Design and use of synthetic regulatory small RNAs

©2013NatureAmerica,Inc.Allrightsreserved.protocol1694|VOL.8NO.9|2013|natureprotocolsIntroDuctIonEfficientandconvenientcontrolovergeneexpressionatagenome-widescaleisessentialinfundamentalandappliedbio-logicalstudies,butitisstillabigchallengebecauseofthedifficul-tiesinconstructinggene-knockoutlibraries.Althoughtherehavebeenafewstudiesontheconstructionofknockoutlibraries,suchhigh-throughputstudiesarerestrictedtothosewhopossessthelibraries1–3.Gene-knockoutexperiments,whichareusuallyusedinmetabolicengineering,aretime-consumingandinvolvethesamelaboriousanditerativeeffortstoconstructlibraries,becausemultiplegeneknockoutsareperformedsequentiallyanditisdif-ficulttoreversedeletions.Therefore,high-throughputstudiesusinggeneknockoutsarelimitedtoafewconstructedlibrarystrains.Thisisachallengeinthemetabolicengineeringfield,asdifferentE.colistrainsshowvaryingperformanceintermsofproducingawiderangeofchemicalsandmaterialsofinterest4,andthusallcandidatestrainsneedtobeengineeredsothattheirperformancecanbeexamined.ThemajorityofsyntheticsRNAsdevelopedsofarfortheeffi-cientregulationofgeneexpressioninEscherichiacoliaresyntheticriboswitches5–7.However,usingsuchcis-actingsRNAscannotbeappliedtometabolicengineering,becausetheymustbeinsertedintothe5′UTRofthechromosomaltargetgenesinordertocon-troltheexpressionofthegeneofinterest.Furthermore,itisdif-ficulttodesignriboswitchsequences,astheyincludesecondarystructuresthatdynamicallychangeuponthebindingofsignalchemicals.Incontrast,Hfqprotein–dependentsRNAsareabletoefficientlycontroltheexpressionoftargetgenesintransthroughbase-paircomplementation.Owingtotheirmodeofaction,sRNAscanbeproducedthroughplasmid-basedexpressiontoregulatechromosomalgeneexpression,withoutdirectmodifi-cationofthechromosomesequence.IthasbeendiscoveredthatHfqproteinsandsRNAsareconservedinprokaryoticspeciesthatareevolutionarilyclosetoE.coli8,9.ThissuggeststhepossibilityofapplyingsyntheticsRNAstoevolutionarilyclosebacterialspeciesinwhichsRNA-mediatedinterferencemechanismisalsoconserved,providedthatappropriatemodificationsaremadeinthesyntheticsRNAdesign.Unfortunately,comparedwiththewell-establishedcharacteris-ticsofeukaryoticRNAinterference,theelusivenessofprokaryoticinterferencebysRNAsmakesitratherdifficulttodeveloptrans-actingsyntheticsRNAs,andthustheapproachesdevelopedsofarhavereliedonrandomscreening10–12.Therefore,anewtypeofsyntheticsRNAthatcanbedesignedwitheaseandthatcanmodulateprokaryoticchromosomalgeneexpressionwithoutcomplicatedengineeringisrequiredforbiologicalandbiotech-nologicalstudies.DevelopmentoftheprotocolRecently,wereportedthedevelopmentofanewgeneexpres-sioncontrolsystemusingsyntheticsRNAsthatwerebasedontheabilitiesandcharacteristicsofnaturalsRNAs13.sRNAsrepressoractivatethetranslationoftargetmRNAsinprokaryoticcells14–16.AmongsRNAs,thetrans-actingandHfq-dependentsRNAspos-sessdistinguishingfeatures,withtheassistanceoftheaccessoryproteinHfq,suchasefficienttargetbinding,facilitatedtarget-mRNAdegradationandaprolongedhalf-life17,18.Theyarecom-posedoftwofunctionalparts:oneisfortarget-mRNAbindingandtheotherisforrecruitingtheHfqproteins.ThesefeaturesshouldbeincludedinsyntheticsRNAsinordertoachieveeffi-cienttranslationrepression.Withthisinmind,weselectedthemostsuitableoftheknownE.colisRNAstouseinoursystem,thussatisfyingallthefollowingconditions:sRNAswhosetarget-bindingsequencesandHfq-bindingsequencesareidentified;sRNAsthatareknowntotargetonlyonemRNAsoastoavoidoff-targetsilencing;andsRNAsthatshowefficientrepressionoftargetgenesexperimentally13.WefurtherdevelopedsyntheticsRNAdesignDesignanduseofsyntheticregulatorysmallRNAstocontrolgeneexpressioninEscherichiacoliSeungMinYoo1,2,DokyunNa1–3&SangYupLee11MetabolicandBiomolecularEngineeringNationalResearchLaboratory,DepartmentofChemicalandBiomolecularEngineering(TheWorldClassUniversityProgram),BioProcessEngineeringResearchCenter,BioinformaticsResearchCenter,CenterforSystemsandSyntheticBiotechnology,InstitutefortheBioCentury,KoreaAdvancedInstituteofScienceandTechnology(KAIST),Daejeon,RepublicofKorea.2Theseauthorsequallycontributedtothiswork.3Presentaddress:CentreforHigh-ThroughputBiology,UniversityofBritishColumbia,Vancouver,BritishColumbia,Canada.CorrespondenceshouldbeaddressedS.Y.L.([e-mail]).Publishedonline8August2013;doi:10.1038/nprot.2013.105Geneknockoutexperimentsareoftenessentialinfunctionalgenomicsandmetabolicengineeringstudies.However,repeatedmultiplegeneknockoutexperimentsarelaborious,timeconsumingandsometimesimpossibletoperformforthosegenesthatareessentialforcellfunction.smallregulatoryrnas(srnas)areshortnoncodingrnasinprokaryotesthatcanfinelycontroltheexpressionoftargetgenesintransatthepost-transcriptionallevel.Herewedescribetheprotocolforsyntheticsrna-basedgeneexpressioncontrol,includingsrnadesignprinciples.customizedsyntheticsrnasconsistofascaffoldandatarget-bindingsequence,andtheycanbecreatedbysimplyreplacinganexistingtarget-bindingsequencewithonethatiscomplementarytothetargetmrnatoberepressed,whileretainingthescaffold.ourplasmid-basedsyntheticsrnasystem