Segmented poly(A) tails significantly reduce recombination of ...

In the case of in vitro-transcribed mRNA, the poly(A) tail can be either encoded into the DNA template (PCR product- or plasmid-based) or added ... Skiptomainpagecontent HOME ABOUT SUBMIT SUBSCRIBE ADVERTISE AUTHORINFO ARCHIVE CONTACT HELP SearchforKeyword: GO AdvancedSearch Segmentedpoly(A)tailssignificantlyreducerecombinationofplasmidDNAwithoutaffectingmRNAtranslationefficiencyor half-life ZeljkaTrepotec1, JohannesGeiger2, ChristianPlank2,3, ManishK.Aneja2and CarstenRudolph1,2 1DepartmentofPediatrics,Ludwig-Maximilian-UniversityofMunich,80337Munich,Germany 2EthrisGmbH,Planegg,82152Planegg,Germany 3InstituteofMolecularImmunologyandExperimentalOncology,KlinikumrechtsderIsar,TechnischeUniversitätMünchen,81675 Munich,Germany Correspondingauthor:rudolph{at}ethris.com  NextSection Abstract ExtensiveresearchinthepastdecadehasbroughtmRNAclosertotheclinicalrealizationofitstherapeuticpotential.One commonstructuralfeatureforallcellularmessengerRNAsisapoly(A)tail,whichcaneitherbebroughtincotranscriptionally viatheDNAtemplate(plasmid-orPCR-based)oraddedtothemRNAinapost-transcriptionalenzymaticprocess.Plasmidscontaining poly(A)regionsrecombineinE.coli,resultinginextensiveshorteningofthepoly(A)tail.Usingasegmentedpoly(A)approach,wecouldsignificantlyreduce recombinationofplasmidsinE.coliwithoutanynegativeeffectonmRNAhalf-lifeandproteinexpression.Thiseffectwasindependentofthecodingsequence. Asegmentedpoly(A)tailischaracterizedinthatitconsistsofatleasttwoA-containingelements,eachdefinedasanucleotide sequenceconsistingof40–60adenosines,separatedbyaspacerelementofdifferentlength.Furthermore,reducingthespacer lengthbetweenthepoly(A)segmentsresultedinhighertranslationefficienciescomparedtohomogeneouspoly(A)tailandreduced recombination(dependinguponthechoiceofspacernucleotide).Ourresultsdemonstratethesuperiorpotentialofsegmented poly(A)tailscomparedtotheconventionallyusedhomogeneouspoly(A)tailswithrespecttorecombinationoftheplasmids andtheresultingmRNAperformance(half-lifeandtranslationalefficiency). Keywords poly(A)tail plasmidrecombination mRNAtherapeutics transcripttherapy PreviousSectionNextSection INTRODUCTION ThepastdecadehaswitnessedtheemergenceandrapidapplicationofinvitrotranscribedmessengerRNA(mRNA)asatherapeutic molecule.ComparedtoclassicalgenetherapywithDNA-basedvectors,useofmRNAoffersseveraladvantages,suchastransient expression(KapeliandYeo2012;Lodish2012),lackofnecessitytoenterthenucleus,andnoriskofchromosomalintegration(CannonandWeissman2002;Hacein-Bey-Abinaetal.2003;Bernal2013).AfterovercomingtheintrinsiclimitationsofthemRNA,namelyimmunogenicityandinstability,usingchemicallymodified nucleotides(Karikóetal.2005,2008;Kormannetal.2011),researchershavesignificantlyimprovedthemRNAmoleculebyoptimizingthestructuralelements,namelycap(Grudzien-Nogalskaetal.2013;Ziemniaketal.2013;Kowalskaetal.2014),5′-and3′-UTRs(Ferizietal.2016;Schrometal.2017;Trepotecetal.2018),andcodingsequence(s)(Thessetal.2015;Schrometal.2017).Increasingitsstabilityandtranslationalyieldshasledtoaprogressfromthefirstuseofencodingapotentiallytherapeutic protein(Wolffetal.1990)topreclinical(Bahletal.2017;Richneretal.2017)andclinicalapplications(Mullard2016). Regardlessofthetargetproteinortissue,allcellularproteinencodingRNAswithsomeexceptions(e.g.,histones)share acommonstructuralfeature,thatis,poly(A)tail.Insidethecellnucleus,thepoly(A)tailisaddedtothemRNAinapost-transcriptional mannerdownstreamfromthegene-encodedpolyadenylationsignal(AATAAA).Thepoly(A)tailisessentialforthestability(Sachs1990;OliveiraandMcCarthy1995)andtranslation(Sachs1990;Wellsetal.1998)ofthemRNA.Thepoly(A)tailinthemRNAisrecognizedbypoly(A)-bindingprotein(PABP)whichinturninteractswitheIF4G ofthetranslationinitiationcomplex,therebyformingaclosedloop(Mangusetal.2003;GoldstrohmandWickens2008)andtheresultingmessengerribonucleoproteinparticle. Inthecaseofinvitro-transcribedmRNA,thepoly(A)tailcanbeeitherencodedintotheDNAtemplate(PCRproduct-orplasmid-based) oraddedenzymaticallytothemRNAinaseparatestepafterinvitrotranscription.Eachoftheabovementionedapproaches hasitsownsetoflimitations.WhilePCRofferstheeaseofhighthroughputandiswidelyusedforsmall-scalemRNAproduction (uptoafewhundredmilligrams),highproductioncosts,andriskofmutagenesisduringPCRamplification(comparedtoplasmid productioninbacteria)limititsusefulnessforlarge-scaleproduction(severalgrams).Plasmidproductionontheotherhand iswellestablished,canbeperformedunderGMPconditions,haslowerproductioncostsandrisksofmutations(inthecoding sequence)whencomparedtoaPCR-basedapproach.However,plasmidDNAencodedhomopolymericstretches[e.g.,poly(A)]recombine duringbacterialamplificationoftheplasmidDNA.Previousstudiesreportedgenerationofspontaneousdeletionmutantsduring amplificationofplasmidsstartingwith∼100bpofpoly(dA:dT)sequences(Preissetal.1998).Forlongerpoly(A)s,forexample,poly(A)150,theinstabilityistoohightoallowisolationofanysinglepositiveclone(Grieretal.2016).Despitethislimitation,template-encodedpoly(A)offerscertainadvantagesoverenzymaticpost-polyadenylationofmRNA, suchasdefinedandreproduciblepoly(A)lengthresultinginahomogenousproduct(Holtkampetal.2006).Althoughenzymaticpost-polyadenylationofmRNAwarrantssufficientlylongpoly(A)tails(CaoandSarkar1992;MartinandKeller1998),thecompositionofthefinalproductduetodifferentpoly(A)lengthsisdifficulttocontrolandthereforemightnotmeet regulatoryrequirements(Weissman2015). Inadditiontotheabovelistedadvantagesofcotranscriptionalpolyadenylation,thereducednumberofstepsinRNAproduction arelikelytotranslatetolowerproductioncosts.Moreover,enzymaticpolyadenylationofmRNAneedstobecarriedoutunder alkalineconditions,astheenzymepoly(A)polymerasehashighestactivityatpH>7.5.mRNAishighlysusceptibletoalkaline hydrolysiswhichinturnresultsinpoorermRNAquality,especiallywithlongertranscripts(>3kb).(VoetandVoet2011) Asanalternativetocircularplasmids,Grieretal.(2016)haveproposedtheuseofalinearplasmidsystem,pEVL,whichallowsstablecloningofpoly(A)sofupto500bp.Asimilar linearvector-basedsystem(pJAZZ)iscommerciallyavailablefromLucigenbutsuffersfromlimitationsoflargevectorsize (>12kb),limitedchoiceforcloningenzymes(availableonlyaseitherSmaIorNotIpredigestedvector)andisaverylow copyvector.Havingaplasmidtemplate-encodedpoly(A)tailwhichisnotpronetorecombinationbutstillsupportsmRNAstability andtranslationalefficiencyincomparablemannertoaconventionalnaturalpoly(A)wouldbeanidealsolutiontotheabovementioned limitations. Themainaimofthepresentstudywastoinvestigateifsegmentationofthepoly(A)tailcouldreducerecombinationofahigh copyplasmidvectorinE.coli.Forthis,themostwidelyusedbutrelativelyunstablepoly(A)tailof∼120A's[poly(A)120]wassplitintoeithertwoorthreesegmentsof40A's[poly(A)3×40]or60A's[poly(A)2×60],respectively.Thesegmentationschemewasdesignedkeepinginmindthefunctional“PABPfootprint”onmRNA.WhilePABP requiresaminimumof12adenosinestobind,proteinoligomerscanbindtothesamepoly(A)stretch,therebyformingarepeating unitof∼27–30nt(BaerandKornberg1980,1983;Wangetal.1999).Moreover,theworkbyPreissetal.showedthatasinglePABPmoleculeboundtomRNA,whileinteractingwiththe5′cap structurewasnotsufficientforpromotingtranslation(Preissetal.1998).Basedonthesedata,ourconstructsweredesignedtoenableatleastoneoligomericstretchofPABPpersegment(30nt). Besidesrecombination,translationefficiencyandmRNAhalf-lifemeasurementsweremadetocomparetheeffectonsegmentation onthesecriticalattributesofthemolecule.Heretoo,poly(A)120wasusedasabenchmarkasithasbeenshowninpreviousstudiestoresultinhighproteinexpression(Holtkampetal.2006;Bangel-Rulandetal.2013).Weshowthatsegmentationofthepoly(A)tail,doesnotnegativelyaffecttranslationalyieldandmRNAhalf-life,buteases thetechnicaldifficultiesconnectedwithrecombinationofhomopolymericpoly(A)stretchesinplasmidvectors. PreviousSectionNextSection RESULTSANDDISCUSSION Thecurrentstudyonsegmentationofpoly(A)intosmallerfragmentsseparatedbyspacerelementswaspromptedbytechnical challengesoftenmetwhiledesigningandproducingplasmidDNAtemplatesforuseininvitrotranscriptiontoproducemRNA foruseastranscripttherapies. Designofmodified/segmentedpoly(A)tails InordertoproducerecombinantRNAtranscriptswithsegmentedpoly(A)tails,thecorrespondingDNAsequenceswerecloned intoaplasmidvectordownstreamfromthegeneofinterest(GOI).Figure1schematicallyshowsthecompositionofdifferentpoly(A)tailsandtheirspacerseparators.Themostconventionallyused