Mind your caps and Poly A tails - NEB

The poly(A) tail can be encoded in the DNA template by using an appropriately tailed PCR primer, or it can be added to the RNA by enzymatic treatment with E. Home Tools&Resources FeatureArticles Mindingyourcapsandtails ApplicationsofsyntheticmRNAhavegrownandbecomeconsiderablydiversifiedinrecentyears.Examplesincludethegenerationofpluripotentstemcells(1-3),vaccinesandtherapeutics(4-5),andCRISPR/Cas9genomeeditingapplications(6-8).ThebasicrequirementsforafunctionalmRNA–a7-methylguanylatecapatthe5´endandapoly(A)tailatthe3´end–mustbeaddedinordertoobtainefficienttranslationineukaryoticcells.Additionalconsiderationscanincludetheincorporationofinternalmodifiedbases,modifiedcapstructuresandpolyadenylationstrategies.StrategiesforinvitrosynthesisofmRNAvaryaccordingtothedesiredscaleofsynthesis.ThisarticlediscussesoptionsfortheselectionofreagentsandtheextenttowhichtheyinfluencesynthesizedmRNAfunctionality. byBretonHornblower,Ph.D.,G.BrettRobb,Ph.D.andGeorgeTzertzinis,Ph.D.,NewEnglandBiolabs,Inc AnascentmRNA,synthesizedinthenucleus,undergoesdifferentmodificationsbeforeitcanbetranslatedintoproteinsinthecytoplasm.ForamRNAtobefunctional,itrequiresmodified5´and3´endsandacodingregion(i.e.,anopenreadingframe(ORF)encodingfortheproteinofinterest)flankedbytheuntranslatedregions(UTRs).ThenascentmRNA(pre-mRNA)undergoestwosignificantmodificationsinadditiontosplicing.Duringsynthesis,a7-methylguanylatestructure,alsoknownasa“cap”,isaddedtothe5´endofthepre-mRNA,via5´→5´triphosphatelinkage.ThiscapprotectsthematuremRNAfromdegradation,andalsoservesaroleinnuclearexportandefficienttranslation. Thesecondmodificationoccursposttranscriptionallyatthe3´endofthenascentRNAmolecule,andischaracterizedbyadditionofapproximately200adenylatenucleotides(poly(A)tail).Theadditionofthethepoly(A)tailconfersstabilitytothemRNA,aidsintheexportofthemRNAtothecytosol,andisinvolvedintheformationofatranslation-competentribonucleoprotein(RNP),togetherwiththe5´capstructure.ThematuremRNAformsacircularstructure(closed-loop)bybridgingthecaptothepoly(A)tailviathecap-bindingproteineIF4E(eukaryoticinitiationfactor4E)andthepoly(A)-bindingprotein,bothofwhichinteractwitheIF4G(eukaryoticinitiationfactor4G),(Figure1,(9)). Figure1.Translationinitiationcomplex. AmaturemRNA,consistingofthe5´and3´untranslatedregions(UTRs)andtheopenreadingframe(ORF),formsa“closed-loop”structureviainteractionsmediatedbyproteincomplexesthatbindthecapstructureandthepoly(A)tail.   RNAcanbeefficientlysynthesizedinvitro(byinvitrotranscription,IVT)withprokaryoticphagepolymerases,suchasT7,T3andSP6.Thecapandpoly(A)tailstructurescharacteristicofmaturemRNAcanbeaddedduringorafterthesynthesisbyenzymaticreactionswithcappingenzymesandPoly(A)Polymerase(NEB#M0276),respectively. ThereareseveralfactorstoconsiderwhenplanningforIVT-mRNAsynthesisthatwillinfluencetheease-of-experimentalsetupandyieldofthefinalmRNAproduct.Thesearediscussedinthefollowingsections. DNAtemplate