CorrespondingauthorEmaildeborahphysicsucsbeduDepartmentofPhysicsUniversityofCaliforniaSantaBarbaraCaliforniaInstituteofTechnologyBiomolecularScienceEngineeringProgramUniversityofCaliforn ID: 183107
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SturdierDNANanotubesviaLigationPatrickO'Neill,PaulW.K.Rothemund,AshishKumar,andD.K.Fygenson*DepartmentofPhysics,UniersityofCalifornia,SantaBarbara,California93106,DepartmentofComputerScienceandComputationalandNeuralSystems,CaliforniaInstituteofTechnology,Pasadena,California91125,andBiomolecularScience&EngineeringProgram,UniersityofCalifornia,SantaBarbara,California93106ReceivedFebruary15,2006;RevisedManuscriptReceivedMay19,2006DNAnanotubesarecrystallineself-assembliesofDNAtiles10nmindiameterthatreadilygrowtensofmicrometersinlength.Easyassembly,programmability,andstiffnessmaketheminterestingformanyapplications,butDNAnanotubesbegintomeltattemperaturesbelow40breakopenwhendepositedonmicaorscannedbyAFM,anddisintegrateindeionizedwater.Theseweaknessescanbetracedtothepresenceofdiscontinuitiesinthephosphatebackbone,callednicks.Thenanotubesstudiedherehavefivenicks,oneinthecoreofatileandoneateachcorner.WereportthesuccessfulligationofallfourcornernicksbyT4DNAligase.Althoughligationdoesnotchangethenanotubes'stiffness,ligatednanotubeswithstandtemperaturesover70C,resistbreakingduringAFM,andarestableinpurewaterforoveramonth.LigatedDNAnanotubesarethusphysicallyandchemicallysturdyenoughtowithstandthemanipulationsnecessaryformanytechnologicalMadeentirelyofDNA,buthundredsoftimesstifferthanordinarydouble-strandedDNA(dsDNA),DNAnanotubescombinethebindingspecificityofnucleicacidswitharigid,linearstructure.ThiscombinationofstructuralandbiochemicalpropertiesmakesDNAnanotubesespeciallyinterestingforapplications.Forexample,theymightbemetallizedorfunctionalizedbysurfaceattachmentofbio-moleculesornanoparticlesandsoserveasinterconnectsinself-assemblednetworks.Alternatively,DNAnanotubesmightbeused,likeactinfilaments,asmechanicalmagnifiersofthenanoscopicmotionofbiomolecules,butwithgreaterchemicalstabilityandversatility.However,thepracticalrealizationoftheseandmanyotherapplicationswillrequireDNAnanotubesthatwithstandconsiderablemechanicalandchemicalmanipulation.Therefore,toenhancethetechno-logicalrelevanceofDNAnanotubes,wesoughttoincreasetheirstabilityvialigation.LigationiscommonlyusedtoestablishthetopologyofprogrammedDNAnanostructures.However,itsef-fectivenesswithinatwo-dimensionalDNAtilearrayhasneverbeenwellcharacterized.Crystalstructuresofligaseenzymessuggestthatligationrequirestheenzymetoaccessalargeportionofthedoublehelicalsurface.Onemightthereforeexpectligationamongthepotentiallyclose-packeddoublehelicesofthenanotubetilelatticetobeparticularlyInthispaper,wereportthatDNAnanotubesareviablesubstratesforT4DNAligase,quantifytheextentofligationateachofthefivenicks,andshowthatligatednanotubesaresignificantlymorestabletopracticalmanipulationthanunligatednanotubes.TheDNAnanotubesthatweuseself-assemblefromasingleDAE-Etypetile(Figure1).ThetileismadeoffivesyntheticDNAoligomersthathybridizeintoarigidrectan-gularcorewithasingle-strandedfive-baseoverhang(stickyend)ateachcorner.Thecoreconsistsoftwodoublehelicesjoinedattwofour-wayjunctions(crossovers).Diagonallyoppositecornerspresentcomplimentarystickyends.Whenthesehybridize,thetilesforma2Dlattice,butthelatticeisnotflat.TheintrinsictwistofdsDNAandthenumberofbasepairs(bp)betweencrossoversinneighboringtilesconstrainthemtojoinataangleabouttheircommondoublehelix,impartinganintrinsiccurvaturetothelatticethatfavorsclosurewithadiameterof10nmEachstickyendjunction,whereDNAstrandsfromadjoiningtilesmeetuponhybridization,isashort(5bp)segmentofdsDNAflankedbyapairofnicks.Nicksaregapsinanotherwisecontinuoussugarphosphatebackbone.Theyarethereforeweakpointsatwhichtiles,orperhapsindividualstrands,dissociateinresponsetochangesintemperature,electrostaticenvironment,andsolventcondi-tions.Thereisalsoanickinthecenterofeachtile,wherethetwoendsofstrandno.3meetupontileformation.Strandno.3isreferredtoasthecorestrand,andtheassociatednickiscalledthecorenick.Theotherfournicksarecalledcornernicks. *Correspondingauthor.E-mail:deborah@physics.ucsb.edu.DepartmentofPhysics,UniversityofCalifornia,SantaBarbara.CaliforniaInstituteofTechnology.BiomolecularScience&EngineeringProgram,UniversityofCalifornia,SantaBarbara.Vol.6,No.710.1021/nl0603505CCC:$33.502006AmericanChemicalSocietyPublishedonWeb06/20/2006 T4DNAligaseisanATP-drivenenzymethatsealsnicks.Moreprecisely,itcatalyzestheformationofacovalentphosphodiesterbondbetweenthe5phosphateofoneDNAstrandandthe3hydroxylofanother.Theterminal5phosphateistypicallyabsentinsyntheticDNA,butcanbeaddedchemically,orbyanotherATP-drivenreactionusingtheenzymeDNAkinase.Onemaythereforecontrolwhichnicksinthelatticecanbeligatedbyselectivelyphospho-rylatingonlyasubsetofthestrandscomprisingthetile.WeusedthiscontroltostudytheextentandconsequenceofligationonDNAnanotubes.Inournanotubelattice,strandno.1ligatestostrandno.2,strandno.2ligatestostrandno.5,strandno.5ligatestostrandno.4,andstrandno.4ligatestotheoriginalstrandno.1.CompleteligationwouldthusresultinameshworkofconcatenatedloopsofssDNA.DNAnanotubesinwhichone,two(strandnos.2and4),three(strandnos.1,2,and4),four(strandnos.1,2,4,and5),orallfivestrandswerechemicallyphosphorylated(onthe5end)arereferredtohereinasone-point,two-corner,three-corner,four-corner,andfive-pointphosphorylatedtubes,respectively(Figure1B).Allfivestrandsweremixedat1.8M(eachstrand)inabufferconsistingof30mMtris-HCl(pH7.8),10mMDTT,1mMATP,and10mMThin-walled200LPCRtubescontaining30ofthestrandmixtureweresubmergedibeakerof90Cwater,whichwasplacedinaStyrofoamboxandlefttocooltoroomtemperatureover48h.Thisprocedure(theanneal)resultsinahighdensityofnanotubes,manyovermlong,asverifiedbyfluorescencemicroscopy(seeForligation,T4DNAligase(FisherCat.No.BP3210-1),ATP,andDTTwereaddedtotheannealednanotubesolution.Finalconcentrationsintheligationmixturewereasfol-lows:[DNA]M(eachstrand),[ligase]0.5units/L,0.9mMmM1.6mM,and8.6mMmM15.3mM.Theupper(lower)boundsassumethatnone(all)oftheATPorDTTinitiallypresentwasdegradedduringtheanneal.Ligationmixtureswereincubatedatroomtemperatureforatleast1daypriortoallexperiments.Toassesstheextentofligationateachofthedifferentnicks,weperformeddenaturinggelelectrophoresisafterligatingnanotubesinwhichonlyoneofthefivestrandswasphosphorylated(Figure2).BandswerequantitatedbasedonthefluorescenceoftheintercalatingdyeSybrgold(MolecularProbesCat.No.S11494).Comparingstrandsfromnanotubesofidenticalcomposition,beforeandafterexposuretoligase,wefindthatligationtakesplaceateachofthefourcornernicks(Figure2,lanes2,4,8,and10),butnotatthenickinthetilecore(Figure2,lane6).Accessibilityofthecornernicks,andinaccessibilityofthecorenick,toligasecanbeexplainedbythespacingbetweencrossoversintheDNAlattice.Eachcornernickliesinaregionofhelixthatislinkedtoneighboringhelicesbycrossoversthataresixhelicalturnsapart.Thisregionmayflexradiallyoutwardorinward,orneighboringhelicesmaybowlaterally,allowingligasetoaccessalargeportionofthedouble-helicalsurface.Thelattersuggestionissupportedbyhigh-resolutionAFMimagesoftilelatticesonmicathatshowrelativelywideseparations(withanapparentwidthof23nanometers)intheseregions.Thecorenicks,bycontrast,eachlieonaregionofhelixconnectedtooneofitsneighborsbycrossoversonlytwohelicalturnsapart.High-resolutionAFMshowsthesepara-tionbetweenhelicesinthisregiontobe0.5nm.Thecore Figure1.SchematicsoftheDNAtileandnanotubeused.