standardpoly(A)tailinplasmidvectors(Holtkampetal.2006;Kormannetal.2011;Vallazzaetal.2015;Balmayoretal.2016,2017;Ferizietal.2016)containing∼120A's[poly(A)120]wasspliteitherintothreesegments,eachcomprising40A'sseparatedbyaNsiIrestrictionsiteof6nt[poly(A)3×40_6]orintotwoequalsegmentsof60A's,alsoseparatedby6nt[poly(A)2×60_6].Thisway,wecouldcontrolthesizeofeachsegmentandstillhaveaphysicalseparatoramongadenosines.Accordingto previousliterature(BaerandKornberg1980,1983;Wangetal.1999),aminimumof12adenosinesareneededforthebindingofasinglePABPmolecule.However,asinglePABPboundtopoly(A) isnotenoughtosupporttranslation,eventhoughitcaninteractwiththeeIF4complex(Preissetal.1998).Thesegmentsofpoly(A)3×40_6andpoly(A)2×60_6arelongenoughtoensurebindingofmorethanthreecopiesofPABPpersegment.Inordertoinvestigatetheroleofthe spacerlengthbetweenthetwoA60segments,besidestheoneseparatedby6nt,fiveadditionalconstructsweresynthesized withaspacerlengthofeither12[poly(A)2×60_12],24[poly(A)2×60_24]nt,or1nt[poly(A)2×60_C,poly(A)2×60_G,poly(A)2×60_T]. Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide FIGURE1. Schematicrepresentationofallcombinationsofpoly(A)modificationstestedinthecurrentstudy.Differenthomo-orheteropolymeric poly(A)stretcheswereinserteddownstreamfromthegeneofinterest(GOI). Segmentedpoly(A)tailsreducerecombinationofpoly(A)-containingplasmidsinE.coli Instabilityofpoly(A)-containingplasmidsinE.colihasbeenpreviouslyreported(KühnandWahle2004;Godiskaetal.2009)andisamajorriskoffailurewhenusingsuchpoly(A)-containingplasmidsforlarge-scalemRNAproduction.Weexamined whethertheuseofsegmentedpoly(A)affectedtherecombinationefficiencyofplasmidspost-transformationintoE.coli.Totestthis,codingregionsfordifferentproteins(d2EGFP,luciferase,andhEPO)wereclonedupstreamofthesepoly(A) formats[poly(A)120,poly(A)2×60_6,poly(A)3×40_6]intoapUC57-Kanamycin(GenScript)vector.Post-transformationintoE.coli,cloneswerescreenedforinsertandpositiveclones(containingthedesiredinsert)wereadditionallyscreenedforthelength ofthepoly(A)region.Foreachofthepoly(A)formats,thepoly(A)regionwasdigestedwithrestrictionenzymesandthedigestions wereresolvedonFragmentAnalyzer(capillarygelelectrophoresis)tomeasurethesizeofthepoly(A)fragment.Asexpected, recombinationinthepoly(A)regionwasobservedformorethan50%oftheclonescontainingahomologouspoly(A),poly(A)120.Theproportionofrecombinationobservedwiththepoly(A)120formatwassequence-independentandcomparabletothevaluesreportedbyGrieretal.(2016).Bysplittingthepoly(A)intoeitherpoly(A)3×40orpoly(A)2×60,recombinationinE.colicouldbereducedwithmoststableclones(<20%recombination)obtainedwithplasmidscontainingpoly(A)2×60_6(Fig.2).Thistrendwasobservedforallthetestedsequencesindicatingthisreductioninrecombinationtobesequence-independent. Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide FIGURE2. Quantificationofpoly(A)tailrecombinationforA120andsegmentedpoly(A)tailsofpoly(A)3×40_6andpoly(A)2×60_6.(n)Totalnumberofclonesofd2EGFP,luciferase,andhEPOsequencestestedwithaparticularpoly(A)format. Effectsofpoly(A)segmentationonmRNAproductivity Encouragingresultsofreducedrecombinationpromptedustoinvestigatetheperformanceofoursegmentedpoly(A)tailswith respecttomRNAstabilityandexpression.Forthis,thecodingregionofd2EGFP(destabilizedEGFPwitharelativelyshortproteinhalf-life)wasclonedintoourdifferentpoly(A)-containingvectorsand chemicallymodifiedmRNA(modification1)wasproducedusingpreviouslydescribedprotocols(Kormannetal.2011;Trepotecetal.2018).TheresultingmRNAsweretransfectedintoA549cellsandatdifferenttimepointspost-transfection(4,24,48,and72 h),bothd2EGFPproteinandmRNAwerequantifiedusingFACSandreal-timereversetranscriptasePCR,respectively(Fig.3).Comparablelevelsofd2EGFPproteinwereobservedforsegmentedpoly(A)constructscomparedtothecontrolA120atall fourtimepoints.Comparablelevelsofd2EGFPmRNAwereobservedforallpoly(A)formats,exceptat24-hpost-transfection, wherelowermRNAamountswerequantifiedforthesegmentedpoly(A)formats.Similartoourpreviouslypublishedwork(Ferizietal.2016),wecalculatedthemRNAproductivity,definedastheamountofprotein(d2EGFPmedianfluorescenceintensity)normalized totheamountofmRNA(quantifiedviaqPCR)forthethreepoly(A)formats.Surprisingly,highermRNAproductivitieswereobserved forsegmentedpoly(A)constructsatearliertimepoints(4and24h)comparedtoA120format. Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide FIGURE3. Determinationofd2EGFPproteinexpressionandmRNAquantificationofdifferentpoly(A)-containingd2EGFPmRNAspost-transfection inA549cells.(A)MeanMFI(medianfluorescenceintensity)at4-,24-,48-,and72-hpost-transfection,measuredbyFACSinA549cells.(B)d2EGFPmRNAquantificationinA549cells.(C)mRNAproductivitywascalculatedbydividingthemeanMFI(FACSdata;A)bythemRNAamounts(real-timePCRdata;B)andnormalizingtheseratiostothoseobservedwithpoly(A)120construct.Valuesrepresentmean±SDofthreereplicates.Statisticalsignificancewasassessedbytwo-wayANOVAtestwith P-values:(*)P<0.5,(**)P<0.01,(***)P<0.001,(****)P<0.0001. Similartotheexperimentswithd2EGFP,luciferaseproteinandmRNAquantificationwasinvestigatedinA549cellsat24-h post-transfectionwithluciferase-encodingmRNA,containingeitherofthepoly(A)formats[poly(A)2×60_6,poly(A)3×40_6vs.poly(A)120].Furthermore,toaddresstheeffectofnucleotidemodifications,luciferasemRNAswereproducedusingeitherunmodified nucleotides,modification1ormodification2nucleotides.Thesemodificationshavebeendescribedpreviously(Trepotecetal.2018).Briefly,modification1included25%5-methylcytidineand25%2-thiouridine.Asforthemodificationset2,35%5-iodouridine and7.5%5-iodocytidinewereusedintheIVTreactionforRNAproduction.Asanadditionalbenchmark,apreviouslypublished construct(Thessetal.2015)comprisinghomopolymericAstretch(A63),homopolymericCstretch(C31)andhistonestem–loop(ACH;Fig.1)wasused.AshistonemRNAslackpoly(A)tails,thefunctionsofpoly(A),thatis,stabilityandtranslationefficiencyare performedbytheconservedstem–loop(WilliamsandMarzluff1995;Zanieretal.2002).Useofsegmentedpoly(A)2×60_6constructsignificantlyincreasedproteinlevelspost-transfectioninamodification-independentmannerwhencomparedto poly(A)120andACHbenchmarks(Fig.4).NodrasticdifferenceswereobservedbetweenthemRNAamountsforthedifferentpoly(A)formatcontainingluciferasemRNAs acrossmodifications.Poly(A)2×60_6constructwasmoreproductivethananyotherpoly(A)formatwhenusingmodificationsets1or2.Theconstructwithhairpin structures(ACH)expressedsignificantlylessamountsofluciferasecomparedtoallotherconstructscontaining120adenosines despitethesethreeconstructsbeingpresentinthecellinsubstantialamounts(qPCRdata).Thereducedtranslationefficiency ofhairpin-containingconstructsisfurtherconfirmedbycalculatingthemRNAproductivities. Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide FIGURE4. DeterminationofluciferaseexpressionandmRNAquantificationofdifferentpoly(A)-containingluciferasemRNA24-hpost-transfection inA549cells.(A)Luciferaseactivity,measuredasrelativelightunits(RLU:arbitraryunits)inproteinlysatesfromA549cellstransfected withdifferentpoly(A)-containingluciferaseRNAmeasured24-hpost-transfection.(B)LuciferasemRNAquantificationinA549cells.(C)mRNAproductivitywascalculatedbydividingtheluciferaseexpressionvalues(RLU;A)bythemRNAamounts(real-timePCRdata;B)andnormalizingtheseratiostothoseobservedwithpoly(A)120construct.Valuesrepresentmean±SDofsixreplicates.Statisticalsignificancewasassessedbytwo-wayANOVAtestwith P-values:(*)P<0.5,(**)P<0.01,(***)P<0.001,(****)P<0.0001. Effectsofpoly(A)segmentationontranslationofphysiologicaltargets Theinitialresultsofreducedrecombinationandcomparable/highermRNAproductivitycomparedtopoly(A)120,withintracellularreporterproteins(d2EGFPandluciferase),promptedustofurthertestthepoly(A)2×60_6formatwithadditionalphysiologicaltargets.TheselectedtargetsvariedinthelengthoftheirmRNAsandcellularlocalization oftheprotein:humanerythropoietin(0.9kb)asaprototypeofasecretoryproteinandhumancysticfibrosistransmembrane conductanceregulator(CFTR;4.5kb)asaprototypeofamembraneprotein. ThecodonoptimizedsequenceencodinghEPOwasclonedintopUC57-Kanamycinvectorupstreamofeitherpoly(A)120orpoly(A)2×60_6.mRNAwasproducedforeachoftheconstructsusingeitherunmodified,modification1ormodification2setsofnucleotides. SinceEPOisprimarilysecretedbykidneycells,transfectionexperiments,attwodoses,wereperformedinhumanHEK293cells. ProteinconcentrationsweredeterminedviaELISAat24-,48-,and72-hpost-transfection(Fig.5).Withsomeexceptions(e.g.,unmodifiedRNAat24hand72handmodification1at72h),nosignificantdifferenceswere observedbetweenthecomparedpoly(A)formatsatanyofthecompareddoses,timepointsormodifications. Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide FIGURE5. QuantificationofsecretedhumanerythropoietinproteinlevelsasmeasuredviaELISAinsupernatantsfromHEK293cellstransfected eitherwithpoly(A)120-orpoly(A)2×60-containingEPOmRNAat24h(A),48h(B),and72h(C)post-transfection.Valuesrepresentmean±SDofthreereplicates.Statisticalsignificancewasassessedbytwo-wayANOVA testwithP-values:(**)P<0.01,(***)P<0.001,n=3. Tofurtherinvestigatetherelationshipbetweenphysiologicalgeneexpressionandpoly(A)tailsegmentation,wefocusedon mRNAconstructsencodinghumanCFTRfurnishedwitheitherpoly(A)2×60_6orpoly(A)120.Bothconstructswereproducedwithunmodifiedsetofnucleotides,andtransfectionexperimentswithCFTRmRNAwereperformed in16HBE14o-cells.OnlyunmodifiedCFTRmRNAwasused,asapreviousstudy(Bangel-Rulandetal.2013)hasdemonstratedfunctionalrestorationofCFTRinhumanCFairwayepitheliaaftertransfectionwithunmodifiedCFTRmRNA containingapoly(A)tailof120A's. At24hand48hpost-transfection,cellswerelysedandwesternblotwasperformedfortheCFTRprotein.Hsp90wasusedas ahousekeeper.Similartoourpreviousresultswithd2EGFP,luciferaseandhEPO,useofsegmentedpoly(A)2×60_6didnotnegativelyaffecttheresultingproteinamountspost-transfectionwhencomparedtotheconventionallyusedpoly(A)120(Fig.6). Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide FIGURE6. RelativequantificationofCFTRproteinin16HBE14o-lysatesasmeasuredviawesternblot.(A)16HBE14o-cellsweretransfectedeitherwithpoly(A)120-orpoly(A)2×60_6-containinghCFTRmRNAandproteinlysatesanalyzedat24and48hpost-transfection.(B)Densitometryanalysisofwesternblotimages.Valuesrepresentmean±SDoftworeplicates.Statisticalsignificancewas assessedbypairedt-testwithP-values:ns=P>0.5. Spacerregionexpansioninpoly(A)2×60 Reducedrecombinationwithsegmentedpoly(A)2×60_6withcomparable(d2EGFP,EPO,CFTR)orhigher(luciferase)translationwithoutsignificanteffectsonmRNAstabilityprompted ustofurtherinvestigatethespacerlengthofthisspecificpoly(A)format.Fortheeaseofexperimentalfeasibility,two newluciferaseconstructsweremadewithlongerspacers[12and24nt:constructpoly(A)2×60_12andpoly(A)2×60_24inFigure1].ThedifferentluciferasemRNAs(unmodified,modification1andmodification2)forthethreepoly(A)formats[poly(A)2×60_6,poly(A)2×60_12,andpoly(A)2×60_24]weretransfectedintoA549cells.At24hpost-transfection,significantlylowerluciferaseexpressionwasobservedwith longerspacersusingunmodifiedandmodification1containingmRNA(Fig.7).Thesemodification-specificeffectscouldbeduetothespacerregion,uponincorporationofchemicallymodifiednucleotides affectingthebindingofPABPtothetwosegmentsofpoly(A).Withtwoexceptions[poly(A)2×60_24unmodifiedandpoly(A)2×60_6modification1],comparablelevelsofluciferasemRNAcouldbequantifiedinthecells.Therefore,increasingthespacer lengthtomorethan6ntinsegmentedpoly(A)2×60_6taildidnotresultinanysignificantadvantage,neitherintranslationnorinmRNAstability. Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide FIGURE7. DeterminationofluciferaseexpressionandmRNAquantificationofdifferentpoly(A)-containingluciferasemRNAat24hpost-transfection inA549cells.(A)Luciferaseactivity,measuredasrelativelightunits(RLU:arbitraryunits),inproteinlysatesfromA549cellstransfected withdifferentpoly(A)-containingluciferasemRNA.(B)LuciferasemRNAquantificationinA549cellstransfectedwithdifferentpoly(A)-containingluciferasemRNA.(C)mRNAproductivitywascalculatedbydividingtheluciferaseexpressionvalues(RLU;A)bythemRNAamounts(real-timePCRdata;B)andnormalizingtheseratiostothoseobservedwithpoly(A)120construct.Valuesrepresentmean±SDofsixreplicates.Statisticalsignificancewasassessedbytwo-wayANOVAtestwith P-values:(*)P<0.5,(**)P<0.01,(****)P<0.0001. Spacerregionreductioninpoly(A)2×60 Thenextsetofexperimentswasaddressedtoexaminingtheeffectofreducingthespacerlengthtoasinglenucleotidein poly(A)2×60segmentedpoly(A)tailonproteinexpressionandmRNAproductivity.Allthreepossibleconstructs(withC,T,orGasaspacer) weremade.Foreachconstruct,unmodified,modification1andmodification2containingmRNAswereproduced.Thedifferent luciferasemRNAswerecomparedinA549cells.Asabenchmark,standardpoly(A)120wasused.Independentoftheusedspacernucleotide/modifications,allthreesegmentedpoly(A)constructsresultedinsignificantly