TheDNAtemplateprovidesthesequencetobetranscribeddownstreamofanRNApolymerasepromoter.Therearetwostrategiesforgeneratingtranscriptiontemplates:PCRamplificationandlinearizationofplasmidwitharestrictionenzyme(Figure2).Whichonetochoosewilldependonthedownstreamapplication.Ingeneral,ifmultiplesequencesaretobemadeandtranscribedinparallel,PCRamplificationisrecommendedasitgeneratesmanytemplatesquickly.Ontheotherhand,iflargeamountsofoneorafewtemplatesarerequired,plasmidDNAisrecommended,becauseoftherelativeeaseofproducinglargequantitiesofhighquality,fullycharacterizedplasmids.TherearedifferentversionsofplasmidsavailablethatallowforpropagationofhomopolymericA-tailsofdefinedlength(1). PCRallowsconversionofanyDNAfragmenttoatranscriptiontemplatebyappendingtheT7(orSP6)promotertotheforwardprimer(Figure2A).Additionally,poly(d)T-tailedreverseprimerscanbeusedinPCRtogeneratetranscriptiontemplateswithA-tails.Thisobviatestheneedforaseparatepolyadenylationstepfollowingtranscription.Repeatedamplificationsshould,however,beavoidedtopreventPCR-generatedpointmutations.AmplificationusingPCRenzymeswiththehighestpossiblefidelity,suchasQ5®High-FidelityDNAPolymerase(NEB#M0491),reducesthelikelihoodofintroducingsuchmutations(2). Figure2.Methodsforgeneratingtranscriptiontemplates. (A)PCRcanbeusedtoamplifytargetDNApriortotranscription.Apromotercanbeintroducedviatheupstreamprimer. (B)WhenusingplasmidDNAasatemplate,linearizewithanenzymethatproducesbluntor5´-overhangingends.UsingatypeIISrestrictionenzyme(e.g.,BspQI)allowsRNAsynthesiswithnoadditional3´-nucleotidesequencefromtherestrictionsite.   ThequalityofthePCRreactioncanbeassessedbyrunningasmallamountonanagarosegel,andDNAshouldbepurifiedbeforeinvitrotranscriptionusingaspincolumnormagneticbeads(e.g.,AMPure®beads).MultiplePCRreactionscanbepurifiedandcombinedtogenerateaDNAstocksolutionthatcanbestoredat-20°Candusedasneededforinvitrotranscription. PlasmidtemplatesareconvenientifthetemplatesequencealreadyexistsinaeukaryoticexpressionvectoralsocontainingtheT7promoter(e.g.,pcDNAvectorseries).Thesetemplatesinclude5´-and3´-untranslatedregions(UTR),whichareimportantfortheexpressioncharacteristicsofthemRNA. PlasmidDNAshouldbepurifiedandlinearizeddownstreamofthedesiredsequence,preferablywitharestrictionenzymethatleavesbluntor5´overhangsatthe3´endofthetemplate.Thesearefavorableforproperrun-offtranscriptionbyT7RNAPolymerase(NEB#M0274),while3´overhangsmayresultinunwantedtranscriptionproducts.ToavoidaddingextranucleotidesfromtherestrictionsitetotheRNAsequence,aTypeIISrestrictionenzymecanbeused(e.g.,BspQI,NEB#R0712),whichpositionstherecognitionsequenceoutsideofthetranscribedsequence(Figure2B,page2).TheplasmidDNAshouldbecompletelydigestedwiththerestrictionenzyme,followedbypurificationusingaspincolumn(e.g.,Monarch®PCR&DNACleanupKit(5μg)NEB#T1030)orphenolextraction/ethanolprecipitation.Althoughlinearizationofplasmidinvolvesmultiplesteps,theprocessiseasiertoscaleforthegenerationoflargeamountsoftemplateformultipletranscriptionreactions. Invitrotranscription