Steric exclusion chromatography (SXC) is a method for separation of large target solutes based on their association with a hydrophilic stationary phase through mutual steric exclusion of polyethylene glycol (PEG). Selectivity in SXC is determined by the size or shape (or both) of the solutes alongside the size and concentration of PEG molecules.
Trang 1j ou rn a l h om ep a ge :w w w e l s e v i e r c o m / l o c a t e / c h r o m a
Alesia Levanova, Minna M Poranen∗
Keywords:
RNA
a b s t r a c t
Stericexclusionchromatography(SXC)isamethodforseparationoflargetargetsolutesbasedontheir associationwithahydrophilicstationaryphasethroughmutualstericexclusionofpolyethyleneglycol (PEG).SelectivityinSXCisdeterminedbythesizeorshape(orboth)ofthesolutesalongsidethesizeand concentrationofPEGmolecules.ElutionisachievedbydecreasingthePEGconcentration.Inthisstudy, SXCapplicabilityfortheseparationandpurificationofsingle-stranded(ss)anddouble-stranded(ds) RNAmoleculeswasevaluatedforthefirsttime.TheretentionofssRNAanddsRNAmoleculesofdifferent lengthsonconvectiveinteractionmedia(CIM)monolithiccolumnswassystematicallystudiedunder variablePEG-6000andNaClconcentrations.Wedeterminedthatover90%oflongssRNAs(700–6374 nucleotides)andlongdsRNAs(500–6374basepairs)areretainedonthestationaryphasein15%
PEG-6000and≥0.4MNaCl.dsDNAanddsRNAmoleculesofthesamelengthwerepartiallyseparatedby SXC.SeparationofRNAmoleculesbelow100nucleotidesfromlongerRNAspeciesiseasilyachieved
bySXC.Furthermore,SXChasthepotentialtoseparatedsRNAsfromssRNAsofthesamelength.We alsodemonstratedthatSXCissuitablefortheenrichmentofssRNA(PRR1bacteriophage)anddsRNA (Phi6bacteriophage)viralgenomesfromcontaminatingcellularRNAspecies.Insummary,SXConCIM monolithiccolumnsisanappropriatetoolforrapidRNAseparationandconcentration
©2018TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/by/4.0/)
1 Introduction
Ribonucleicacid(RNA) and deoxyribonucleicacid(DNA) are
macromoleculesbuiltfromnucleotides,whichconsistofa
hete-rocyclicnitrogenousbase,a phosphate,and a pentosesugar In
aqueoussolutions, duplex DNA is primarily in B-conformation,
whileRNAismostlyasingle-strandedmoleculeadoptingcomplex
three-dimensionalstructures.WhenRNAduplexesareobserved,
theyare predominantly in anA-form [1 DNA and RNA differ
inoneoffourbases;DNAcontainsthymine,whileRNAcontains
uracil, a demethylated derivativeof thymine Furthermore,the
pentosesugarinRNAisribosebutdeoxyriboseinDNA,whichis
derivedfromribosebythelossofa2’oxygenatom[2 Thisloss
providesDNAwiththestabilitynecessarytofulfillitsmain
bio-logicalfunctionasakeeperofgeneticinformation[3 Oneofthe
numerousfunctionsofRNAmoleculesistoserveasa mediator
in the flow of genetic information fromDNA toproteins
Fur-thermore,RNAitselfcarriesgeneticinformationinmanyviruses
eitherasasingle-stranded(ssRNA)oradouble-stranded(dsRNA)
molecule.BiochemicalandstructuralstudiesofviralRNA biosyn-thesisrequirelargeamounts of pureRNA molecules.For some applications,ssRNAanddsRNAcanbesimplysynthesizedinvitro [4] and purified from thereaction components However,viral RNAsareheavilymodified[5 Accordingly,tostudytheeffectof thesemodificationsRNAspeciesmustbeisolateddirectlyfromhost cellswithsubsequentremovalofcellularRNA,separationofviral ssRNAfromdsRNA,andsize-separationofRNAmolecules Thereareanumberofbiochemicalmethodsandtheir modifi-cationsdevelopedforRNAisolation,purification,andseparation [6 However, all of these methods have certain limitations While isopycnic centrifugation yields RNA molecules of high purity [7 the method is laborious, time-consuming, requires expensive equipment, and hence is rarely used The most commonlyusedlaboratorymethodforRNAisolationisacid guani-diniumthiocyanate-phenol-chloroformextraction[8,9 whichis unsurpassed in terms of pure RNA yields and provides excel-lent protection from RNases due to protein denaturation by guanidiniumthiocyanate.However,themethodhasserious dis-advantagesasitistime-consumingandrequiresnoxiousreagents Althoughfastandeasy,solidphaseextractionbyimmobilizingRNA
ona specificsupport(silica,glass,magneticbeads, polysterene-latex)inthepresenceofchaotropicsaltsgenerallyresultsinlow
https://doi.org/10.1016/j.chroma.2018.08.063
Trang 2yieldsof RNA and requires DNasetreatment[6 Recently, two
alternativemethodsofRNAisolationhavebeenproposed,namely
RNAsnap[10]andRNASwift[11].Bothmethodsdonotinvolvetoxic
reagentsandtotalRNAcanberecoveredbyisopropanol
precipita-tionafterasingleroundofdifferentialcentrifugationofcelllysates
Furthermore,amino-acidbasedaffinitychromatography(histidine
affinitychromatographyorarginineaffinitychromatography)can
besuccessfullyappliedtoisolatetotalRNAorcertainfractionsof
smallRNAs[12–14]
Allthe methods listedabove lead to isolation of total RNA,
whichisasubjectoffurtherfractionation.Preparative
polyacry-lamide gel electrophoresis (PAGE) allows RNA separation with
single-baseresolutionforssRNAmoleculesuptoapproximately
1000ntin denaturingconditions [15] However, themethod is
extremelylengthyandprovidesonlylowyieldsofRNAmolecules
withacrylamidecontaminants,whicharedifficulttoremovefrom
thesample.TheRNApurifiedunderdenaturingconditionsmustbe
refolded,whichisnotalwayspossibleormightresultin
unproduc-tiveconformationswiththelossofbiologicalactivity[16,17].LiCl
fractionationcanselectivelyseparatelongandshortRNAaswell
asssRNAanddsRNAmoleculesfromimpurities[18].However,the
procedureisnotveryefficientandmightrequireadditionalsteps
toobtainRNAofrequiredpurityandtoremoveLiCl,whichmight
interferewithdownstream applications.A separation ofdsRNA
fromssRNA and DNA can beachieved onCF11 cellulose using
ethanolintheelutionbuffer[19].Thischromatographicmethod
iscommonlyusedtoisolateviraldsRNAfromplantsand fungi
Nonetheless, the procedure is laborious, lengthy, and requires
considerableamountsofinitialsample.Furthermore,itdoesnot
provideseparationofviraldsRNAsegmentsofdifferentlengths
Fastprocessingand superiorresolution ofRNA moleculesof
differentlengthscanbeachievedbyion-pairreversed-phase
chro-matography[20,21].Inthischromatographymodeanion-pairing
reagentformsionpairswithphosphatesofnucleicacid.The
result-ingcomplexisstronglyretainedinthecolumnviahydrophobic
interactions[22].However,toachieveagoodseparationof
differ-entRNAspecies,thistechniquerequiresdenaturingconditionsthat
canresultinirreversiblelossofRNAsecondarystructure.While
ion-pairreversed-phase chromatographyis anexcellent
analyt-ical tool, its application for preparative purposes is limited as
someion-pairing reagentsandtoxicsolvents(acetonitrile)used
intheprocedurearedifficulttoremovefromthepurifiedsample
Furthermore,resinshaveonlymoderateloadingcapacity.Anion
exchangechromatography(AEX)isbasedprimarilyonreversible
electrostaticinteractionsbetweennegativelychargedphosphates
ofnucleicacidsandthepositivelychargedstationaryphase
Sta-tionary phasesare alsodesigned tohave somehydrophobicity,
whichcontributetotheseparation AEXis a robustandwidely
appliedpreparativemethodtoobtainpureRNAmolecules[23].In
ourlaboratory,wehavedevelopedanon-denaturingAEXprotocol
topurifyenzymaticallyproducedsiRNAmoleculesonamonolithic
stronganionexchangeQAcolumn[24].Accordingtoour
obser-vations[24],itwasnotpossibletoresolveRNAmoleculeslonger
than1000basepairs(bp)byAEX.SinceviralRNAgenomesrange
insizefrom1700nucleotides(nt;humanhepatitisDvirus[25])to
32000nt(coronoviruses[26]),wesoughtalternativemethodsto
separateRNAmoleculeslargerthan1000ntundernon-denaturing
conditions
A novel mode of steric exclusion chromatography (SXC)
was recently described as a tool to separate and purify large
biomolecules[27] It should benoted thatthe term“SXC”was
previouslyusedtorefertosizeexclusionchromatography(SEC)
[28].AlthoughbothSXCandSECdonotrelyonanydirect
chemi-calinteractionsandtheseparationofsamplecomponentsisbased
ondifferencesintheirsizeorshape (orboth),theSXCmethod
developed by Lee et al in 2012 substantially differs from SEC
sinceitissuitableonlyforlargesoluteswhoseretentionstrongly dependsonthemobilephasecompositionandisfeasibleonlyin hydrophilicstationaryphase.Inthisapproach,asampleismixed withaspecificsizeandconcentrationofpolyethyleneglycol(PEG) andisimmediatelyloadedontoahydroxyl-functionalized mono-lithiccolumn.ThemutualstericexclusionofPEGfromboththe largetargetsolutes(proteins,virusparticles)andthehydrophilic stationaryphaseresultsintheirassociationwithoutdirectchemical interactions,whilelowermolecularweightmoleculesarewashed away.TheelutionisachievedbyreducingPEGconcentration[27] Thismethodis mechanisticallysimilar tothesizefractionation
of nucleic acids[29] and proteins[30] withPEG, in which the effectivenessofPEGincreasesalongwiththesizeofthepolymer andlargermoleculesprecipitateatlowerPEGconcentrations.The phenomenonisbasedonthestericexclusionofchemically non-reactivesolutes[31,32].Accordingtothistheory,PEGandbiological solutesarestericallyexcludedfromeachother,whichresultsin theformationofaPEG-deficientzonearoundasoluteandcreates discontinuitybetweenthePEG-deficientzoneandhigh-PEGbulk solution.Thisdiscontinuityresultsinanunfavorableincreasein freeenergy.Groupingofthesolutesfollowedbytheir precipita-tionleadstoreductioninthecontactareabetweenPEG-deficient and high-PEGbulksolvents, andthusdecreasesthe discontinu-ityandfreeenergyofthesystem[27,31,32].In thecaseofSXC, biomoleculesaccretetotheinerthydrophilicsupportinsteadof formingprecipitates
Ithasbeendemonstratedthatmonolithiccolumnscanbe suc-cessfully appliedfor SXC since their performance is minimally affectedbyviscosity(duetothelargesizeoftheinterconnected pores)andconvectivemasstransfer[33].Thedescribedmodeof SXCwasappliedforthepurificationofimmunoglobulinM[27] andimmunoglobulinGmonoclonalantibodies[34],virusparticles [27,35],andfortheseparationofserumproteins[36].However,the potentialofSXCtoseparatenucleicacidmoleculeshasnotbeen investigated
Inthisstudy,weevaluatedthebindingandelutionbehaviorof nucleicacidmoleculesduringSXCwithafocusonRNA,we deter-minedtheconditionssuitableforseparationofssRNAanddsRNA moleculesofdifferentlengths,andpurifiedviralRNAgenomes
2 Material and methods
2.1 Stericexclusionchromatography
A Convective Interaction Media (CIM) OH 1ml tube mono-lithic column inpolypropylene housingwasobtained fromBIA Separations(Ajdovˇsˇcina,Slovenia).Thispolyglycidyl methacrylate-co-ethylenedimethacrylate-basedmonolithhasanaveragepore sizeof1.3m,outerdiameter18.6mm,innerdiameter6.7mm, length 4.2mm, and bed volume 1.0ml The matrix is highly hydrophilicduetothehighdensityofhydroxylgroupsthat origi-natefromhydrolysisofepoxyligands
Allchromatographyexperimentswereperformedatroom tem-perature usinganÄKTA Purifier10 UPCliquid chromatography system (GE Healthcare) operated by Unicorn 5.2 software (GE Healthcare).ThechromatographysystemconsistedofpumpP-900, mixerM-925,monitorUPC-900,andfractioncollectorFrac-920 Theabsorbanceat260nmwasmonitoredduringchromatography and0.5-mlfractionscorrespondingtopeakareaswerecollected automatically
Allreagentsusedinthisstudywereofanalyticalgrade
PEG-4000andPEG-8000werefromFlukaandPEG-6000waspurchased fromUbichem.Weobservedthattoobtainreproducibleresultsand minimizedatavariabilitybetweendifferentexperiments,it was required tousePEGfrom thesamemanufacturer Trizmabase,
Trang 3Fig 1.Chromatography system setup for the in-line sample injection.
