Large-Scale in Vitro Transcription, RNA Purification and ...

Methods: Here we present an optimized protocol for analyzing the structure of any RNA, including in vitro transcription, size-exclusion ... × Title (Title) Mr Ms Dr. Professor FirstName* LastName* Company/Institution E-Mail* Message *indicatesrequiredfields ThissiteisprotectedbyreCAPTCHAandtheGooglePrivacyPolicyandTermsofServiceapply. 0 0 × × Pleasenote:Karger.comwillundergoscheduledmaintenanceonMonday,September05between06:00and08:00CET. OurServices OurContent PublishWithUs AboutUs Login 0 0 JournalMenu JournalHome Contents:allyears × × Pleasenote:Karger.comwillundergoscheduledmaintenanceonMonday,September05between06:00and08:00CET. CellularPhysiologyandBiochemistry DownloadFulltextPDF OriginalPaper Large-ScaleinVitroTranscription,RNAPurificationandChemicalProbingAnalysis KanwalF.a·ChenT.a·ZhangY.a·SimairA.a·RujieC.a·Sadaf ZaidiN.S.c·GuoX.a·WeiX.a·SiegelG.b·LuC.a Authoraffiliations aCollegeofChemistry,ChemicalEngineeringandBiotechnology,DongHuaUniversity,Shanghai,ChinabDepartmentofOrthopaedicSurgery,MusculoskeletalOncologyDivision,UniversityofMichiganMedicalSchool,AnnArbor,MI,USAcDepartmentofIndustrialBiotechnology,Atta-ur-RahmanSchoolofAppliedBiosciences(ASAB),NationalUniversityofSciencesandTechnology(NUST),Islamabad,Pakistan CorrespondingAuthor ChangruiLu,PhDCollegeofChemistry,ChemicalEngineeringandBiotechnology,DongHuaUniversity2999NorthRenMinRoad,Shanghai201620,(China)Tel.86-21-67792740,Fax86-21-67792740,[email protected] KeyWords:RNARegulatoryRNARNAanalysisInvitrotranscriptionRNApurificationSize-exclusionFPLCSHAPERNAmodificationChemicalprobing RelatedArticlesfor"" CellPhysiolBiochem2018;48:1915–1927 https://doi.org/10.1159/000492512 Abstract FullText PDF References Extras:6 Abstract Background/Aims:RNAelementssuchascatalyticRNA,riboswitch,microRNA,andlongnoncodingRNA(lncRNA)playcentralrolesinmanycellularprocesses.StudyingdiverseRNAfunctionsrequirelargequantitiesofRNAforprecisestructureanalysis.CurrentRNAstructureandfunctionstudiescanbenefitfromimprovedRNAquantityandquality,simplerseparationprocedureandenhancedaccuracyofstructuralanalysis.Methods:HerewepresentanoptimizedprotocolforanalyzingthestructureofanyRNA,includinginvitrotranscription,size-exclusionchromatography(SEC)baseddenaturingpurificationandimprovedsecondarystructureanalysisbychemicalprobing.Results:WeobservedthathigherMg2+,nucleosidetriphosphate(NTP)concentrationsandlongerreactiondurationcanimprovetheRNAyieldfrominvitrotranscription,specificallyforlongerandmorecomplicatedconstructs.OurimprovedSEC-baseddenaturingRNApurificationeffectivelyhalvedtheexperimentdurationandlaborwithoutintroducinganycontaminant.Finally,thisstudyincreasedtheaccuracyandsignal-to-noiseratio(SNR)ofselective2′-hydroxylacylationanalyzedbyprimerextension(SHAPE)chemicalprobingforanalyzingRNAstructure.Conclusion:PartorallofourmodifiedmethodcanimprovealmostanyRNA-relatedstudyfromprotein-RNAinteractionanalysistocrystallography. ©2018TheAuthor(s).PublishedbyS.KargerAG,Basel IntroductionSincethediscoveryofregulatoryRNAelementsinthe1990s,RNAhasemergedacentralroleinrecentstudiesofgeneregulation.ThesenovelregulatorsincludebutarenotlimitedtocatalyticRNA,riboswitch,microRNA,andlncRNA.SomeevidenceevensuggeststhatRNAjumpstartedlifeonEarthandgaverisetoallcellularprocessestoday[1,2].FunctionthisdiverserequirespreciseunderstandingofthestructuralelementsofRNAandwebelievethemannerinwhichRNAfolds,isakeycomponentandwarrantsfurtherinvestigation.CompletebiochemicalanalysesofRNA-relatedtopicsoftenincludestructuraldeterminationanddynamicstudiesthatstressuponboththequantityandqualityofRNAsamples.DespiteadvancementsinoverexpressionofRNAincells[3,4],invitrotranscriptionisstillthepredominantmethodduetoitssimplicityandbroadapplicability[5-8].However,thequalityandquantityofRNAproducedinaninvitrotranscriptionreactioncanvarydependinguponmanyfactorsincludingtheconcentrationandratioofindividualcomponents,incubationtimeandtemperature.Additionally,sinceribozymesarefrequentlyincludedtogeneratehomogeneous3′and5′ends[9-11],theircleavageefficiencyuponcompletionoftranscriptionalsowarrantsfurtheroptimizationtoincreaseyieldandpurity.Afterthetranscriptionandribozymecleavage,themixturetypicallyrequireslengthyseparationtoproducefull-lengthmatureRNA[5,9,11].Traditionally,thepurificationrequiresdenaturingpolyacrylamidegelelectrophoresis(urea-PAGE)toachieveseparationofthetargetRNAfromRNAP,DNAtemplate,uncleavedprecursorRNAs,abortivetranscriptsandNTPs.Althougheffective,thisprocesshasseveralmajordrawbacks.First,itrequiresminimumtwodaystocomplete,makingittheratelimitingstep.Second,theelutionstepfrequentlyleavessomeRNAtrappedinthegel,loweringthetotalyield.Thirdly,theeluateoftencontainsacrylamide,loweringthepurityofthefinalproduct[8,12-15].Thealternativeapproachesofurea-PAGEincludesprotein-assistedaffinitypurification[16,17]andfastperformanceliquidchromatography(FPLC)basedmethods[18-22].Affinitytagpurificationmethodstypicallyrequirearibozymecleavabletag(i.e.tandemMS2tags)thatbindstohis-taggedMS2proteins.FollowedbyadditionalSEC,thisreliabletechniquecanproducemilligramsofcrystallographicqualityRNAofsmalltomediumsize.LargertargetRNA(200+nt)significantlyreducestheefficiencyofthistechnique.Alternatively,recentSEC-basedmethodseffectivelyseparatetargetRNAwithunwantedproductsandreactantsundernativeconditions.However,thistechniquecurrentlyrequireslengthypretreatmentstepstoremoveRNAPanddesaltthetranscriptionmix.AnewervariationoftheFPLC-basedmethodusesweak-anion-exchangechromatographywithoutremovingtheRNAP.Despiteprovidingasingle-step,rapidpurificationoftargettranscriptathighefficiency,thismethodcannotseparatemultipleribozymecleavageproductswiththetargetRNA.AfterisolatingthetargetRNA,thenextgoalistocomprehenditsstructure-functionrelationship.Anumberoftechniquesincludingbutnotlimitedtocrystallography,NMRandvariouschemical/inlineprobingassaysanalyzeRNAstructuralandfunctionalproperties.Amongthem,SHAPEchemistryallowsquantificationoflocalnucleotideflexibilityatsinglenucleotideresolutioninanyRNA.SHAPEchemistryexploitsthereactivityoftheRNAribose2′-OHgroupusingsmallhydroxyl-selectiveelectrophilicreagents.Incontrasttootherchemicalprobingtechniques,SHAPEreactivitiesdetectandquantifylocalnucleotideflexibilityanddynamics,ignoringbaseidentity[23-30].Suchreactivityprofilecorrelateswithsecondary/tertiarystructureelements,ligandbindingpocketsandproteinrecognitionsites[31-35].CurrentSHAPEprotocol,optimizedagainstRNAtrascriptslessthan200nucleotides,lackstheSNRforlongerRNAtemplates,possiblyduetorelativelylowprocessivityofthereverse-transcriptase[27,36].RecentstudiescircumventedthisissueonlargerRNAtemplatesbyperformingmultipleSHAPEassayswithdifferentprimerstocovertheentirelengthofRNA[35,37,38].WhilethisappoachiseffectiveatobtainingsufficientSNR,itrequiresextensiveandcarefulnormalizationofSHAPEreactivitytoavoidartifactsfrommultipleindependentreverse-transcription(RT)reactions.Currently,SHAPEexperimentsproduceweaksignalsthosetypicallyneedhighlysensitiveequipmentsuchascapillarysequencersorexpensivelaserfluorescentscanners.TocombatthesedifficultiesinanalyzingRNA,wepresenthereacompleteprotocoloptimizationofRNAanalysis,includinginvitrotranscription,RNApurificationandSHAPEchemicalprobing(Fig. 1).First,ourmodifiedinvitrotranscriptionmethodoptimizedboththetranscriptionandribozymecleavagereaction,increasingthetargetRNA(upto450nt)yieldbyalmosttwofold.Next,ourSEC-baseddenaturingpurificationmethodreducedboththenumberofstepsandtotaltimeofpurificationwhilemaintaininghighyieldandpurity.Lastly,wemodifiedthecurrentSHAPEprotocoltoenhancetheaccuracyandSNRofSHAPEreactivitiesforlargerRNAconstructs.Overall,ournewprotocolforproducing,isolatingandchemicallyprobingRNAsubstantiallyincreasesRNAyield,purityandconfidenceinstructuralanalysis,consequentlymakingtheprotocolmorerobustandreliabletoawiderrangeofRNAmolecules.Fig. 1.Optimizationschemaforlarge-scaleinvitrotranscription,RNApurificationandchemicalprobinganalysis.MaterialsandMethodsGeneralPlasmidDNAwasobtainedbygrowingE.colistrainDH5αat37°ConstandardLBagarplatesorinaeratedLBliquidmediumwithproperantibiotics.TheoligonucleotidesforthisworkwereallobtainedfromSangonBiotech.TheenzymesandRTkitswerepurchasedfromInvitrogen(ThermoFisher),andstandardchemicalswereobtainedfromAmbion,Inc.andSigma-Aldrich.RNApurificationwasconductedonaGEAktaPurifier100FPLCsystem(GEHealthcare)withSuperdex-75andSuperdex-200filtrationmediapackedinto890/10mmandXK16/1000mmcolumns(GEHealthcare).ThebufferusedforpurificationwasTris-ethylenediaminetetraaceticacid(EDTA)containing8Mureaand100mMNaCl.ConstructionandpreparationofDNAtemplateThedesignofRNAinvitrotranscriptionconstructsfollowedstandardprotocol[11,39].TheaptamerdomainoftheB.subtilisyitJS-boxriboswitch(S-box;119nt)wasinsertedintoapUC57plasmidunderthecontrolofaT7RNAPpromoter;thehepatitisdeltavirus(HDV)ribozymewaspositionedatthe3′-endandthehammerhead(HH)ribozymeatthe5′-end[9,10].WeusedEcoRIandBamHIrestrictionsites.TheS-boxRNAforchemicalprobingexperimentswasconstructedtobeflankedbya5′-T7RNAPpromoteranda3′-RTstartsiteusingoverlappingPCRandinsertedintopUC57vector[30,40].Aftersequenceverification,theSHAPEcassetteDNAtemplateswerePCRamplifiedusingterminalprimersanduseddirectlytoproduceRNAsforSHAPEanalysisbyinvitrotranscriptionasexplained[39,41].TheDNAtemplatesfortranscriptionweregeneratedbyPCR[1mL;containing20mMTris(pH8.4),50mMKCl,2.5mMMg2+,200µMeachdNTP,500nMeachforwardandreverseprimer,5pMtemplate,and0.025units/µLofTaqpolymerase;(denaturationat94°C,20s;annealing60°C,25s;andelongation72°C,30s;35cycles).InvitrotranscriptionofRNARNAwaspreparedbyastandardT7RNAPrun-offtranscriptionreactionusingPCRproductfromthepreviousstepasatemplateandpurifiedbyurea-PAGEasdescribed[5,8].TheRNAyieldfrominvitrotranscriptionwasoptimizedforeachindividualDNAtemplatein25μLtrialreactionsbyvaryingtheconcentrationofMg2+,NTPs[22]andincubationtime.Atypicallarge-scale10mLtranscriptionreactionmixturecontained30mMTris(pH8.1at37°C),15mMMg2+,10mMdithiothreitol(DTT),2mMspermidine,0.01%(v/v)TritonX-100,4mMeachNTP,1mLofPCR-generatedDNAtemplate,and0.1mg/mLofT7RNAP[6,8].After2.5hofincubationat37°C,pyrophosphate,whichformsduringinvitrotranscriptionreaction,waspelleteddownbycentrifugation,andadditionalMg2+addedtothereaction.Thereactioncontinuedtill5h.Toconcentratethereactionmixture,weusedMilliporecentrifugalfilterunitswithappropriateMWCO.Thetranscriptionreactionscreeningwascomposedofonevariablecomponentatatimewiththerestofcomponentsfixed.ThetestedMg2+concentrationsincluded5mM,15mM,25mM,35mM,45mM,55mM,65mM,75mM,85mMand95mMwhileNTPsconcentrationswere1mM,2mM,3mM,4mM,5mM,6mM,7mM,8mM,9mMand10mM.Theincubationtimefortranscriptionat37°Cwastestedat5h,6h,7h,8h,9hand10h.Theresultswereevaluatedthroughimagequantification(BioRad)oftargetRNAbandon12%TAEurea-PAGE.Size-exclusionFPLCbasedRNApurificationRNAsamplepreparationforchromatographicpurificationTranscriptionmixturesfortheS-boxRNAwereheatedto90°Cfor5min,immediatelytransferredtoicefor5minandthenpelleteddown(centrifugedat16,000gfor10min).Thesupernatantwascollectedandmixedas1:4withTris-EDTAbuffercontaining8Mureaand100mMNaClbeforeloadingontosize-exclusionFPLC(SEC)columns.Samplepre-loadtreatmentwasoperatedtoremovetheT7RNAP,residualpyrophosphatesandaggregatesformedduringinvitrotranscription.DenaturingchromatographicpurificationTheglasscolumns(890/10mm)and(xk16/1000mm)werepackedaccordingtomanufacturer’sspecificationswithgelfiltrationmatrix(Superdex-75,Superdex-200)andequilibratedinacoldroom(4°C)withseveralcolumnvolumes(CV)ofspecifiedbuffer.ThetranscriptionmixturefromthepreviousstepwasloadeddirectlyontotheequilibratedSECcolumn,andthechromatographywasperformedat1mL/minflowrate,collecting2mLfractions.Fractionswereanalyzedbydenaturingurea-PAGE.S-boxRNAcontainingfractionswerecombinedandconcentratedto0.5mLusingMilliporecentrifugalfilterunits(EMDMillipore),washedtwicewiththeappropriatebufferforchemicalprobinganalysis.Allexperimentswereperformedat4°C.TheefficiencyofproposedpurificationsystemwasconfirmedthroughSHAPEexperimentsforS-boxRNA.OptimizingRNAchemicalprobing(SHAPE)TheS-boxRNAwasfirstoptimizedatconcentrationsof0.2μM,0.4μM,0.8μM,1μM,1.5μM,2.0μM,3.0μMand4.0μMthroughRTbyemploying1µLreversetranscriptase;analyzedthroughaBioRadFXscannerandcapillaryelectrophoresis.Structure-sensitiveRNAmodificationandPrimerExtensionTheS-boxRNAwassubsequentlydilutedintoRNAfoldingbuffer[111mMHepes(pH8.0),6.7mMMg2+,111mMNaCl]to2.0μM.Immediatelypriortochemicalprobing,thedilutedRNAwasrefoldedaspreviouslydescribed[41]duringtheannealingprocess.SHAPEchemicalprobingwasperformedasdescribedpreviously[29,30].Refolded10μLofthe2.0μMS-boxRNAwaspre-equilibratedusingagradientPCRmachineto65°Cfor10minandsubsequentlymixedwith0.1µL,0.5µLand1µLof130mMN-methylisatoicanhydride(NMIA)inanhydrousdimethylsulfoxide(DMSO)forbestmodificationoptimizationbyinitiating2′-ribosehydroxylalkylation.Thereactionsproceededtocompletionbyincubatingforfivehalf-livesofNMIA;laterquenchedwith900μLofprecipitationbuffercontaining80%EtOH,45μMNaCl,0.45μMEDTA,and2μLglycoblue(15mg/mL).TheRNAwasprecipitatedbyincubationto-80°Cfor30min,followedbycentrifugationat16,000gto4°Cfor45min.Thepelletwasair-driedandre-suspendedin10μLof0