higherluciferaseexpressionwhencomparedtopoly(A)120(Fig.8).Withafewexceptions,mRNAlevelswerecomparableforthedifferentluciferasemRNAs.Irrespectiveofthespacernucleotide, segmentedpoly(A)constructsweremoreproductivethanthestandardpoly(A)120.Amongthethreespacernucleotides,nosignificantdifferencescouldbeobserved.SincethemRNAstabilitywasnotaffected, itisverylikelythatsegmentedpoly(A)withasinglenucleotidespaceraugmentedtranslation.Besidesincreasingtranslation, incorporationofasingleGasaspacerfurtherreducedrecombinationfrom20%[asobservedwithpoly(A)2×60_6]tozero(Fig.9).AspacerwithasinglenucleotideofTrecombinedin10%ofcases,andtheonewithaCasaspacernucleotiderecombined in50%ofcases,whichinturniscomparabletorecombinationobservedwithA120(Fig.2).Identificationofthemechanismsunderlyingtheobservedreducedrecombinationandenhancedtranslationwithsegmented poly(A)2×60_1comparedtoclassicalpoly(A)120willbethesubjectoffuturestudies. Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide FIGURE8. DeterminationofluciferaseexpressionandmRNAquantificationofdifferentpoly(A)-containingluciferasemRNAat24hpost-transfection inA549cells.(A)Luciferaseactivity,measuredasrelativelightunits(RLU:arbitraryunits),inproteinlysatesfromA549cellstransfected withdifferentpoly(A)-containingluciferasemRNA.(B)LuciferasemRNAquantificationinA549cellstransfectedwithdifferentpoly(A)containingluciferasemRNA.(C)mRNAproductivitywascalculatedbydividingtheluciferaseexpressionvalues(RLU;A)bythemRNAamounts(real-timePCRdata;B)andnormalizingtheseratiostothoseobservedwithpoly(A)120construct.Valuesrepresentmean±SDofsixreplicates.Statisticalsignificancewasassessedbytwo-wayANOVAtestwith P-values:(*)P<0.5,(****)P<0.0001. Viewlargerversion: Inthiswindow Inanewwindow DownloadasPowerPointSlide FIGURE9. Quantificationofpoly(A)tailrecombinationrateforsegmentedpoly(A)tailswithasinglenucleotidespacer.(n)Totalnumberofclonesofluciferasetestedwithaparticularpoly(A)format. Theseresultsallowustorecommendasegmentedpoly(A)region[poly(A)2×60]witheithera6orasinglenucleotide(G/T)spacerforuseinplasmid-basedvectorsforRNAproduction.Usingsuchasegmented poly(A)didnothaveanynegativeeffectonproteinexpressionandmRNAhalf-lifebutreducedrecombinationofplasmidsin E.coli. PreviousSectionNextSection MATERIALSANDMETHODS Plasmidpreparation Thesyntheticpoly(A)sequenceswereintroducedtothevectorbackboneeitherasannealedcomplementaryoligonucleotidesor fragmentscreatedbyPCR(Table1).Forsequencescomprisingof2×60,3×40,andACH,specificsetsofcomplementaryoligonucleotidesweresynthesizedand annealed.Thesyntheticpoly(A)fragmentsofA120,2×60_1,2×60_12,and2×60_24werecreatedbyPCR. Viewthistable: Inthiswindow Inanewwindow TABLE1. Segmentedpoly(A)sequencesandtheircorrespondingcloningstrategyusingeitherPCRprimersetsoroligonucleotides Annealingofcomplementaryoligonucleotideswasperformedasfollows:100µMofeacholigonucleotideweremixedwith40µL annealingbuffer(10mMTris-HCl,50mMNaCl,1mMEDTA,pH7.5)andincubatedfor5minat95°C.Subsequently,themixture waslettocooldowntoroomtemperaturebeforeproceedingwithrestrictiondigestion(BglII-BstBI). ForthehighperformanceofPCRreaction,PhusionHigh-fidelityPCRmastermix(ThermoFisherScientific)wasused.Tothe mastermix,whichcontains2×PhusionDNAPolymerase,nucleotidesandoptimizedreactionbufferincludingMgCl2,0.5µMofforwardandreverseprimer,3%DMSO,and1ngoftemplateDNAwereaddedtothereaction.Thetotalvolumeof 25µLperreactionwasinitiallydenaturedat98°Cfor30sec,followingby30cyclesat98°Cfor10sec,annealingat72°C for30sec,andextensionat72°Cfor30sec/kb.Thefinalextensionwasperformedat72°Cfor10min.ThesizeofthePCR productwasconfirmedon1%agarosegelandthedesiredbandwaspurifiedusingNucleoSpinGelandPCRclean-upkit(Macherey Nagel).PurifiedPCRproductwasdigestedwithNheI-BstBIandstoredat−20°Ctillfurtheruse. DigestedproductsofannealedoligonucleotidesandPCRproductswereclonedintoaccordinglydigestedpUC57-Kanavector(GenScript) containingthedesiredcodingsequences(fireflyluciferase,d2EGFP,humanEPO,andhumanCFTR). GenerationofmRNA TogenerateinvitrotranscribedmRNA,plasmidswerelinearizedbyBstBI(ThermoFisherScientific)digestionandpurified bychloroformextractionandethanolprecipitation.Purifiedlinearplasmidswereusedasatemplateforinvitrotranscription. Plasmidtemplates(0.5µg/µL)weresubjectedtoinvitrotranscriptionusing3U/µLT7RNApolymerase(ThermoFisherScientific), transcriptionbufferII(EthrisGmbH),1U/µLRiboLockRnaseinhibitor(ThermoFisherScientific),0.015U/µLinorganicpyrophosphatase 