siRNADesign

RNAitargetselectionrules:

1. TargetedregionsontheCDNAsequenceofatargetedgeneshouldbelocated50-100ntdownstreamofthestartcodon(ATG).
2. SearchforsequencemotifAA(N₁₉)TTorNA(N₂₁),orNAR(N₁₇)YNN,whereNisanynucleotide,Rispurine(A,G)andYispyrimidine(C,U).
3. Avoidtargetingintrons,sinceRNAionlyworksinthecytoplasmandnotwithinthenucleus.
4. Avoidsequenceswith>50%G+Ccontent.
5. Avoidstretchesof4ormorenucleotiderepeats.
6. Avoid5URTand3UTR,althoughsiRNAstargetingUTRshavesuccessfullyinducedgeneinhibition.
7. Avoidsequencesthatshareacertaindegreeofhomologywithotherrelatedorunrelatedgenes.

HowtoobtainacDNAsequencefortargetselection

BeforefindingaRNAitargetonthegeneofyourinterest,firstyouhavetogetitsmRNAsequenceorsequenceaccessionnumberassomesiRNAdesigntoolscantakeaccessionnumberasinput.Itisrecommendedtousethegene"sRefSeqfromNCBI,sincetheRefSeqrepresentsnon-redundant,curatedandvalidatedsequences.RefSeqmRNAsequenceshaveuniqueaccessionnumberswhichstartwithNMorXM,followedby6digits.Forexample,NM_123456(curatedmRNAsequence)orXM_0123456(modelmRNAsequencepredictedbygenomesequenceanalysis).ThereareseveralwaysofqueryingRefSeq.

1. SearchLocusLinkbygenenameorsymbolathttp://www.ncbi.nlm.nih.gov/LocusLink/.Oncethelocusofyourgeneisfound,scrolldowntothe"NCBIReferenceSequence(RefSeq)"sectionandlookformRNA.
2. SearchEntrezGeneathttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene,andselecttherightgeneofdesiredorganism.Oncethepageforthegeneisshown,scrolldowntothe"NCBIReferenceSequence(RefSeq)"andlookformRNA.
3. SearchNucleotidedatabaseusingEntrezquerytoolathttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=NucleotideanduseEntrezLimitssettingstorestrictyourquerytotheRefSeqdatabaseonly
   • select"RefSeq"fromthe"Onlyfrom"menu,thisrestrictsthequerytotheRefSeqcollection
   • select"mRNA"fromthe"Molecule"menu,thisrestrictsthequerytomRNARefSeqrecords

HomologysearchTheRNAitargetedregiononthemRNAsequenceofageneshouldnotsharesignificanthomologywithothergenesorsequencesinthegenome,therefore,homologysearchisessentialtominimizeoff-targeteffects.AlthoughmostsiRNAdesigntoolsprovideBLASToption,somesimplyuseNCBIBLASTtoolswhichsometimesarequiteslow.HerearesomeBLASTtoolsforhomologysearch.

• NCBIBlasttool:Nucleotide-nucleotideBLAST(blastn)orSearchforshort,nearlyexactmatches
• BlattoolonUCSCGenomeWebsitehttp://genome.ucsc.edu/cgi-bin/hgBlat
• EnsemblBlasthttp://www.ensembl.org/Multi/blastview

ExamplesofRNAitargetselection

References

1.ElbashirSM,HarborthJ,LendeckelW,YalcinA,WeberK,TuschlT.Duplexesof21-nucleotideRNAsmediateRNAinterferenceinculturedmammaliancells.Nature.2001May24;411(6836):494-8.2.ElbashirSM,LendeckelW,TuschlT.RNAinterferenceismediatedby21-and22-nucleotideRNAs.GenesDev.2001Jan15;15(2):188-200.

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