.5×TEbuffer[39].Theprimerextensionprotocolwassimilartothatdescribedpreviously[29].AfluorescentlylabeledDNAprimer(6-FAM-GAACCGGACCGAAGCCCG;0.5μL,50μM)wasannealedtotherecoveredRNA(10μL)byincubationat65°Cfor2min,then35°Cfor5min,followedbysnapcoolingto4°Cforanother2min.Theresulting10.5μLofRNA-primermixturewasmixedwith6μLofRTbuffer[167mMTris(pH8.3),250mMKCl,10mMMg2+,1.67mMofeachdNTP],preheatedto52°Cfor1min,andthenincubatedwith1μLofSuperscriptIII(Invitrogen,200units)at52°Cfor20min.Thereactionswerequenched,andtheRNAtemplatewasdestroyedbyadditionof1μLof5MNaOH,followedbyheatingto90°Cfor4min.Acidstopmix[4:25(v/v)mixtureof1MunbufferedTris–HClandstopsolution(85%formamide,0.5×TBE,50mMEDTA,pH8.0);29μL]wasaddedtoeachreaction;thereactionwasincubatedforanadditional5minto90°Candthencooledto-20°C.Nointernaltrackingdyewasusedinthereactiontoavoidinterferencewithfragmentanalysis.Eachsetofprimerextensionreactionswasfirstevaluatedby12%TAEurea-PAGEfollowedbysequencingofbandintensities,performedatSangonBiotech.EachSHAPEreactionproductwasanalyzedusingprimerslabeledwithdistinctfluorophoresasdescribedby[27,35,42].ResultsIncreasingRNAyieldthroughinvitrotranscriptionoptimizationTypicalRNAstructuralandfunctionalstudiesrequiremilligramsofRNAfromaninvitrotranscriptionassay,anditsyielddependsonMg2+concentration,NTPconcentrationsandincubationtime[18,19,21,43].Weoptednottousepyrophosphataseinourprotocolduetoitshighcostandnegligibleimpactonthetranscriptionyieldforlarge-scaleexperiments(datanotshown).Thisstudysystematicallyvariedtheseparameterstomaximizethetranscriptionefficiency.WeusedapreviouslycharacterizedS-boxRNA(119nt)asthetargetRNA.ThistargetRNAisflankedbytworibozymesthatself-cleavesaftertranscriptiontogivethefinalproductasdescribed[9,10,39].SystematicvariationofMg2+,NTPconcentrationsandreactiondurationrevealedthattheoptimumvalueliesoutsidethestandardcondition.ThetargetRNAyieldpeaksat180%ofstockcondition,around45mMMg2+(Fig. 2A).Thisincreaseinyieldalsocoincideswiththedecreaseintheuncleavedprecursortranscript(topband),whichsuggeststhattheRNAself-cleavagereactionrequireshigherMg2+concentration.FurtherincreaseinMg2+beyond45mMleadstodiminishedRNAproduction(Fig. 2A,lane5-10).Fig. 2.TheS-boxRNA(119nt)invitrotranscriptionoptimization.(A)OptimizingMg2+concentration(mM).Left:VariousMg2+concentrations(lanes1-10)forinvitrotranscriptionreactionseparatedby12%TAEurea-PAGE.ArrowsindicatetargetRNAandribozymes.S-boxRNAintensitygraduallyincreasedfrom5mMto45mM(lane1-5),attainingthemaximumtargetRNAyieldat45mM,followedbygradualdecrease(lane6-10).Right:Quantifiedtranscriptionprofilerevealed80%increaseinS-boxRNAsynthesisat45mMMg2+concentration.(B)EffectofindividualNTPconcentrations(1mMto10mM).Left:Transcriptionefficiencyplateauedaround8mMindividualNTPconcentrationvisualizedby12%TAEurea-PAGE.Right:Quantifiedtranscriptionprofilerevealedalmost100%increaseinS-boxRNAproductionat8mMindividualNTPconcentrations.(C)Optimizingincubationtime(h)at37°C.Left:Optimizedincubationhours(lanes1-6)forinvitrotranscriptionreactionsseparatedby12%TAEurea-PAGE.AgradualincreasewasobservedinS-boxRNAintensity(lane1-5),attainingthemaximumtargetRNAyield(lane5)followedbygradualdecreaseintargetRNAsynthesis(lane6).Right:Quantifiedtranscriptionprofilerevealed20%increaseinS-boxRNAsynthesisat9hincubationtime.(D)Comparisonofcombinedpre-andpost-optimizationstepsdepictedthrough12%TAEurea-PAGE.LaneMisRNAmarker.Left:TheS-boxRNAinvitrotranscriptionprofilebyfollowingstandardmethod(lane1)andcumulativeeffectofoptimizedprotocol(lane2).Right:QuantificationofoptimizedtargetRNAyieldthroughbarchartdepictednearlytwofoldincreaseinaveragetargetRNAintensity.ThetargetRNAandribozymesarelabeled.WethenindependentlyvariedtheindividualNTPconcentrationcenteredonthestockconcentrationof4mMeach.Ingeneral,theRNAyieldincreasesfrom1mMupto10mMNTP,plateauingaround8mMeach(Fig. 2B,lane8).Atconcentrationsbeyond4mM,weobservedincreasedprecursorRNAaswellasincreasedtargetRNA(Fig. 2B,lane5-10).QuantificationofthegelimageshowsthatthetargetRNAyieldincreased100%at8mMindividualNTPsconcentration.Lastly,weextendedthereactiontimeandobservedasteadyincreaseofproductuntil9hours.At37°Creactiontemperature,thereactioncontinuedtoproduceincreasedRNAproductcomparingwiththestandard5hoursreactiontime(Fig. 2C).ImagequantificationofthetargetRNAyieldshows20%increasecomparedtothestandardconditionwithasubsequentdecreaseafterwards.Combiningallthreeexperimentsabove,wetestedthecumulativeeffectofallthreenewconditions.Fig. 