity.NucleicAcidsRes.28,2862-2865.14.Chalk,.M,SonnhammerE.1.1.(2002)Computationalantisenseoligopredictionwithaneuralnetworkmodel.BioinformaticslS,15671575.15.Resultsofmultiplesequencealignmentof19completeSARScoronavirusgenome,at/news/news.jsp?id=114.16
35、.IshikawaM.,Mcshi,T.Ohno,T,OkadaY.etal.(1991)SpecificCessationofMinusStrandRNAccumulationatanEarlyStageofTMVInfection.J.Virol.65,861-868.7Table1siRNAtargetsequenceinminus-strandSARS-CoVtargetsequence53,positionlengthappear.reducedAOAAmut.rateappear,rate8eff5site(10)endscoreUUUCUUGAAUUCCGCGACUACAAUUG
36、AUCUAAGAGUAAAAUUGUCAACCAGUUCACCCUCCCUucGaauuguuauaguaagACaucAAAAACaAAAGUGACCCUCUAAGCUCACCCUUGUGAGUACCCAUGCAUCACGACCACACACACCUAGUAUAGGUCGGUUUUUUCCUUUUCUUCCUUCAAUACUAAAUUUUCAAGCUACACAGAUUUUGUU1041-10644446-4467242221212121212121212121897+-1-106.3-4.4-1.22.612902-1292217025-17045202922031221671-2169122
37、788-2280824177-2419725280-2530025327-2534728430-2845029662296825.613710.44.90-5.0-1.2+011101211101088.80100121.7010.68.6-2.20+010-1.4-1.207.808Table2siRNAtargetsequenceinplus-strandSARS-CoVlengthappear.reduced0Amut.targetsequence5-3,endpositionrateappear,rate8eff.5site(10)scoreAAACAAUAAUAAAUUUUACUGU
38、UGUUUCUGUUACCUUCUCUUAAUCAUUAUUAAAGACUGUA?UCCCGUCCUCGAUUUUUCUCCCUUUUUCACAAAUUGCCUUUCUUUUACUAUUGACUACAAGGGCCCCACCUUCUCCCCUCUUUUCUUAUUAUUUCUUACUCAUUAUUAACAAUUCUACUUCCUUGCUCUAUUUUUUCCACAGUUUAUGAUAAUAUGAGCAAUAUAUUAAAUACGACUCUUUUUAUUCGCUGAAAUUAUUCUCUU128-1482121212321212122212121212121248-4.21.8+00000000000000011107-1112713921-1394115861-1588317699-177191929119311198091982920567-2058821499-2151921837-2185724327-2434724834-2485425051-2507126347-2636727266272891214109129.810-3.08.17.26.4118+8-2.0-2.00.91112131312811.810.79.110.86.49-3.0-1.2+910109