Therearetwooptionsfortheinvitrotranscription(IVT)reactiondependingonthecappingstrategychosen:standardsynthesiswithenzyme-basedcappingfollowingthetranscriptionreaction(post-transcriptionalcapping)orincorporationofacapanalogduringtranscription(co-transcriptionalcapping)(Figure3).MethodselectionwilldependonthescaleofmRNAsynthesisrequiredandnumberoftemplatestobetranscribed. Figure3.Invitrotranscriptionoptionsbaseduponcappingstrategy Enzyme-basedcapping(top)isperformedafterinvitrotranscriptionusing5´-triphosphateRNA,GTP,andS-adenosyl-methionine(SAM).Cap-0mRNAcanbeconvertedtoCap-1mRNAusingmRNAcap2´-O-methyltransferase(MTase)andSAMinasubsequentorconcurrentreaction.ThemethylgrouptransferredbytheMTasetothe2´-Oofthefirstnucleotideofthetranscriptisindicatedinred.Conversionof~100%of5´-triphosphorylatedtranscriptstocappedmRNAisroutinelyachievableusingenzyme-basedcapping. Co-transcriptionalcapping(bottom)usesanmRNAcapanalog,showninyellow,inthetranscriptionreaction.ForARCA(anti-reversecapanalog)(left),thecapanalogisincorporatedasthefirstnucleotideofthetranscript.ARCAcontainsanadditional3´-O-methylgrouponthe7-methylguanosinetoensureincorporationinthecorrectorientation.The3´-O-methylmodificationdoesnotoccurinnaturalmRNAcaps.Comparedtoreactionsnotcontainingcapanalog,transcriptionyieldsarelower.ARCA-cappedmRNAcanbeconvertedtocap1mRNAusingmRNAcap2´-O-MTaseandSAMinasubsequentreaction.CleanCapReagentAG(right)usesatrinucleotidecapanalogthatrequiresamodifiedtemplateinitiationsequence.AnaturalCap-1structureisaccomplishedinaco-transcriptionalreaction.   Transcriptionforenzyme-basedcapping (post-transcriptionalcapping) StandardRNAsynthesisreactionsproducethehighestyieldofRNAtranscript(typically≥100μgper20μlina1hrreactionusingtheHiScribe™QuickT7HighYieldRNASynthesisKit,NEB#E2050S).Transcriptionreactionsarehighlyscalable,andcanbeperformedusinganall-inclusivekit(e.g.,HiScribekits),orindividualreagents.MoreinformationontheHiScribekitscanbefoundlaterinthearticle. Followingtranscription,theRNAistreatedwithDNaseI(NEB#M0303)toremovetheDNAtemplate,andpurifiedusinganappropriatecolumn,kitormagneticbeads,priortocapping.ThismethodproduceshighyieldsofRNAwith5´-triphosphateterminithatmustbeconvertedtocapstructures.Intheabsenceoftemplate-encodedpoly(A)tails,transcriptsproducedusingthismethodbear3´terminithatalsomustbepolyadenylatedinaseparateenzymaticstep,asdescribedbelowin“Post-transcriptionalcappingandCap-1methylation”. Transcriptionwithdinucleotideco-transcriptionalcapping Inco-transcriptionalcapping,acapanalogisintroducedintothetranscriptionreaction,alongwiththefourstandardnucleotidetriphosphates,inanoptimizedratioofcapanalogtoGTP4:1.ThisallowsinitiationofthetranscriptwiththecapstructureinalargeproportionofthesynthesizedRNAmolecules.Thisapproachproducesamixtureoftranscripts,ofwhich~80%arecapped,andtheremainderhave5´-triphosphateends.DecreasedoverallyieldofRNAproductsresultsfromthelowerconcentrationofGTPinthereaction(Figure4). Thereareseveralcapanalogsusedinco-transcriptionalRNAcapping(3,4).Themostcommonarethestandard7-methylguanosine(m7G)capanalogandanti-reversecapanalog(ARCA),alsoknownas3´O-me7-meGpppGcapanalog.ARCAismethylatedatthe3´positionofthem7G,preventingRNAelongationbyphosphodiesterbondformationatthisposition. Figure4.StructureofCleanCapReagentAG   