Tris-HCI,andNaCI werepurchasedfromSigma-Aldrich.Freshly
preparedautoclavedMilli-Q(Millipore)waterwasusedforbuffer
preparation.Thiswaterwasdeterminedtobenuclease-freebyour
internallaboratorytests
ThebufferAusedforchromatographywascomposedof50mM
Tris-HCl(pH8.0).BufferBcontaineddifferentconcentrationsofPEG
andNaClin50mMTris-HCl(pH8.0).Allbufferswerevacuum
fil-teredthrough0.22-mbottle-topfilters(ThermoFisherScientific)
anddegassedbysonicationfor18minpriortouse.Freshbuffers
werepreparedeveryweekandusedwithin4–5days
Abasicchromatographicprocedureusedpreviously[27]was
adaptedasdescribedbelow:(1)Systemequilibration.Thestandard
setupoftheÄKTA Purifierwaschanged tomakeit suitable for
SXCsothatonlybufferAflewtotheinjectorvalve,whilebuffer
Bbypasseditandwentdirectlytothemixer(Fig.1).Thecolumn
wasplacedin-lineandequilibratedwith20%bufferA:80%buffer
B.Unlessotherwiseindicated,thebufferA:bufferBratiowaskept
thesameforsimplicity,whiletheconcentrationsofPEGandNaCl
inbufferBwereoptimizedtoachievesufficientretentionofnucleic
acids.Furtherinthetext,onlythefinalconcentrationsoftwo
chem-icalsareindicated(i.e.thoseinamixtureof20%bufferAand80%
bufferB).TocalculatetheinitialconcentrationsofPEGandNaCl
inbufferB,thetargetfinal concentrationsweredivided by0.8
(2)Sampleloading.Samplesforchromatographyweredilutedwith
bufferAandcentrifuged(11,000×g,10min)toremove
particu-latematter.Theinjectionvolumewas0.25mlandflowratewas
2.5ml/minor3ml/mindependingonthesampleamountandPEG
concentrationinthesystem.Flowratesbelow2.5ml/minresulted
inpre-columnsampleprecipitationandchromatographyfailure
(3)Washing.ThesystemwaswasheduntiltheUVabsorbanceof
columneffluentreachedbaseline.(4)Elution.ThebufferA:bufferB
ratiowasreducedstepwiseorlinearly.(5)Cleaninginplace(CIP)
RemovalofresidualRNAfromthecolumnmatrixwasachievedby
washingwith1MNaOHsolutionfollowedby1MNaClin50mM
Tris-HCl(pH8.0).CIPwasperformedaftereachchromatography
experimenttoavoidcross-contaminationofsubsequentanalyses
Eachchromatographyexperimentwasrepeatedtwotosixtimes
TocalculatetheretentionofnucleicacidsontheCIM-OHmatrix,
the molecules were precipitated from the fractions and
flow-through(see2.5fordetails).Concentrationwasmeasuredwitha
NanoDrop2000c(ThermoFisherScientific)andthepercentageof
RNAorDNAelutedbythegradientwasthencalculatedusingthe
formula:NA,%= b
a+b×100,whereNAisthefractionofnucleicacid retainedinthecolumn(%);aistheamountofunboundsamplein
theflow-throughsolution(g),andbistheamountofthesample
elutedundergradientconditions(g).Therecoveryofnucleicacids
wascalculatedaccordingtotheformula:Recovery,%= Sl
Se×100%, whereSlistheamountofloadedsample(g)andSeistheamount
ofelutedsample(g).Theaveragerecovery±standarddeviation
(S.D.) wascalculated in Excel based onthedata frommultiple
experiments
2.2 Preparationofnucleicacidmoleculesforchromatography
BacteriophagegenomicDNA(48502bp)wasobtainedfrom
Fermentas and dissolved in nuclease-free Milli-Q water DNA
molecules ranging in length from 88 to 1800 bps were
pre-pared byPCR amplificationusing Phusion HFDNA polymerase (Finnzymes),deoxynucleotidetriphosphates(ThermoFisher Scien-tific),andpLM659plasmidcontainingacomplementaryDNAcopy
ofPseudomonasphagePhi6genomesegmentS[37].Theforward primersforPCRamplificationcontainedtheT7polymerase pro-motersequence(5’-TAATACGACTCACTATAGGG-3’)followedbya sequenceof17to21ntcomplementarytothePhi6S-segmentat positions80,100,200,500,700,or1800ntascountedfromthe3’ end.Thereverseprimerwascomplementarytothevery3’endof thePhi6 S-segment (5’-GGAAAAAAAGAGAGAGAGCCCCCGAAGG-3’)andcontainedthePhi6polymerasepromotersequenceatthe 5’end (underlined) A list of primer sequences has been pub-lished [38] Plasmids pLM659 [37], pLM656 [39], and pLM687 [40] were used as templates for the production of full-length complementaryDNAcopiesofphagePhi6S(2948bp),M(4065 bp),andL(6374bp)genomesegments,respectively.Thereverse primerwasthesameasdescribedaboveandtheforwardprimer containedtheT7polymerasepromoter sequence followedby a homologoussequence(5’-GGAAAAAAACTTTATATA-3’)presentat the5’-endofallthreegenomesegmentsofPhi6[41].PCR prod-uctsof thecorrectsize wereexcisedfromthe geland purified withNucleoSpinExtractIIkit(Macherey-Nagel)accordingtothe manufacturer’sinstructions.PureDNAmoleculeswereelutedin
30lMilli-Qwater.ThePCR-generatedDNAmoleculeswereused
astemplatesforRNAsynthesis.ssRNAswereproducedbyinvitro transcriptionwithT7RNApolymerase(ThermoFisherScientific)
as previously described [42] dsRNA molecules were also gen-eratedinvitro usinga single-tube transcriptionand replication reactioncatalyzed bytheT7 and Phi6 RNApolymerases [4,42] TherecombinantPhi6polymerasewasexpressedandpurifiedas described[42] Nucleoside5’-triphosphates wereobtainedfrom Thermo FisherScientific Enzymaticallysynthesized ssRNA and dsRNA molecules were isolated with TRIzure reagent (Bioline) andchloroform(Merck)accordingtothemanufacturer’s instruc-tions,followedbyprecipitationofT7-generatedtranscriptsin3M sodiumacetate(Merck)orstepwisefractionationofdsRNAwith LiCl(Merck)(seebelow).AllRNAswerewashedwith70%ethanol anddissolvedinsterilenuclease-freewater
2.3 LiClprecipitation ContaminatingssRNAmoleculeswereremovedfromthedsRNA synthesisreactionsbyLiCl precipitation.ssRNAmoleculeswere firstprecipitatedbyincubationat−20◦Cfor30minin2MLiCl
fol-lowedby20mincentrifugation at13,000×gat4◦C dsRNAwas collectedfromtheresultingsupernatantbyrepeatingthe proce-durein 4MLiCl.ThedsRNApelletwaswashedtwicewith70% ethanol and dissolvedin sterile nuclease-free water In case of unsatisfactoryseparationofdsRNAsfromssRNAs,theprocedure describedabovewasrepeated