1(ThermoFisherScientific)withadefinedchoiceofnaturalandchemicallymodifiedribonucleotides(JenaBiosciences). Themodificationset1wassynthetizedusing5-methylcytidine(25%)and2-thiouridine(25%),inadditiontounmodifiednucleotides. Formodificationset2,insteadof5-methylcytidine(25%)and2-thiouridine(25%),5-iodouridine(35%),and5-iodocytidine (7.5%)wereused.ThecompleteIVT-mixwasincubatedat37°Cfor2h.Afterwards,0.01U/µLDNaseI(ThermoFisherScientific) wasaddedforanadditional45minat37°Ctoremovetheplasmidtemplate.RNAwasprecipitatedwithammoniumacetateata finalconcentrationof2.5mM,followedbytwowashingstepswith70%ethanol.Thepelletwasre-suspendedinaquaadinjectabilia. AC1-m7Gcapstructurewasaddedenzymaticallyby0.5mMVacciniaVirusCappingEnzyme(NewEnglandBiolabs)tothe5′end ofthepreviouslydenaturedtranscript(1mg/mL)at80°Cfor5min.Thecappingreactionmixalsocontained1×cappingbuffer (NewEnglandBiolabs),0.5mMGTP(NewEnglandBiolabs),0.2mMS-methyladenosine(NewEnglandBiolabs),2.5U/µLmRNACap 2′-O-Methyltransferase(NewEnglandBiolabs),and1U/µLRiboLockRNaseInhibitor(ThermoFisherScientific).Thecapping mixturewasincubatedfor60minat37°C,followedbyRNAprecipitationwithammoniumacetateatafinalconcentrationof 2.5mMandtwowashingstepswith70%ethanol.Thepelletwasre-suspendedinaquaadinjectabilia. RNAqualityandconcentrationweremeasuredspectrophotometricallyonaNanoDrop2000C(ThermoFisherScientific).Itscorrect sizeandpurityweredeterminedviaautomatedcapillaryelectrophoresis(FragmentAnalyzer,AdvancedAnalytical). Cellculture A549(ACC-107)andHEK293(ACC-305)cellswerepurchasedfromDSMZ.16HBE14o-cellswerekindlyprovidedbyProfessorWeber (UniversityofMuenster,Germany). Allcellswerecultivatedinminimumessentialmedia(MEM)withGlutamax(Gibco/LifeTechnologies).Mediaweresupplemented with10%heat-inactivatedfetalbovineserum(FBS;Gibco/LifeTechnologies)and1%penicillin/streptomycin(Gibco/LifeTechnologies). Cellswereculturedinahumidified5%CO2incubatorat37°C. Invitrotransfection A549andHEK293cellswereseededatthedensityof2×104cells/welland4×104cells/well,respectively,ina96-wellplate,forthepurposeoffireflyluciferase,FACSmeasurementsandEPOELISAassay. 16HBE14o-cellswereseededina6-wellplateatthedensityof7.5×105cells/well,forthepurposeofwesternblotanalysis.At24-hpost-seeding,cellsweretransfectedusingthecommercialtransfection reagentLipofectamine2000(ThermoFischerScientific).Complexeswerepreparedataratioof2µLLipofectamine2000per 1µgmRNA.A549andHEK293cellsweretransfectedwith250ng/welland250and125ng/wellmRNA,respectively.Forexperiments inA549andHEK293cells,requiredamountsofmRNAweredilutedinwaterandtheneededamountsofLipofectamine2000inserum-free MEM.mRNAwasaddedtotheLipofectamine2000solutionfollowedby20minincubationatRT.Theconcentrationofthefinal mRNA/Lipofectamine2000solutionwas25ng/µL.Tenmicrolitersofthecomplexsolutionwasaddedtothecellsandcellswere incubatedfor24h.ForeverymRNAconstruct,replicatesofthreeorsixwereprepared.For16HBE14o-cells,Lipofectamine MessengerMaxwasusedduetoitssuperiortransfectionefficiency(datanotshown).Fortransfection,7.5µgmRNAwasdiluted in125µLwater,and11.25µLLipofectamineMessengerMaxseparatelyin125µLserum-freeMEM.ThemRNAsolutionwasadded totheLipofectamineMessengerMaxsolutionfollowedby5minincubationtimeatRT.Atotalvolumeof250µLofthelipoplex solutionwasaddedtothecellscontaining2mLnormalgrowthmedia.Themediawaschanged4haftertransfection. Flowcytometryanalysisford2EGFP CellswerewashedwithPBS,detachedwithTrypLE(Gibco/LifeTechnologies),andre-suspendedinflowcytometrybuffer(PBS supplementedwith10%FBS).Shortlybeforemeasurement,cellswerestainedwithpropidiumiodidefordiscriminationbetween liveanddeadcells(1µg/mL;SigmaAldrich).AnalysiswasperformedonanAttuneAcousticFocusingCytometer(LifeTechologies) withAttuneCytometricSoftware(version2.1;LifeTechnologies)andFlowJo(version10). Fireflyluciferaseassay Fordetectionoffireflyluciferaseactivity,theassaywasperformed24hpost-transfection.CellswerewashedwithPBS, followedbyadditionof100µLlysisbuffer(25mMTris-HCl,0.1%TritonX-100,pH7.4).Cellswereshakenfor20minatroom temperature.Afterlysis,50µLofthecelllysatewasusedtomeasureluciferaseactivityviaphotonluminescenceemission for5secusingInfiniteR200PRO(Tecan).Theproteinamountineachsamplewasquantifiedin5µLofthecelllysatewith