2Dshowsthecomparisonofouroptimizedtranscriptionprotocolwiththestandardconditionthrough12%urea-PAGEanditsimagequantification.Inlane1,beforeoptimization,theinvitrotranscriptionproductappearsmuchweakeronthegelthantheoptimizedconditioninlane2.Quantificationrevealsthattheoptimizedconditions(45mMMg2+,8mMindividualNTPand9hoursreactiontime)increasethetargetRNAproductionbytwofold.WefurthertestthisconditiononvariousotherRNAconstructsrangingfrom200to700nucleotidesandobtainedsimilarresults(datanotshown).OptimizingSE-FPLCbasedpurificationoftranscriptionproductsPreciseanalysisofRNArequiresremovalofunwantedcomponentssuchastheDNAtemplate,T7RNAP,unincorporatedNTPs,smallabortiveRNAtranscriptsandvariousribozymefragmentsfromthetranscriptionmix.Sincethosecomponentsvaryinsize,weaimtoefficientlyseparatethemregardlessoftheirshapeandchargeproperties.Inordertoavoidintroducinganyacrylamideorothercontaminants,wechosetousesize-exclusionchromatographyunderdenaturingconditions.First,weboiledthetranscriptionmixandkeptthesupernatant.ThisstepeffectivelyremovedallRNAPfromthemixaswedetectednoRNAPinanysubsequentstepsthroughSDSgel(datanotshown).UsingSuperdex-75and-200(16/890)prepgradecolumns,weobtainedanelutionprofileoftargetRNAwithdistinctpeaks.ForasimpleRNAtranscriptionreactionwhereonlyonetargetRNAisexpected,figure3Achromatogramshowstwomajorpeaksaftereachinjection,100μLand500μLrespectively.ThefirstpeakcontainsthetargetS-boxRNAwithinoneCVofelution,andthesecondpeakcontainedunincorporatedNTPsand/orsmallabortivetranscripts.TargetRNAwaspooledoutintotal8fractionscontaining2mLeachwhileresidual/deactivatedT7RNAPandtemplateDNAwerenotdetectedinanyfractionscollected(datanotshown).ThisispossiblebecausethemajorityofRNAPwereremovedduringthefirstdenaturingandprecipitationstep,andtheremainingRNAPexitedwiththetemplateinthevoidfraction.Theinsetinfigure3AshowsSE-FPLCpurifiedS-boxRNAfractions(lanes1-8)visualizedthrough12%TAEurea-PAGE.Fig. 3Bshowsthechromatogramofafull-scalepurificationwith5mLsampleloading(3%CV).OurresultshowsthatwithinthelimitsoftheSECcolumn,theelutionprofilesareconsistent.ThefractionscontainingthedesiredRNAwerecombined,concentratedandbufferexchangedtoappropriateconditionsusingMilliporecentrifugalfilterunits.Ourpurificationmethodyields>99%puretargetRNAwithoutNTPs,abortivetranscriptsoracrylamidecontaminants.Wealsoshortenedthepurificationprocesstoeighthoursorless,avoidinglengthygelseparationandextractionthattypicallytakestwodaystocomplete,allusingstandardFPLCsystemandoff-the-shelfSECcolumnsandparts.Experimentdurationcanbefurthercompressedusingpre-packedcolumnsathigherflowrates.SinceourSEC-basedprotocoldoesnotinvolveurea-PAGE,wecanalsoimproveyieldbynotrelyingongelelution.Asaresult,ourRNAyieldhasasevenfoldimprovement(31mg/mL)ascomparedtotheconventionalPAGEpurificationmethod(4.2mg/mL)duringparallelexperiments.Fig. 3.SE-FPLCbasedRNApurification.(A)PurificationofS-boxRNA(fromasimpletranscriptionmixture).SE-FPLCwasperformedat1mL/minat4°C.Elutionprofileontheleftwasobtainedfrom100μLsampleloadvolume,andtherightonewith500μLsampleloadvolume.Theinsetin(A)showsSE-FPLCpurifiedS-boxRNAfractions(lanes1-8)visualizedthrough12%TAEurea-PAGE.(B)Ascale-uppurificationprofileofS-boxRNAthroughSE-FPLCbyloading5mLoftranscriptionmixturesample.Inadditiontopurifysimpleinvitrotranscriptionreaction,wealsotestedourprotocolagainstmorecomplexRNAcrystallizationconstructs.WeusedapreviouslycharacterizedS-boxRNAconstructcontaininga5′-HHand3′-HDVribozymeusingaSuperdex-200(xk16/1000)SECcolumn.Chromatographywith100μLsamplevolumewasperformedat1mL/min(4°C),collecting2mLfractions.Fig. 4Ashowsanelutionprofilewiththreedistinctpeaks,theshoulderbeforethefirstdistinctpeakcontainedtheunsplicedprecursorRNAtranscript(277nt),andthefirstpeakcontainedthetargetS-boxRNA(119nt)splicedfrombothribozymes.ThesecondpeakcontainedtheHDVribozyme(67nt)whilethethirdpeakcontainedsmallerfragmentssuchasHHribozyme(51nt),NTPsandothersmallabortiveproducts.Oursubsequenturea-PAGEonthefirsttwopeaksconfirmedtheidentityofthepurifiedtargetRNAwithafivefoldimprovement(14.8mg/mL)ascomparedtoconventionalPAGEpurificationmethod(2.7mg/mL)duringparallelexperiments.Fig. 4.SE-FPLCbasedRNApurificationfromamorecomplextranscriptionmixture.