2.4 Preparationofbacteriophagegenomesforchromatography PseudomonasphagePhi6[43]waspropagatedandpurifiedas previouslydescribed[44].Thesameprotocolwasusedtorecover PseudomonasphagePRR1[45].Briefly,virionswerecollectedfrom thelysatesofinfectedPseudomonassyringaeHB10YorPseudomonas aeruginosaPAO1byprecipitationwith10%PEG-6000and0.5M NaCl.Toopentheviralcapsids,halfoftheprecipitatewastreated withproteinaseK(ThermoFisherScientific)ataconcentrationof
1mg/mlin10mMTris-HCl(pH7.5)buffercontaining5mMEDTA (Merck)and1%sodiumdodecylsulfate(SDS,Merck)at50◦Cfor
1h.Fromtheremainderoftheprecipitate,totalRNAwasextracted usingTRIzurereagent(Bioline)accordingtothemanufacturer’s instructions
Trang 42.5 AnalysisofdsRNAintegrityandpurity
RNAwasrecoveredfromthecollected0.5-mlchromatography
fractionscorrespondingtothepeakareasonchromatogramsby
overnightprecipitationat−20◦Cin0.3Msodiumacetate(pH6.5)
and67%ethanol,followedbycentrifugationat10,000×gfor30min
TheRNApelletswerewashedwith70%ethanol,airdried,and
dis-solvedin10lofsterilenuclease-freewater.A5-lsamplewas
mixedwith2×Uloadingbuffer(10mMEDTA[pH8.0],0.2%SDS,
0.05%bromphenolblue,0.05%xylenecyanol,6%[v/v]glycerol,8M
urea).RNAmoleculeswereseparatedin an0.8%agarosegelby
electrophoresisinTris-borate-EDTA(TBE)buffer(6.6gTrizmabase,
3.9gboricacid,0.93gEDTA).Forelectrophoresis,anEPS301
elec-trophoresispowersupply(GEHealthcare)andanOwlEasyCastB2
minigelelectrophoresis system(ThermoFisherScientific)were
used.Adetailedprotocolhasbeendescribedpreviously[46]
2.6 TreatmentofthechromatographyfractionswithRNaseor
DNase
ThefractionscollectedaftertheseparationofDNAanddsRNA
onCIM-OHcolumn (Section2.1)wereprecipitatedasdescribed
(Section2.5).5.5lfromthecombinedfractions1and2;6lfrom
thecombinedfractions3and4;5lfromthecombinedfractions5
and6;and6.5lfromthecombinedfractions7to9were
sub-jectedtoRNase Atreatment, DNasetreatment, ornotreatment
(control).The reactionvolume was20l For RNase treatment,
1gofnucleicacidwasincubatedwith0.1gofRNase A
(Fer-mentas)in0.1×SSCbuffer(3Msodiumchloride,0.3Msodium
citrate[pH7.0])for15minatroomtemperature.DNasetreatment
wasperformedusingRQ1DNase(Promega;1unit/gnucleicacid)
at37◦Cfor30min.Allsamples(includingcontrols)weredesalted
withIllustra MicroSpinG25columnsaccordingtothe
manufac-turer’sinstructions(GEHealthcare)andresolvedwithagarosegel
electrophoresisinTBEbuffer(seeSection2.5)
3 Results and discussion
3.1 Setupforstericexclusionchromatographyfornucleicacid
molecules
Themonolithic chromatographic supportischaracterizedby
veryhighporosity,exceptionalchemicalstability,andflow
char-acteristics, which makes it a valuable tool for separation or
purificationoflargebiomolecules[47].CIMmonolithiccolumns,
atrademarkofBIASeparations(Slovenia),areavailablein
differ-entformatswiththesamestructureofthemonolithandligand
density,whichensurescalability.Inourwork,weusedaCIM-OH
columnwithacolumnvolumeof1ml.Achromatographic
proce-dureusedpreviouslyforproteinpurification[27]wasadoptedhere
forseparationofnucleicacidmolecules
To keep the pressure below 1.8MPa (the pressure limit for
theCIM-OH1ml column),weapplieda maximumflow rateof
3ml/minandthefinalconcentrationofPEGdidnottypicallyexceed
15%.TominimizePEG-inducedprecipitationofnucleicacidsbefore
entranceintothecolumn,weusedthein-linedilutiontechnique
wheresamplewasinjectedinbufferAfollowedbymixingwitha
PEG-containingbufferBdirectlyintheM-925mixer(Fig.1).The
gradientdelayvolume(the volumebetweenthemixerandthe
column)wasalsokeptaslowaspossible(here29l).Thus,ata
3ml/minflowrateittookonly34.8sforthesampletoenterthe
column
3.2 SuitabilityofCIM-OHcolumnfornucleicacidseparation High-capacitybindingofnucleicacidsunderSXCconditionshas notbeenobservedundertheconditionsusedpreviously[27]and suitabilityofthis chromatography modefor nucleicacid purifi-cationremainedunclear.Therefore,theinitialexperimentswere performedwithdsDNAmolecules,whicharemorerobustand eas-iertopreparethanRNAmolecules.WeusedthreedifferentdsDNA species,namely500bp,1800bp,and48502bpinlength(see2.2 fortheoriginofdsDNAs).Weestablishedthatretentionofnucleic acidsonthecolumnmatrixrequiredthepresenceofatleast0.4M NaCl(Fig.S1)andwasnegligible(0.5–3%)whenbufferBcontained onlyPEG-6000(upto20%PEG-6000wastested;datanotshown) ThisobservationisconsistentwiththePEGfractionationmethod,
inwhich15%PEG6000at0.55MNaClprecipitatesessentiallyall DNAinthesizerangeof100to46500bp[29].TheshorterdsDNA molecules(500bpand1800bp)wereelutedalmostcompletely fromthecolumnatreduced PEG-6000andNaClconcentrations However,about25%ofthe48502-bplongbacteriophageDNA wasirreversiblyretainedonthecolumn
TherequirementofNaClforefficientretentionofnucleicacids underSXC conditionsis likelyconnectedtothemodificationof stericeffectsbytherepulsiveforcesbetweennegativelycharged chemicalgroupsofthenucleicacidmolecules[31,48,49].Although PEGchangeselectrostaticinteractionsinsolutionsandthemolar conductivityofNaCl-containingbufferdecreaseswithincreasesin PEGconcentration,allnucleicacidmoleculesbecomehydrated.In thisaqueousphase,NaClionizesandformsionpairswith phos-phatesofnucleicacids.Therefore,toneutralizethenegativecharge andtodecreasetheelectrostaticrepulsionforcesbetweennucleic acidmolecules,weusedNaCl-containingbuffersinallsubsequent experiments.Thisresultedinsubstantiallybetterbindingofnucleic acidstothecolumnmatrix(seebelow).Therequirementto neu-tralize chargesfor optimalSXCperformance wasalsoobserved previously for protein samples; the bestresults were achieved whenproteinswereneartheirisoelectricpoint[27]