Thus,transcriptssynthesizedusingARCAcontain5´-m7Gcapstructuresinthecorrectorientation,withthe7-methylatedGastheterminalresidue.Incontrast,them7Gcapanalogcanbeincorporatedineitherthecorrectorthereverseorientation. HiScribeT7ARCAmRNASynthesiskits(NEB#E2060and#E2065)containreagents,includinganoptimizedmixofARCAandNTPs,forstreamlinedreactionsetupforsynthesisofco-transcriptionallycappedRNAs.   TranscriptionwithCleanCap®reagentAGco-transcriptionalcapping TheuseofCleanCapreagentAGresultsinsignificantadvantagesovertraditionaldinucleotideco-transcriptionalcapping.CleanCapReagentAGisatrinucleotidewitha5´-m7Gjoinedbya5´-5´triphosphatelinkagetoanAGsequence.Theadeninehasamethylgrouponthe2´-Oposition(Figure4).TheincorporationofthistrinucleotideinthebeginningofatranscriptresultsinaCap-1structure. InordertouseCleanCapReagentAGinaninvitrotranscriptionreactionthetemplatemustcontainanAGinplaceofaGGfollowingtheT7promoterintheinitiationsequence. Unliketraditionalco-transcriptionalcapping,reductionofGTPconcentrationisnotrequiredandthereforeyieldishigherandhighcappingeffiencies,>95%,areachieved(Figure5). Figure5.ComparisonofRNAyieldsfrominvitrotranscriptionreactions. Allreactionswereperformedwith5mMCTP,5mMUTPand6mMATP.StandardIVTreactionsincluded5mMGTPandnocapanalog.ARCAreactionscontaineda4:1ratioofARCA:GTP(4mM:1mM).IVTwithCleanCapReagentAGcontained5mMGTPand4mMCleanCapReagentAGandwasperformedasdescribedbelow(StandardmRNASynthesis).Reactionswereincubatedfor2hoursat37°C,purifiedandquantifiedbyNanoDrop®.   Transcriptionwithcompletesubstitutionwith modifiednucleotides RNAsynthesiscanbecarriedoutwithamixtureofmodifiednucleotidesinplaceoftheregularmixtureofA,G,CandUtriphosphates.Forexpressionapplications,themodifiednucleotidesofchoicearethenaturallyoccurring5´-methylcytidineand/orpseudouridineintheplaceofCandU,respectively.ThesehavebeendemonstratedtoconferdesirablepropertiestothemRNA,suchasincreasedmRNAstability,increasedtranslation,andreducedimmuneresponseinthekeyapplicationsofproteinreplacementandstem-celldifferentiation(1).ItisimportanttonotethatnucleotidechoicecaninfluencetheoverallyieldofmRNAsynthesisreactions. FullysubstitutedRNAsynthesiscanbeachievedusingtheHiScribeT7mRNAKitwithCleanCapReagentAG(NEB#E2080),HiScribeT7High-YieldRNASynthesisKit(NEB#E2040)orHiScribeSP6RNASynthesisKit(NEB#E2070)inconjunctionwithNTPswiththedesiredmodification.Transcriptsmadewithcompletereplacementofoneormorenucleotidesmaybepost-transcriptionallycapped(seenextsection),ormaybeco-transcriptionallycappedbyincludingCleanCapReagentAG,ARCAoranothercapanalog,asdescribedpreviously. Ifpartialreplacementofnucleotidesisdesired,theHiScribeT7ARCAmRNASynthesisKits(NEB#E2060and#E2065),maybeusedwithaddedmodifiedNTPs,toproduceco-transcriptionallycappedmRNAs,asdescribedabove.Alternatively,theHiScribeT7QuickRNASynthesisKit(NEB#E2050)maybeusedtopreparetranscriptsforpost-transcriptionalcapping.  