(A)PurificationofS-boxRNA(119nt)fromRNAcrystallizationconstruct.Elutionprofile,obtainedthroughSuperdex-200(xk16/1000)columnwithsampleloadvolumeof100μLhasthreedistinctpeaks.TheshoulderofthefirstpeakcontainedtheunsplicedprecursorRNAtranscript(277nt).FirstpeakcarriedoverS-boxRNA(119nt)splicedfrombothribozymes,andtheHDVribozyme(67nt)pooledoutinsecondpeak.Whereas,NTPsandsmallabortivetranscriptionproductsincludingHHribozyme(51nt)elutedoutinthirdlargepeak.(B)A12%TAEurea-PAGEstainedwithethidiumbromideconfirmedtheidentityofeachpurifiedfraction.LaneMisRNAmarker,lane(1-2)representsunsplicedprecursorS-boxRNA(277nt),lane(3-5)containspurifiedS-boxRNA(119nt)andlane(6-8)indicatestheidentityof67ntlongHDVribozyme.SHAPEoptimizationforRNAsecondarystructureanalysisAfterobtainingpuretargetRNAinlargequantities,thenextlogicalprocessistoanalyzeitsstructureandfunction.HereweaimtooptimizethequantitativeRNAstructureanalysisatsinglenucleotideresolutionthroughSHAPE.Inourstudy,wefocusedontheRTstepsincewehaveseenmajoradvancementsinthecommercialreversetranscriptiondevelopmentinrecentyears.Wefocusedat8RTexperimentswiththeS-boxRNAconcentrationsof0.2μM,0.4μM,0.8μM,1μM,1.5μM,2μM,3μMand4μMwithoutanychemicalmodificationontheRNA.Fig. 5Ashowstheurea-PAGEresultofthereversetranscriptionusing6-FAM-labeledprimer.Quantificationofthebandintensities(Fig. 5B)showsthattheresultingcDNAmaxedoutat2μMRNAconcentration.Addingmorethan2μMRNAoffersnoadditionalincreaseastheRTasehasbeensaturated.Comparedwiththestandardprotocolof0.2μM,weobserveda10.5-foldincreaseincDNAyieldfromreversetranscriptase.Thelinearincreaseinbandintensityupto2μMsuggeststhatusingcurrentgenerationofsuperscriptreversetranscriptase,theenzymewasinexcessandmoretemplateRNAcanincreasethesignallinearlyupto2μM.Fig. 5.OptimizingS-boxRNARTstepbeforechemicalprobing.(A)Theurea-PAGEillustrationofS-boxRNART,analyzedthroughaBioRadFXscanner.Lane1showsthecDNAsignalat0.2μMRNAconcentration,whereassubsequentlanes(2-8)showsgraduallyimprovedcDNAsignalwiththeincreasingRNAtemplateconcentrations.(B)TheconfirmationofoptimizedRNAconcentrationresultsforRTthroughimagequantification.SincewechangedtheRNAconcentrationintheRTstep,theSHAPEreagentalsoneedscorrectiontoadjustfortheRNAquantitytoensureatmostonemodificationperRNAmolecule.WethereforefixedtheRNAconcentrationat2μMandexploredmultipleNMIAconcentrationsandnoticedthatreportedconcentrationof13mMorhigherintroducedsignificantendbias(datanotshown).LoweringtheNMIAconcentrationten-foldto1.3mMresolvedtheissue.Fig. 6AshowstheunmodifiedrawsequencingtraceofthecDNAfrommodificationandsubsequentreversetranscriptionduringparallelexperiments.Theredtracefromthe1.3mMNMIAconcentrationhashigherSNRthan13mMNMIA(cyan)(Fig. 6A;inset).Pre-andpost-optimizationforNMIAconcentrationcorrespondingtotheS-boxRNAP2domain(118-149nt)showsanaverageof13-foldincreaseineffectivesignalsat1.3mMNMIAconcentrationwhereas13mMNMIAconcentrationresultedinmarginallydetectablesignalusingthesameequipmentandparameters.Similarly,lower(1.3mM)NIMAalsogeneratedmorefull-lengthcDNAproductmanifestedbymuchhighersignalinFig. 6B.ThisresultsignifieslessmodificationontheRNA(preferablyoneperRNAchain),reducingsignalbiastowardsthe3′ofthetargetRNA[29].Wefurthertesttheseconditionsonvariousotherriboswitchesandobtainedsimilarresults(datanotshown).Ourdataindicate[NMIA(mM)/RNA(μM)=13/20]isthebestworkingratiotoevaluateS-boxRNAsecondarystructurethroughSHAPE.Fig. 6.SHAPEanalysisusingupdatedRNAconcentrationcoupledwithdifferentNMIAratios.(A)NMIAreactivityofS-boxRNAP2domaincorrespondingtonucleotidepositions(118-149)at1.3mM(red,post-optimized)and13mM(cyan,pre-optimized),Inset:RAWtraceoftheSHAPEreactionfromcapillaryelectrophoresisoftheabove-mentionedregion.(B)RawtracesignalcorrespondingtothefulllengthcDNApre-andpost-optimization.ColoringisconsistentthroughoutthisFig.DiscussionCurrentstructuralandbiochemicalstudieshavedemonstratedthatRNAactivelyparticipatesinallaspectsofcellularprocesses.Fast,efficientRNApreparationandanalysisgreatlyfacilitateRNAstructureandfunctionalstudies.ThispaperprovidesanimprovedguidetoanalyzethestructureofanyRNA,fromgeneratingtheRNAtosecondarystructureanalysisbySHAPE.Duringtranscriptionoptimization,weobservedthathigherthanreportedMg2+,NTP,concentrationsandlongerdurationcanimprovetheRNAyieldfrominvitrotranscription,specificallyforlongerandmorecomplicatedconstructs.TheTris-HClbuffer(40mM,pH8.1at37°C),5mMDTT,0.01%Triton-X100and1mMspermidinewereselectedastheoptimaltranscriptionreactionconditions[44].RaisingtheconcentrationsofMg2+upto45mMincreasedtheamountoftranscribedtargetRNA.Sinceweoptednottousepyrophosphatase,magnesiumpyrophosphateprecipitatesoutofthesolutionasaresultofNTPincorporationanddrivethereactionforward.Additionally,HHandHDVribozymesbothrequiremagnesiumtocatalyzetheirself-cleavage[45-49].Consequently,wefoundthathigherinitialconcentrationoffsetsthelossofmagnesiumionstothepyrophosphateprecipitatesandfacilitatesribozymecleavage.Similarly,duringourtrials,weobservedalinearincreaseoftranscriptionefficiencyandnoapparentadverseeffectswithincreasedNTPconcentrationsupto8mM(32mMtotal),twotosixteen-foldthatofpreviouslyreportedrangesbetween0.5to4mM[11,18-20,22,50,51].Wespeculatethatthisdiscrepancyresultsfromthemuchlargervolumesofourreactions(5to15mL)comparing10to50μLreportedreactionvolumes.Theincreasedreactionvolumemaydecreaseeffectivelocalreactantconcentrations,reducingeffectivecollisionbetweenactivereactants.Therefore,higherinitialreactantconcentrationsofNTPsandMg2+bothpositivelyaffectinvitrotranscriptionyield,consistentwithpreviousreports[18,19,21,43].Usingthestandardurea-PAGE,isolatingthetargetRNAtypicallytakestwoormoredaysandmaycontainunwantedacrylamide[22].Alternativeapproachessuchasionexchange[18,19]requirespecificconditionsfordifferentRNAmolecules.PreviousSEC-basedstudies[20,21,52]requireeithertoxicphenol/chloroformextractionoradditionofproteinaseKtoremoveRNAP.Ourimprovedmethodbasedondenaturinggel-filtrationeffectivelyhalvedtheexperimentdurationandthenumberofstepswithoutintroducinganycontaminant.Inaddition,themethoddescribedaboveallowsribozymemodificationsforcrystallographicstudiesandisinsensitivetotargetRNAconformation.Overall,thisimprovedmethodshouldapplytomostmediumtolargeRNAsforbiophysicalorbiochemicalstudies.Additionalscaling-uponlyrequiresimilarmodifications(i.e.slightlyhigherreactantsconcentration)andhighercapacityoff-the-shelfSECcomponents.Finally,thisstudyincreasedtheSNRofSHAPEchemicalprobingforanalyzingRNAstructure.SHAPEtechniquehasseengenerationsofimprovementssincefirstintroduction[30],suchasnewprobingreagent[42],bettertimeresolution[53,54]andinvitroprobingtechniques[26,55].ThisstudyprovidesafurtheroptimizationthatbenefitsallSHAPEtechniques.WefocusedontwoSHAPEreactioncomponents,RNAandNMIAconcentrations.IncreasingRNAconcentrationsupto2μMwiththeconstantamountofreversetranscriptaseoffersamarkedincreaseincDNAsynthesis.TolimitonlyonemodificationperRNAforunbiasedSHAPEresult[30,56],additionalRNAwarrantsalteredconcentrationsofmodifyingreagentNMIAtoyield2′-O-adductsatflexiblenucleotidepositions.Initially,wespeculatethatmoreNMIAisrequiredtomaintainaconstantratiobetweenRNAandNMIAasreported[29].However,theten-foldRNAincreaserequiredmuchlower(alsoten-fold)NMIAconcentration.WespeculatethatincreasedRNAconcentrationalsoincreasestheprobabilityofRNA-NMIAcollision,out-competingtheparallelNMIAhydrolysisreaction.Therefore,furtherincreasingRNAconcentrationinSHAPEmayrequirelowerNMIAtoavoid3′biascausedbymultiplemodifications.OurdatasuggestthatminimizingsecondaryNMIAadditiontothetargetRNAmayrequiremaintainingaconstantRNA-NMIAreactionrate.Therefore,higherRNAconcentrationwarrantslowerNMIAconcentration(rate=k[RNA]m[NMIA]n).ThelineardecreaseofNMIAconcentration,weobservedisconsistentwithpreviousobservationthatlongerRNAsneedasimilarlineardecreaseofNMIAconcentration[29].SincelongerRNAshavemoresitesavailableforNMIAattack,loweringreactiondurationand/orNMIAconcentrationwillsignificantlyimprovemodificationsignalsandeliminateendbias[29,57].Consequently,individualexperimentswithdifferentRNAlengthsmayrequireanadditionaltweakofNMIA.WerecommendkeepingtheRNAconcentrationconstantat2μMforoptimalresult,andincaselowerRNAamountisused,slightlyhigherNMIAmaybedesired.Weconsiderthisfast,efficientRNApreparationandanalysisguideasaworthwhileapproachforRNAresearchersatalmostallstepsstudyingRNAstructure/function.OurimprovedinvitrotranscriptionconditionsnoticeablyamplifythemagnitudeofRNA.Amodifiedgel-filtrationbasedpurificationsystemseparatesthetargetRNAwithtranscriptionmixturecomponents,beingusefulasacomparativelyrapid,less-laboriousandcontamination-freesystem.Finally,anoptimizedmethodincreasestheaccuracyandSNRofSHAPEchemicalprobingforanalyzingRNAstructure.Inshort,ourimprovedRNAstudyguidecanbenefittheresearchworldforanyRNA-relatedstudyfromprotein-RNAinteractionanalysistocrystallography.AcknowledgementsWethankDr.Chen,Peiran,andDr.MuhammadAqeelforhelpfuldiscussions.FundingwasprovidedbytheNationalNaturalScienceFoundationofChina(No.31300603),ProgramforProfessorofSpecialAppointment(EasternScholar)atShanghaiInstitutionsofHigherLearning(No.2012-28),FundamentalResearchFundsfortheCentralUniversities(No.15D110527,No.15D110508,No.13D110522,No.15D110568,No.15D310523),theNationalCollegeStudentInnovationExperimentProgram(No.14T10501,17D210502),GeneralFinancialGrantfromtheChinaPostdoctoralScienceFoundation(2015M571455).WealsoacknowledgetheChinaScholarshipCouncil(2014GXY252)forsponsoringthePhDfellowship.DisclosureStatementTheauthorsdeclarenoconflictsofinterest. 