WealsoinitiallytestedPEGmoleculesofdifferentmolecular weights (4000,6000,and 8000g×mol−1).PEG-6000performed betterintermsofRNAbindingandelutionandingenerated back-pressure.Therefore,inoursubsequentexperimentsweusedonly PEG-6000
3.3 OptimizingSXCforssRNAanddsRNAmolecules 3.3.1 InfluenceofNaClconcentrationonRNAretentionin CIM-OHcolumnmatrix
AftersuccessfulexperimentswithdsDNA,wetestedthe pos-sibilitytouseSXC fortheseparationofRNAmoleculesand the influenceofNaClandPEG-6000concentrationsonRNAretention and subsequent elution.We used 300-bp, 1800-bp or 6374-bp dsRNAmoleculesand initiallykeptthePEG-6000concentration constant(15%)whilesystematicallyvaryingthesaltcontentofthe buffers(Fig.2A).SmallfractionsoftheappliedRNAswereretained
ontheCIM-OHcolumnmatrixinthepresenceof0.2MNaCl How-ever, about75% of the 300-bp dsRNAand 50%of the 1800-bp and6374-bpdsRNAselutedintheflow-through(Fig.2A).After increasingtheNaClconcentrationto0.4Morabove,noRNAwas detectedbyagarosegelelectrophoresisintheflow-through sam-plesofthe1800-bpand6374-bpdsRNAs.Forthe300-bpdsRNA,an evenhighersaltconcentration(0.8–1M)wasrequiredforefficient bindingtothecolumnmatrix.Athighersaltconcentrations,PEG moleculesareknowntoadoptamorecompact,coiledstructure [50].ThisdecreasesthehydrodynamicradiusofPEGand conse-quentlyitsstericexclusioneffects,whichshouldleadtoreduced retentionofnucleicacidsonthecolumnmatrix[50].Inprotein SXC[27],retentionofIgMdiminishedrapidlytozerowhentheNaCl
Trang 5Fig 2.NaCl (A) and PEG-6000 (B, C) dependence of RNA binding to the CIM-OH
concentrationexceededcertainthresholdvalues(0.4MNaClat8%
PEG-6000or0.9MNaClat10%PEG-6000).However,inour
experi-mentsahigherPEG-6000concentrationandupto1MNaCldidnot
showasubstantialnegativeeffectonthenucleicacidretention
3.3.2 InfluenceofPEGconcentrationonssRNAanddsRNA
retentioninCIM-OHcolumnmatrix
Inthenextseries ofexperiments,wekepttheNaCl
concen-trationconstant (0.8M)andfollowedhowchangesinPEG-6000
concentrationaffectedthebindingandelutionbehaviorofssRNA
anddsRNAmolecules(Fig.2Band2C).IrrespectiveofthessRNAor
dsRNAlength,nobindingwasachievedwhenthePEG-6000
con-centrationwas7%orless.SincethevolumeofthePEG-deficient
zonemustbeproportionaltothesizeofthetargetbiomolecules
[31],largerspeciesofssRNAmoleculesboundatlowerPEG
con-centrations(Fig.2B).Accordingly,35%of6374-nt,20%of4065-nt,
and7%of2948-ntssRNAswereretainedonthecolumnin8%
PEG-6000.NoneoftheseRNAspeciesweredetectedintheflow-through
in11%or12%PEG-6000.ssRNAsof1800ntand700ntrequiredat
least10%PEG-6000forbindingtothecolumnmatrix.About95%of
thesessRNAspecieswereretainedin15%PEG-6000.Some
reten-tionoftheshorterssRNAmoleculesusedinthestudy(≤500nt)
wasobservedin11%PEG-6000.However,evenin15%PEG-6000, lessthan80%ofssRNAsof500to100ntwasretained
SmallpercentageofdsRNAmoleculesintherangeof700–6374
bpaccretedtothecolumnmatrixalreadyin8%PEG-6000,while retentionof86%–100%wasachievedin12%PEG-6000(Fig.2C) dsRNAmoleculesof500bpstartedtoaccretetothecolumnmatrix
in10%PEG-6000;fullretentionwasaccomplishedin15%PEG-6000 CompleteretentionofdsRNAshorterthan500bpwasnotachieved undertheconditionstested(Fig.2C)
InthecaseoflongerdsRNAmoleculescorrespondingtophage Phi6genome segments,wedidnot observea clearrelationship betweenmoleculesize and thePEG concentrationrequired for binding We calculated the lengths of these dsRNA molecules, assumingthat1bpofdsRNAcorrespondsto0.29nm[51].Thus, theestimatedlengthofPhi6L-,M-,andS-segments(6374,4065, and2948bp,respectively)wereapproximately1.8m,1.2m,and
854nm,respectively.Thestructure ofdsRNAmoleculesismore rigidcomparedtossRNAs,anddsRNAsmightnotcondensetothe sameextentasssRNAsofthesamelengthinPEG/NaClsolution.This couldhamperthemigrationofthelongdsRNAmoleculesthrough theporesofthecolumnmatrix,whichareonly1.3mindiameter Alternatively,cryogelmonoliths[36]withsuperporousstructures (10–100m)couldbetestedfortheseparationofthelongerdsRNA molecules
Thedynamicbindingcapacity forRNAsamplescouldnotbe determined,asuponloading>250gofdsRNAin13%PEG-6000the backpressureincreasedbeyondacceptablelimits.TheRNA recov-eryunderoptimalgradientconditionswas80±9%.Ourpreliminary experimentsdemonstratedthatforeffectiveelutionofRNAspecies,
ahigh-amplituderapidchangeinPEGconcentrationisrequired Converselywithlong lineargradients (>18CV), RNAmolecules werespread innumerous fractions,which resultedin low ulti-mateRNArecoveryandpoorseparation(Fig.S2).Thus,toachieve satisfactoryresultsashortlinearorastepwisegradientshould pref-erentiallybeapplied(seeFigs.3–6fortheexamplesofoptimized gradientconditions)
3.4 SeparationofDNAanddsRNAmolecules dsDNAanddsRNAspeciesofthesamelengthcouldbepartially resolvedbySXConaCIM-OHcolumn(Fig.3).dsDNAwaselutedfirst whilethemajorityofdsRNAmoleculeswereretainedlongerand elutedinthesecondpeak.Similarresultswereobtainedfornucleic acidmoleculesof1800bpand500bp.Dataontheseparationof 1800-bpdsRNAanddsDNAareshowninFig.3.Inadditiontothe sizeofthenucleicacidmolecule(Fig.2 differencesintheir con-figurationandchemistrymightplayanimportantroleinsample retention.ComparedtotheB-formofdsDNA,theA-formofdsRNA duplexisshorterandwiderwithadeepermajorgroove Monova-lentanddivalentcationspenetrateintothemajorgroovesofdsRNA, whichresultsinmoreefficientshieldingofdsRNAchargecompared withdsDNA[52].Intermolecularandintramolecularrepulsion van-ishesatalowercationconcentrationand,therefore,dsRNAmight associatewitheachotherandthecolumnmatrixmoreefficiently thandsDNA