Post-transcriptionalcappingand Cap-1methylation Post-transcriptionalcappingisoftenperformedusingthemRNAcappingsystemfromVacciniavirus.Thisenzymecomplexconvertsthe5´-triphosphateendsofinvitrotranscriptstom7G-cap(Cap-0)requiredforefficientproteintranslationineukaryotes.TheVacciniaCappingSystem(NEB#M2080)comprisesthreeenzymaticactivities(RNAtriphosphatase,guanylyltransferase,guanineN7-methyltransferase)thatarenecessaryfortheformationofthecompleteCap-0structure,m7Gppp5´N,usingGTPandthemethyldonorS-adenosylmethionine.Asanaddedoption,theinclusionofthemRNACap2´O-Methyltransferase(NEB#M0366)inthesamereactionresultsinformationoftheCap-1structure(m7Gppp5´Nm),anaturalmodificationinmanyeukaryoticmRNAsresponsibleforevadingcellularinnateimmuneresponseagainstforeignRNA.Thisenzyme-basedcappingapproachresultsinahighproportionofcappedmessage,anditiseasilyscalable.TheresultingcappedRNAcanbefurthermodifiedbypoly(A)additionbeforefinalpurification.  A-tailingusingE.coliPoly(A) Polymerase Thepoly(A)tailconfersstabilitytothemRNAandenhancestranslationefficiency.Thepoly(A)tailcanbeencodedintheDNAtemplatebyusinganappropriatelytailedPCRprimer,oritcanbeaddedtotheRNAbyenzymatictreatmentwithE.coliPoly(A)Polymerase(NEB#M0276).ThelengthoftheaddedtailcanbeadjustedbytitratingthePoly(A)Polymeraseinthereaction(Figure6). TheimportanceoftheA-tailisdemonstratedbytransfectionofuntailedvs.tailedmRNA.WhenluciferaseactivityfromcellstransfectedwithequimolaramountsoftailedoruntailedmRNAswerecompared,asignificantenhancementoftranslationefficiencywasevident(Figure6).HiScribeT7ARCAmRNASynthesisKit(withtailing)(NEB#E2060)includesE.coliPoly(A)Polymerase,andenablesastreamlinedworkflowfortheenzymatictailingofco-transcriptionallycappedRNA. FormRNAsynthesisfromtemplateswithencodedpoly(A)tails,theHiScribeT7ARCAmRNASynthesisKit(NEB#E2065)providesanoptimizedformulationforco-transcriptionallycappedtranscripts. Figure6.AnalysisofcappedandpolyadenylatedRNA (A)Agilent®Bioanalyzer®analysisofcappedandpolyadenylatedRNA.Longertailsareproducedbyincreasingtheenzymeconcentrationinthereaction.CalculatedA-taillengthsareindicatedovereachlane.Lanes:L:sizemarker,1:Nopoly-Atail,2:5units,3:15units,4:25unitsofE.coliPoly(A)Polymeraseper10μgCLucRNAina50μlreaction. (B)EffectofenzymaticA-tailingontheluciferasereporteractivityofCLucmRNA.   AnalysisofcappedRNAfunctionintransfectedmammaliancells (A)SchematicrepresentationofreportermRNAtransfectionworkflow. (B)ExpressionofCypridinaluciferase(CLuc)aftercappingusingdifferentmethods.HighactivityfromallcappedRNAsisobserved.   