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NilsenTW,RioDC,AresM:High-yieldsynthesisofRNAusingT7RNApolymeraseandplasmidDNAoroligonucleotidetemplates.ColdSpringHarbprotocDOI:10.1101/pdb.prot078535. ChillónI,MarciaM,LegiewiczM,LiuF,SomarowthuS,PyleAM:NativepurificationandanalysisoflongRNAs.MethodsEnzymol2015;558:3-37. MortimerSA,WeeksKM:Time-resolvedRNASHAPEchemistry.JAmChemSoc2008;130:16178-16180. MortimerSA,WeeksKM:Time-resolvedRNASHAPEchemistry:quantitativeRNAstructureanalysisinone-secondsnapshotsandatsingle-nucleotideresolution.NatProtoc2009;4:1413. KnappG:EnzymaticapproachestoprobingofRNAsecondaryandtertiarystructure.MethodsEnzymol1989;180:192-212. WilkinsonKA,MerinoEJ,WeeksKM:RNASHAPEchemistryrevealsnonhierarchicalinteractionsdominateequilibriumstructuraltransitionsintRNAAsptranscripts.JAmChemSoc2005;127:4659-4667. SeetinMG,KladwangW,BidaJP,DasR:MassivelyparallelRNAchemicalmappingwithareducedbiasMAP-seqprotocol.MethodsMolBiol2014;1086:95-117. AuthorContacts ChangruiLu,PhDCollegeofChemistry,ChemicalEngineeringandBiotechnology,DongHuaUniversity2999NorthRenMinRoad,Shanghai201620,(China)Tel.86-21-67792740,Fax86-21-67792740,[email protected] Article/PublicationDetails First-PagePreview Received:May30,2018Accepted:July30,2018Publishedonline:August09,2018 Issuereleasedate:August2018 NumberofPrintPages:13 NumberofFigures:6 NumberofTables:0 ISSN:1015-8987(Print)eISSN:1421-9778(Online) Foradditionalinformation:https://www.karger.com/CPB OpenAccessLicense/DrugDosage/Disclaimer ThisarticleislicensedundertheCreativeCommonsAttribution-NonCommercial-NoDerivatives4.0InternationalLicense(CCBY-NC-ND).Usageanddistributionforcommercialpurposesaswellasanydistributionofmodifiedmaterialrequireswrittenpermission.DrugDosage:Theauthorsandthepublisherhaveexertedeveryefforttoensurethatdrugselectionanddosagesetforthinthistextareinaccordwithcurrentrecommendationsandpracticeatthetimeofpublication.However,inviewofongoingresearch,changesingovernmentregulations,andtheconstantflowofinformationrelatingtodrugtherapyanddrugreactions,thereaderisurgedtocheckthepackageinsertforeachdrugforanychangesinindicationsanddosageandforaddedwarningsandprecautions.Thisisparticularlyimportantwhentherecommendedagentisanewand/orinfrequentlyemployeddrug.Disclaimer:Thestatements,opinionsanddatacontainedinthispublicationaresolelythoseoftheindividualauthorsandcontributorsandnotofthepublishersandtheeditor(s).Theappearanceofadvertisementsor/andproductreferencesinthepublicationisnotawarranty,endorsement,orapprovaloftheproductsorservicesadvertisedoroftheireffectiveness,qualityorsafety.Thepublisherandtheeditor(s)disclaimresponsibilityforanyinjurytopersonsorpropertyresultingfromanyideas,methods,instructionsorproductsreferredtointhecontentoradvertisements. 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HeusHA,PardiA:Nuclearmagneticresonancestudiesofthehammerheadribozymedomain:secondarystructureformationandmagnesiumiondependence.JMolBiol1991;217:113-124. PerreaultJP,LabudaD,UsmanN,YangJH,CedergrenR:Relationshipbetween2’-hydroxylsandmagnesiumbindinginthehammerheadRNAdomain:amodelforribozymecatalysis.Biochemistry1991;30:4020-4025. LafontaineDA,AnanvoranichS,PerreaultJ-P:Presenceofacoordinatedmetalioninatrans-actingantigenomicdeltaribozyme.NucleicAcidsRes1999;27:3236-3243. VeeraraghavanN,GangulyA,GoldenBL,BevilacquaPC,Hammes-SchifferS:MechanisticstrategiesintheHDVribozyme:chelatedanddiffusemetalioninteractionsandactivesiteprotonation.J.PhysChemB2011;115:8346-8357. TanakaY,TagayaM,HoriT,SakamotoT,KuriharaY,KatahiraM,UesugiS:CleavagereactionofHDVribozymesinthepresenceofMg2+isaccompaniedbyaconformationalchange.GenesCells2002;7:567-579. MilliganJF,UhlenbeckOC:SynthesisofsmallRNAsusingT7RNApolymerase.MethodsEnzymol1989;180:51-62. 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