3.5 SeparationofRNAmoleculesofdifferentsizes
WedidnotachieveanysatisfactoryseparationofRNAmolecules
ofdifferentsizes(eitherssRNAordsRNA)despitesignificant(2 timesorgreater)differencesinsize(datanotshown).Only sep-arationof RNAmolecules shorter than 100nt fromlongerRNA specieswaseasilyachievedbySXC(Fig.S3).Thisisbecausethe shortmoleculesarenotretainedonthecolumnmatrix,whereas thelongeronesareretainedundertheconditionsapplied(Fig.2)
Trang 6Fig 3.Chromatographic separation of dsRNA and dsDNA molecules of 1800 bp using a CIM-OH column A sample containing 15 g of dsRNA and 15 g of dsDNA molecules of
3.6 SeparationofdsRNAandssRNAmoleculesofthesamelength
WehavedevelopedamethodforinvitroproductionofdsRNA
moleculesofdifferentlengthsandsequences[4 Forsubsequent
biochemicalapplications,thedsRNAmoleculesmustbepurified
from contaminating ssRNAs of the same length, abortive
tran-scripts,NTPs,andenzymes.AstepwiseLiClprecipitationcanbe
appliedforroutineuse However,thismethoddoesnotprovide
efficientremovalofssRNAs.WhenssRNAmoleculesinterferewith
a subsequentapplication, anAEXcan beused topurifydsRNA
[24].WeappliedSXCasanalternativemeanstoseparatedsRNA
fromcontaminatingssRNA.Sincethedifferenceinelectrophoretic
mobilitybetweenssRNAanddsRNAspeciesissubstantialonlyfor
longmolecules(inourstudy≥700nt),wetestedRNAmolecules
of700nt,1800nt,and4063nt(Fig.4)toverifytheefficiencyof
SXCseparationusingnon-denaturingagarosegelelectrophoresis
analysisofthesamples
Generally,theseparationofssRNAanddsRNAmoleculesbySXC
improvedasthesizeofthemoleculesincreased;thebest
separa-tionandhighestpuritywasobtainedfordsRNAsof1800bpand
4063bp,whereasafterSXCdsRNAof700bpcontaineda
substan-tialamountofssRNAof700nt.Inallexperiments,ssRNAmolecules
wereretainedonthecolumnmatrixmorestronglythandsRNAsof
equallength(Fig.4).Thus,onthebasisofagarosegel
electrophore-sisanalysisoftheelutionfractions,apurefractionofdsRNAwas
attainablefromamixtureofdsRNAandssRNAusingSXC.However,
subsequentfractionsalwayscontaineddsRNAmoleculesin
addi-tiontossRNA.ThismightbeduetothepresenceofdsRNAinthe
intersticesofthedenselypackedssRNAprecipitatesonthecolumn
stationaryphasesurface
The observed separation efficiency of SXC is comparable to
thatobtainedbyAEX[24].Moreover,thewholeprocesstookonly
30min(20minforsystempreparationand10minforsample
injec-tion and elution) Achievement of similar resolution with AEX
requirestheuseofsubstantiallylongergradients(100CV)[24]
Accordingly,fortheseparationoflongdsRNAmoleculesfroma
mixtureofssRNAanddsRNA,SXCisaneffectiveandefficient
alter-nativetoAEX
3.7 PurificationofssRNAanddsRNAvirusgenomesbySXC
3.7.1 PurificationofphagePRR1genomefromhostcelllysate
Weappliedbothlinearandstepwisegradientstoseparatethe
3574nt-longgenomicssRNAofPseudomonasphagePRR1directly
fromlysateofinfectedbacterialcells(Fig.5AandB).Short
bac-terialssRNAmolecules(<300nt)didnotbindtothecolumnand
wererecoveredintheflow-through.AdecreaseinPEG concen-trationresultedintheelutionofthephagegenometogetherwith contaminatinghostRNAs.HostplasmidDNAwasalsodetectedin someofthefractionscontainingthephagegenome(Fig.5Aand B).Moreover,someproteinsco-elutedwiththeviralRNA(Fig.S4), whichwasexpectedsincetheseparationissize-dependentand largeimpuritiescanco-precipitatewiththetargetmolecules.To increasethepurityoftheviralgenome,weextractedthetotalRNA fromthebacteriallysatebyTRIzurereagenttoremoveproteinsand cellularDNAmoleculesfromthesample(see2.4).Usingastepwise gradientofPEG-6000weobtained85%purephagePRR1genome,
asdeterminedbyagarosegelelectrophoresisanalysis(Fig.5C) TheCIM-OHcolumnconcentratedviralRNAmoleculessothat evenminorRNAspeciescouldbedetectedinsomeofthefractions
byagarosegelelectrophoresis(Fig.5A).Thisconfirmedprevious observations[27]thatunlikePEGprecipitation,bindingefficiency duringSXCisunaffectedbylowtargetconcentration.Thus,SXC couldpotentiallybeusedasananalyticaltoolforcharacterization
ofcomplexRNAmixtures
3.7.2 PurificationofphagePhi6genomefromhostcelllysate
APseudomonasphagePhi6-infectedbacteriallysatewasused
toevaluatethepossibilitytopurifyaviraldsRNAgenomefrom infectedcellsandtoseparatetheindividualgenomesegments.We firstappliedthelysatefromaPhi6-infectedbacterialcultureontoa CIM-OHcolumnafterproteinaseKandSDStreatment(see2.4).In thiscase,thebestseparationwasachievedwithastepwise gra-dient(Fig.6A).However,bacterialplasmidDNA co-eluted with both dsRNAandssRNA species.Withthisapproach itwas pos-sibletoobtainfractionssignificantlyenrichedwithPhi6genomic dsRNAdirectlyfromthehostlysatewithoutmajorprotein contami-nants(Fig.S4).However,wewereunabletoseparatecontaminating ssRNAmoleculesfromtheviralgenome.Wewerealsounableto separatethethreeviralgenomesegmentsfromeachother
ToimprovetheSXC-basedpurificationofthedsRNAgenome
ofbacteriophagePhi6,weisolatedthetotalRNAfromthelysateof Phi6-infectedbacteriausingTRIzurereagent.Alineargradient(12% PEG-6000,0.6MNaClto0%PEG-6000in10CV)providedgood sepa-rationofthedsRNAgenomefromcontaminatingssRNAmolecules (Fig.6B).PuredsRNAgenomewaselutedat7.5%PEG-6000and 0.38MNaCl
TherecoveryandefficiencyofdsRNApurificationwitha
CIM-OH columnwascompared withtheLiClfractionation routinely usedin ourlaboratory Sixidentical samplesoftotal RNA after phenol-chloroformextractionfromP.syringaelysates were pre-pared.ThreesampleswereappliedontoaCIM-OHcolumnandthe
Trang 7Fig 4. Chromatographic separation of dsRNA from contaminating ssRNA of the same size using a CIM-OH column and a linear (A, C, E) or stepwise (B, D, F) PEG-6000 gradient.