TheeffectofcappingcanbestudiedbydeliveringthemRNAtoculturedmammaliancellsandmonitoringitstranslation.UsingRNAencodingsecretedluciferases(e.g.,Cypridinaluciferase,CLuc)thetranslationcanbemonitoredbyassayingitsactivityinthecellculturemedium(Fig.A). CLucmRNAwassynthesizedandcappedpost-transcriptionally(Cap0orCap1)orco-transcriptionally(asdescribedabove)usingstandard(7mG)oranti-reversecapanalog(ARCA).Forconsistency,themRNAswerepreparedfromtemplatesencodingpoly-Atailsofthesamelength. Aftercapping,themRNAwaspurifiedusingmagneticbeadsandquantifiedbeforetransfectionintoU2OScellsusingtheTransIT®mRNAtransfectionreagentfollowingthemanufacturer’sprotocol.CLucactivitywasmeasured16hrsaftertransfectionusingtheBioLux®CypridinaLuciferaseAssayKit(NEB#E3309). VirtuallynoluciferasereporteractivitywasobservedinconditionswhereuncappedRNAwastransfected(Fig.B).Incontrast,robustactivitywasdetectedfromcellstransfectedwithRNAcappedusingthemethodsdescribedabove.Asanticipated,loweractivitywasobservedfromcellstransfectedwithmRNAcappedusingthe7mGcapanalogascomparedtoARCA-cappedmRNA.   Summary Insummary,whenchoosingtherightworkflowforyourfunctionalmRNAsynthesisneeds,youmustbalanceyourexperimentalrequirementsforthemRNA(e.g.,internalmodifednucleotides)withscalability(i.e.,ease-of-reactionsetupvs.yieldoffnalproduct). Ingeneral,co-transcriptionalcappingofmRNAwithtemplateencodedpoly(A)tailsorpost-transcriptionaladditionofpoly(A)tailisrecommendedformostapplications.Thisapproach,usingtheHiScribeT7mRNAKitswithCleanCapReagentAG(NEB#E2080),enablesthequickandstreamlinedproductionofoneormanytranscriptswithtypicalyieldsof≥90μgperreaction,totaling~1.8mgperkit. Post-transcriptionalmRNAcappingwithVacciniaCappingSystemiswellsuitedtolargerscalesynthesisofoneorafewmRNAs,andisreadilyscalabletoproducegram-scalequantitiesandbeyond.ReagentsforinvitrosynthesisofmRNAareavailableinkitformorasseparatecomponentstoenableresearchandlarge-scaleproduction. ProductsfromNEBareavailableforeachstepoftheRNASynthesisProductWorkflow.GMP-gradeproductssuitableformanufactureoflargescalemanufactureoftherapeuticmRNAareavailablethroughourCustomizedSolutionGroup. ViewaPDFofthisfeaturearticle References: Warren,L.,etal.(2010)CellStemCell,7,618-630. Angel,M.andYanik,M.F.(2010)PLoSOne,5:e11756. Yakubov,E.,etal.(2010)Biochem.Biophys.Res.Commun.394,189. Geall,A.J.,etal.(2012)Proc.Natl.Acad.Sci.USA,109,14604-14609. Ramaswamy,S.,etal.(2017)Proc.Natl.Acad.Sci.USA,114,E1941-E1950. Ma,Y.,etal.(2014)PLoSOne,9:e89413. Ota,S.,etal.(2014)GenesCells,19,555-564. Bassett,A.R.,etal.(2013)CellRep.4,220–228. Wells,S.E.,etal.(1998)MolecularCell2,135–140. Chooseyourcountry NorthAmerica Canada UnitedStates Europe France Germany UnitedKingdom Asia-Pacific Australia China Japan NewZealand Singapore Ifyoudon'tseeyourcountryabove,pleasevisitour internationalsite SessionExpired Youhavebeenidleformorethan20minutes,foryoursecurityyouhavebeenloggedout.Pleasesignbackintocontinueyoursession. SignIn InstitutionChanged YourprofilehasbeenmappedtoanInstitution,pleasesignbackforyourprofileupdatestobecompleted. SignIn SignintoyourNEBaccount Tosaveyourcartandviewpreviousorders,signintoyourNEBaccount.Addingproductstoyourcartwithoutbeingsignedinwillresultinalossofyourcartwhenyoudosigninorleavethesite. SignIn SignIn ContinueasGuest Don'tshowmeagain