Bio-Radproteinassay(Bio-Rad),usingbovineserumalbuminasastandard.Luciferasevalueswerenormalizedtotheprotein concentrations. WesternblotanalysisforhumanCFTR CellswerelysedwithRIPAbuffer(50mMTris,pH8.0,150mMNaCl,1%TritonX-100,0.5%sodiumdeoxycholate,and0.1%sodium dodecylsulfate).Totalproteinamountwasdeterminedbythebicinchoninicacid(BCA)assay,followingmanufacturer'sinstructions (ThermoFisherScientific).Celllysateswereseparatedon3%–8%TRIS-Acetategels(ThermoFisherScientific)andtransferred topolyvinylidenefluoride(PVDF)blottingmembranes(Bio-Rad).MembraneswereblockedinWesternBreezeblockingbuffer(Thermo FisherScientific)andprobedwithantibodiesagainstCFTR(R&DSystemsMAB25031;1:2.000)andHsp90(OrigeneTA500494;1:15.000). Horseradishperoxidase(HRP)-conjugatedanti-mouseantibody(1:10.000;ab6820;Abcam)wasusedassecondaryantibody.Blots ofCFTRweredevelopedusingSuperSignalWestFemto(ThermoFischerScientific),andofHsp90usingLuminataForteWestern HRPSubstrate(Millipore). Enzyme-linkedimmunosorbentassayforhEPO QuantificationofhEPOproteinincellsupernatantswasperformedusinghumanErythropoietinQuantikineIVDELISAkit(R&D Systems)followingmanufacturer'sinstructions. RNAisolationandreversetranscription RNAwasisolatedatdifferenttimepointspost-transfectionusingSingleShotCellLysiskit(Bio-Rad)followingmanufacturer's protocol.PriortoRNAextraction,thecellculturemediawasremovedandcellswerewashedtwicewithPBSbeforebeinglysed inrespectiveRNAisolationbuffer.Fromthelysates(1µgofRNA),cDNAwassynthesizedusingiScriptSelectcDNASynthesis kit(Bio-Rad)witholigo(dT)primersfollowingmanufacturer'sinstructions.ThesynthesizedcDNAwasstoredat−20°C. Quantitativereal-timepolymerasechainreaction(qPCR) Real-timeqPCRwasperformedwithshorthydrolysisprobesford2EGFPandLuciferasetargets(UniversalProbeLibrary#37and #29;Roche)onaRocheLightCycler96(RocheDiagnostics).Ford2EGFP,thefollowingprimerswereused:5′-cctgaagttcatctgcacca-3′ and5′-ctcgtgaccaccctgacc-3′.LuciferasemRNAwasquantifiedusingthefollowingprimers,5′-acgccgagtacttcgagatg-3′and5′-attcagcccatagcgcttc-3′. AbsolutemRNAvalueswerecalculatedbyinterpolationfromthestandardcurve. Statisticalanalysis Eachexperimentwasperformedwithatleastthreetechnicalreplicatespersample.Resultsareshownasmeans±SDunless otherwisestated.StatisticalanalysiswasperformedusingGraphPadPrismsoftware(version6).Dataweretestedfornormal distributionusingD'Agostino-Pearsonomnibusnormalitytest.Multiplecomparisonswereconductedbytwo-wayANOVA,followed bySidak'stest(pairwisecomparison)orDunnett'stest(many-to-onecomparison).AP-value≤0.05wasconsideredstatisticallysignificant. PreviousSectionNextSection ACKNOWLEDGMENTS TheprojectwassupportedbytheNIMExcellenceCluster. PreviousSectionNextSection Footnotes Articleisonlineathttp://www.rnajournal.org/cgi/doi/10.1261/rna.069286.118. ReceivedOctober18,2018. AcceptedDecember22,2018. ©2019Trepotecetal.;PublishedbyColdSpringHarborLaboratoryPressfortheRNASociety ThisarticleisdistributedexclusivelybytheRNASocietyforthefirst12monthsafterthefull-issuepublicationdate(seehttp://rnajournal.cshlp.org/site/misc/terms.xhtml).After12months,itisavailableunderaCreativeCommonsLicense(Attribution-NonCommercial4.0International),asdescribed athttp://creativecommons.org/licenses/by-nc/4.0/. 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ArticlesbyRudolph,C. Searchforrelatedcontent PubMed/NCBI PubMedcitation ArticlesbyTrepotec,Z. ArticlesbyRudolph,C. Share CiteULike Delicious Digg Facebook Reddit Twitter What'sthis? NavigateThisArticle Top Abstract INTRODUCTION RESULTSANDDISCUSSION MATERIALSANDMETHODS ACKNOWLEDGMENTS Footnotes REFERENCES CurrentIssue September2022,28(9) FromtheCover StructureofthedenguevirusRNApromoter AdualrolefortheRNAhelicaseDHX34inNMDandpre-mRNAsplicing High-throughputimagingofmRNAatthesingle-celllevel Alertmetonewissuesof RNA AdvanceOnlineArticles SubmitaManuscript EditorialBoard AuthorInformation E-mailAlerts&RSSFeeds RecommendtoYourLibrary JobOpportunities MolecularCloningTheNewEdition BuyNowandSave30% Paperbackonly(limitedtimeoffer) MolecularCloning:ALaboratoryManual(FourthEdition)   Home About Submit Subscribe Advertise AuthorInfo Archive Contact Help Copyright©2022bytheRNASociety PrintISSN: 1355-8382 OnlineISSN: 1469-9001