Trang 8Fig 5. Separation of Pseudomonas phage PRR1 ssRNA genome from contaminating cellular nucleic acid molecules Bacterial lysate containing PRR1 virions was treated with
remainingsampleswereprecipitatedwithLiCl.AfterLiCl
precipi-tation,7.05±1%ofthetotalRNAwasprecipitatedasdsRNA.While
slightlylessRNA(5.4±1.6%)wasrecoveredinthedsRNA-enriched
fractionselutedfromthecolumn,thepuritysignificantlysurpassed
thatobtainedusingasinglecycleofLiClfractionation(Fig.6B)
4 Conclusions
WeevaluatedthesuitabilityofSXCfortheseparationand
purifi-cation of ssRNA and dsRNA moleculesof different lengths We
determined theconditions under which efficient retention and
elutionofnucleicacids(bothDNA andRNA)couldbeachieved
(Fig.2,Fig.S1).Retentionofnucleicacidsrequiredupto1MNaCl
dependingonthemoleculelengthandabove7%PEG-6000.We
demonstratedthatSXConCIMmonolithiccolumnscanbeapplied
toseparatedsRNAfromssRNAandthattheresolutionisbetterfor
longer(>700bp)dsRNAmolecules(Fig.4).Nevertheless,theuse
ofSXCfortheseparationofRNAsofdifferentlengthsislimited, and onlyshortRNAmolecules(<100nts)canbeeasilyresolved fromlongerRNAspecies(Fig.S3).SXConaCIM-OHcolumnhas thepotentialtoseparatedsDNAanddsRNAmoleculesofthesame length (Fig 3) due tothe structural differences between these molecules
Although separation of viral genome segments was not achieved,SXCcouldseparateandpurifywholeviralssRNAand dsRNAgenomesfromcontaminatingcellularRNAs(Fig.5and6).In termsofrecovery,SXCsurpassedAEXonCIM-QA,CIM-DEAE,and Gen-PakFAXcolumnsbyatleast25%.Furthermore,SXCisof gen-eralutilityforconcentratingRNAvirusgenomes.Thisisespecially usefulfor low-abundanceRNA species,suchas viralreplicative formsandmutualisticviruses
Trang 9Fig 6.Separation of Pseudomonas phage Phi6 dsRNA genome from contaminating cellular nucleic acid molecules (A) Bacterial lysate containing Phi6 virions was treated
Declarations of interest
None
Acknowledgements
WethankDr.SebastijanPeljhanforhisvaluableadviceonSXC
setupof a chromatography systemand Tanja Westerholm and
HirnouScottforexcellenttechnicalassistance.Thisworkwas
sup-portedbytheAcademyofFinland[grant272507],theSigridJusélius
Foundation,Helsinki,Finland,theJaneandAatosErkkoFoundation,
Helsinki,Finland(toM.M.P),andtheFinnishCulturalFoundation,
Helsinki,Finland(toA.L.).Theauthorsacknowledgetheuseofthe
UniversityofHelsinkiInstruct-HiLIFEBiocomplex unit(member
oftheBiocenterFinlandandInstruct-FI)andAcademyofFinland
support[grant1306833]fortheunit
Appendix A Supplementary data
Supplementarymaterialrelatedtothisarticlecanbefound,in
theonlineversion,at doi:https://doi.org/10.1016/j.chroma.2018
08.063
References
[1] A Sedova, N.K Banavali, RNA approaches the B-form in stacked single strand
[2] M.M Cox, D.L Nelson, Nucleotides and nucleic acids, in: Lehninger Principles
of Biochemistry, W.H Freeman and Company, New York, 2005, pp 273–305.
[3] A Travers, G Muskhelishvili, DNA structure and function, FEBS J 282 (2015) 2279–2295.
[4] A.P Aalto, L.P Sarin, A.A van Dijk, M Saarma, M.M Poranen, U Arumae, D.H Bamford, Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage 6 RNA-dependent RNA polymerase, RNA 13 (2007) 422–429.
[5] E.M Kennedy, D.G Courtney, K Tsai, B.R Cullen, Viral epitranscriptomics, J Virol (2017) 91.
[6] R Martins, J.A Queiroz, F Sousa, Ribonucleic acid purification, J Chromatogr.
A 1355 (2014) 1–14.
[7] V Glisin, R Crkvenjakov, C Byus, Ribonucleic acid isolated by cesium chloride centrifugation, Biochemistry 13 (1974) 2633–2637.
[8] P Chomczynski, N Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal Biochem 162 (1987) 156–159.
[9] P Chomczynski, N Sacchi, The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on, Nat Protoc 1 (2006) 581–585.
[10] M.B Stead, A Agrawal, K.E Bowden, R Nasir, B.K Mohanty, R.B Meagher, S.R Kushner, RNAsnap: a rapid, quantitative and inexpensive, method for isolating total RNA from bacteria, Nucleic Acids Res 40 (2012), e156.
[11] A.O Nwokeoji, P.M Kilby, D.E Portwood, M.J Dickman, RNASwift: a rapid, versatile RNA extraction method free from phenol and chloroform, Anal Biochem 512 (2016) 36–46.
[12] R Martins, C.J Maia, J.A Queiroz, F Sousa, A new strategy for RNA isolation from eukaryotic cells using arginine affinity chromatography, J Sep Sci 35 (2012) 3217–3226.
[13] R Martins, J.A Queiroz, F Sousa, A new affinity approach to isolate Escherichia coli 6S RNA with histidine-chromatography, J Mol Recogn 23 (2010) 519–524.
[14] R Martins, J.A Queiroz, F Sousa, Histidine affinity chromatography-based methodology for the simultaneous isolation of Escherichia coli small and ribosomal RNA, Biomed Chromatogr 26 (2012) 781–788.
Trang 10[15] A Petrov, T Wu, E.V Puglisi, J.D Puglisi, RNA purification by preparative
polyacrylamide gel electrophoresis, Methods Enzymol 530 (2013) 315–330.
[16] J.S Kieft, R.T Batey, A general method for rapid and nondenaturing
purification of RNAs, RNA 10 (2004) 988–995.
[17] O.C Uhlenbeck, Keeping RNA happy, RNA 1 (1995) 4–6.
[18] J.R Diaz-Ruiz, J.M Kaper, Isolation of viral double-stranded RNAs using a LiCl
fractionation procedure, Prep Biochem 8 (1978) 1–17.
[19] R.M Franklin, Purification and properties of the replicative intermediate of
the RNA bacteriophage R17, Proc Natl Acad Sci U S A 55 (1966) 1504–1511.
[20] A Azarani, K.H Hecker, RNA analysis by ion-pair reversed-phase high
performance liquid chromatography, Nucleic Acids Res 29 (2001) E7.
[21] M.J Dickman, Effects of sequence and structure in the separation of nucleic
acids using ion pair reverse phase liquid chromatography, J Chromatogr A
1076 (2005) 83–89.
[22] J.A Thompson, R.D Wells, HPLC in nucleic acids research, Nature 334 (1988)
87–88.
[23] J Koubek, K.F Lin, Y.R Chen, R.P Cheng, J.J Huang, Strong anion-exchange
fast performance liquid chromatography as a versatile tool for preparation
and purification of RNA produced by in vitro transcription, RNA 19 (2013)
1449–1459.
[24] A Romanovskaya, L.P Sarin, D.H Bamford, M.M Poranen, High-throughput
purification of double-stranded RNA molecules using convective interaction
media monolithic anion exchange columns, J Chromatogr A 1278 (2013)
54–60.
[25] C.R Huang, S.J Lo, Evolution and diversity of the human hepatitis d virus
genome, Adv Bioinformatics (2010), 323654.
[26] A.E Gorbalenya, L Enjuanes, J Ziebuhr, E.J Snijder, Nidovirales: evolving the
largest RNA virus genome, Virus Res 117 (2006) 17–37.
[27] J Lee, H.T Gan, S.M Latiff, C Chuah, W.Y Lee, Y.S Yang, B Loo, S.K Ng, P.
Gagnon, Principles and applications of steric exclusion chromatography, J.
Chromatogr A 1270 (2012) 162–170.
[28] Steric exclusion chromatography, in: P N.A (Ed.), J Chromatogr Library, 1984,
pp 253–283.
[29] J.T Lis, R Schleif, Size fractionation of double-stranded DNA by precipitation
with polyethylene glycol, Nucleic Acids Res 2 (1975) 383–389.
[30] A Polson, G.M Potgieter, J.F Largier, G.E Mears, F.J Joubert, The fractionation
of protein mixtures by linear polymers of high molecular weight, Biochim.
Biophys Acta 82 (1964) 463–475.
[31] T Arakawa, S.N Timasheff, Mechanism of poly(ethylene glycol) interaction
with proteins, Biochemistry 24 (1985) 6756–6762.
[32] R Bhat, S.N Timasheff, Steric exclusion is the principal source of the
preferential hydration of proteins in the presence of polyethylene glycols,
Protein Sci 1 (1992) 1133–1143.
[33] A Strancar, P Koselj, H Schwinn, D Josic, Application of compact porous
disks for fast separations of biopolymers and in-process control in
biotechnology, Anal Chem 68 (1996) 3483–3488.
[34] P Gagnon, P Toh, J Lee, High productivity purification of immunoglobulin G
monoclonal antibodies on starch-coated magnetic nanoparticles by steric
exclusion of polyethylene glycol, J Chromatogr A 1324 (2014) 171–180.
[35] P Marichal-Gallardo, M.M Pieler, M.W Wolff, U Reichl, Steric exclusion
chromatography for purification of cell culture-derived influenza A virus
using regenerated cellulose membranes and polyethylene glycol, J.
Chromatogr A 1483 (2017) 110–119.
[36] C Wang, S Bai, S.P Tao, Y Sun, Evaluation of steric exclusion chromatography
on cryogel column for the separation of serum proteins, J Chromatogr A 1333 (2014) 54–59.
[37] P Gottlieb, J Strassman, X Qiao, M Frilander, A Frucht, L Mindich, In vitro packaging and replication of individual genomic segments of bacteriophage
6 RNA, J Virol 66 (1992) 2611–2616.
[38] M Jiang, P Osterlund, L.P Sarin, M.M Poranen, D.H Bamford, D Guo, I Julkunen, Innate immune responses in human monocyte-derived dendritic cells are highly dependent on the size and the 5’ phosphorylation of RNA molecules, J Immunol 187 (2011) 1713–1721.
[39] V.M Olkkonen, P Gottlieb, J Strassman, X.Y Qiao, D.H Bamford, L Mindich,
In vitro assembly of infectious nucleocapsids of bacteriophage 6: Formation
of a recombinant double-stranded RNA virus, Proc Natl Acad Sci U S A 87 (1990) 9173–9177.
[40] L Mindich, X Qiao, S Onodera, P Gottlieb, M Frilander, RNA structural requirements for stability and minus-strand synthesis in the dsRNA bacteriophage 6, Virology 202 (1994) 258–263.
[41] M Szekeres, B.H Brownstein, H.R Revel, R Haselkorn, Terminal sequences of the bacteriophage 6 segmented dsRNA genome and its messenger RNAs, Virology 142 (1985) 1–11.
[42] E.V Makeyev, D.H Bamford, Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage 6, EMBO J 19 (2000) 124–133.
[43] A.K Vidaver, R.K Koski, J.L Van Etten, Bacteriophage phi6: a lipid-containing virus of Pseudomonas phaseolicola, J Virol 11 (1973) 799–805.
[44] D.H Bamford, P.M Ojala, M Frilander, L Walin, J.K.H Bamford, Isolation, purification, and function of assembly intermediates and subviral particles of bacteriophages PRD1 and 6, in: K.W Adolph (Ed.), Methods in Molecular Genetics, Academic Press, San Diego, 1995, pp 455–474.
[45] R.H Olsen, D.D Thomas, Characteristics and purification of PRR1, an RNA phage specific for the broad host range Pseudomonas R1822 drug resistance plasmid, J Virol 12 (1973) 1560–1567.
[46] D.C Rio, M Ares Jr., G.J Hannon, T.W Nilsen, Nondenaturing agarose gel electrophoresis of RNA, Cold Spring Harb Protoc (2010), pdb.prot5445.
[47] M Krajacic, M Ravnikar, A Strancar, I Gutierrez-Aguirre, Application of monolithic chromatographic supports in virus research, Electrophoresis 38 (2017) 2827–2836.
[48] A.P Minton, The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media, J Biol Chem.
276 (2001) 10577–10580.
[49] D.H Atha, K.C Ingham, Mechanism of precipitation of proteins by polyethylene glycols Analysis in terms of excluded volume, J Biol Chem 256 (1981) 12108–12117.
[50] C Tan, J.G Albright, O Annunziata, Determination of preferential interaction parameters by multicomponent diffusion Application to poly(ethylene glycol)-salt-water ternary mixtures, J Phys Chem B 112 (2008) 4967–4974.
[51] J.A Abels, F Moreno-Herrero, T van der Heijden, C Dekker, N.H Dekker, Single-molecule measurements of the persistence length of double-stranded RNA, Biophys J 88 (2005) 2737–2744.
[52] S.A Pabit, X Qiu, J.S Lamb, L Li, S.P Meisburger, L Pollack, Both helix topology and counterion distribution contribute to the more effective charge screening
in dsRNA compared with dsDNA, Nucleic Acids Res 37 (2009) 3887–3896.