(A)Doublehelicalrepresentationofasingletile.Arrowheadsmarkthe3endofeachstrand.(B)Sequenceandnumberingschemeforeachstrandinthetile.(C)Smallportionoflattice,consistingofseventiles.Thedashedboxframesaregionwherecrossoverssixhelicalturnsapartconnectneighboringhelices.Regionswhereneighboringhelicesareconnectedbycrossoversonlytwohelicalturnsapartarefoundatthelocationofeachstrandno.3.(D)Shortnanotubesegmentwitharedregioncorrespondingtotheseven-tilearraydepictedinC. Figure2.DenaturingpolyacrylamidegelcomparingstrandsfromligatedandunligatedDNAnanotubeswithdifferentstrandsphosphorylated.Lanes110:uppermostbandscorrespondto63baselongligationproduct(eitherno.14,45,52,or21)asdeterminedbyextrapolationbasedontheknownlengthsofstrandsinthelower(unligated)bands(nos.2and4:26bases,nos.1and5:37bases,no.3:42bases).Noslowlymigratingbandcorrespondingtoacyclizedstrandno.3isdetected(lane6).Notethateachcornerligationinvolvesonlyoneofthetwo26basestrands(lanes2,4,8,and10).Lanes11and12:uppermostbandcorrespondstoa126basestrand,generatedwhenthreecontiguousnicksareligatedtoformonelargestrand(no.5412).Strandsof100bases(no.521)and89bases(no.214)aregeneratedwhenonlytwocontiguousnicksareligated.NanoLett.,Vol.6,No.7, nickfacesthissmallintratilegap.Accordingly,wefoundthatthecorenickisinaccessibletoligaseeveninisolatedtiles.Tileswith5-phosphorylatedstrandno.3andfournoncomplimentarystickyendswereexposedtoligase,andtheresultingstrandswereseparatedonadenaturinggel(notAgain,nobandcorrespondingtocyclizedstrandno.3wasdetected.Wesuspectthecorenickwouldbeligatedifitfacedthelargerintertilegap.Ligationofsuchoutward-facingcorenickshasbeendemonstrated,albeitnotinatwo-dimensionaltilelattice.QuantitativeinterpretationofgelbandintensitiesiscomplicatedbythefactthatSybrgoldisthreetimesmorefluorescentwhenboundtodsDNAascomparedtossDNA.Wemustthereforeconsiderthepossibilitythattheligationproductisnotcompletelydenatured.Assumingthattheligationproducthaseitheradopteditslowestenergysecond-arystructureorremainedentirelyunhybridized,weplaceconservativelowerandupperboundsontheextentofligationateachcornernick:5285%,4674%,5182%,and5482%fornicksatthe5endsofstrandnos.1,2,4,and5,respectively.(SeetheSupportingInformationfordetailsofthecalculations.)Asimilaranalysisoftheligationproductofthree-cornerphosphorylatednanotubes,assumingligationoccurswithequalprobabilityateachnick,placestheextentofligationbetween68%and77%pernick.Theconsistencyofthesevaluessuggeststhatcornernicksareligatedindependently.Interestingly,thereisnoindicationthatligasedistinguishesbetweencornernicksontheinterior(atthe5endsofstrandnos.1and5)orexterior(atthe5endsofstrandnos.2and4)faceofananotube.(SeetheSupportingInformationofref1for3Dgraphicsdepictingallstrandsinthecontextofananotube.)Clearly,notallstickyendswereligated.Forexample,ligationofthree-cornerphosphorylatednanotubesyieldsone-cornerandtwo-cornerligationproductsaswell(Figure2,lane12).Althoughthismayreflectfinitereactionconditionsorincompletephosphorylation,itmayalsobearesultofdefectsinthenanotubelatticethatleavesomestickyendsunhybridized.Thus,ifcareistakentoensurethattheligationreactiongoestocompletion,theextentofligationmightbeusedasasimpleassayforthedensityofdefects.Giventhehighproportionofligatedcornernicks,itisreasonabletoexpectqualitativechangesinnanotubeproper-ties.Welookedforchangesinthermalandmechanicalstability,stiffness,andbuffercompatibilityuponligation.Asignificantincreaseinthermalstabilityisevidentbyfluorescencemicroscopy(Figure3,seetheSupportingInformationforexperimentaldetails).Fluorescencecomesfromanorganicdye(FAM)covalentlyattachedtothe5endofstrandno.3.Foursolutionsofnanotubes,differingonlyinthenumberof5phosphorylatedstrandspertile,wereexposedtoligase.Theywerethenheldatafixedtemperaturefor30minandimagedinfluorescence.Theimagesarequalitativelydividedintothreegroups:normal(toprow),deformed(middlerow),andmelted(bottomrow).Forthetemperaturessampled,onlynormalandmeltedtubeswereseenforone-cornerandthree-cornerligatedtubes.Eachadditionalligatedtilecornerenhancedthethermalstabilityofthenanotubes(lefttoright).Atthetwoextremes,unligated(nonphosphorylated)tubesmeltcompletelyby46whereasfour-cornerligatedtubesappearunaffectedattemperaturesupto73Increasedmechanicalstabilityisevidentbyatomicforcemicroscopyunderbuffer(Figure4).Aftersettlingonthemicasubstrate,unligatednanotubesusuallyopenwithinminutes,revealingthetilelattice.Underidenticalcondi- Figure3.Fluorescentmicrographsshowingtheincreaseinthermalstabilityasthenumberofligationsitesisincreased.Allofthenanotubeshavebeenexposedtoligase,theydifferonlyinthenumberof5phosphorylatedstrandspertile.Phosphorylatedstrands:One-corner(strandno.1),two-corner(strandnos.2and4),three-corner(strandnos.1,2,and4),four-corner(strandnos.1,2,4,and5).Toprow:imagesfromthehighesttemperaturessampledatwhichheatedtubeswereindistinguishablefromunheatedtubes.Middlerow:imagesfromintermediatetemperaturesatwhichtubesweredeformedbutnotcompletelymelted.Forone-cornerandthree-cornerligatedtubes,wedidnotfindanysuchintermediatestabilityatthetemperaturessampled.Bottomrow:imagesfromthelowesttemperaturessampledatwhichtubeswerecompletelymelted.Imagesare50monaside. Figure4.AtomicforcemicrographsofDNAnanotubesdepositedonmicaandimagedintappingmodeunderTAE/Mg1Xbuffer.Aligatednanotube(A)retainsitsstreakyappearanceand5nmthicknessfortheentiretimeofimaging(1h).Anunligatednanotube,initiallyclosed(B,right),lookssimilar,butopens(C,right)intoa2-nm-thicktilelatticewithinminutesofdeposition.Thedisplacementoftubesatthetopofimage(B)withrespecttoimage(C)isanAFMartifact.Imagesbandcareadaptedfromref3.Scalebar:500nm.NanoLett.,Vol.6,No.7, tions,four-cornerligatednanotubesremainintactforatleastanhour.Notevenaggressiveimagingathighforcewasabletotearopentheligatedtubes.Thisenhancedmechanicalstabilityraisesthequestionofwhetherligationchangestheoverallstiffnessofananotube.Wemeasuredthestiffnessofligatedandunligatednanotubesbyobservingthermallyexcitedbendingfluctuationsoffree-floatingnanotubesconfinedto2D(seetheSupportingInformation).Thecorrelationbetweenagiventube'saveragesquaredend-to-enddistance,,anditscontourlength,relatestoitsstiffness,intermsofthepersistencelength,accordingtothewormlikechainmodel:Aplotofforligatedandunligatednanotubesfitswelltothisfunctionwithasafreeparameter(Figure5).Theresultingpersistencelengthsofligated(28.6m)andunligated(26.2m)nanotubesareindistin-guishableandcomparabletotheexpectedvaluefornano-tubes12or14doublehelices(6or7tiles)incircumference.Thesepersistencelengthsareseveraltimeslongerthanthoseobservedpreviously.HereweusedhigherDNAconcentrationsandaslowerthermalanneal,bothofwhichshouldreducetheoccurrenceoflatticedefectsandfavorlargertubediameters.Theobservationthatligationdoesnotnoticeablyinfluencestiffnessatroomtemperaturereinforcesthenotionthatlatticestructure,ratherthanstickyendstability,istheprimarydeterminantofnanotubestiffness.Withapersistencelengthoftensofmicrometers,stiffnessisnotamajorpracticallimitation.Morelimitingwouldbesensitivitytochangesinsolutionconditions.Inparticular,highsaltconditionsarenotcompatiblewithmanychemicalreactions.Charge-stabilizedcolloidsthatonemightwishtoattachtothenanotubes,includingmanytypesofsemicon-ductorquantumdots,aggregateinthepresenceofexcess.BufferexchangemaythereforeberequiredforanumberofchemistriesaimedatfunctionalizingDNAnano-Wesoughttoremoveallpotentiallyundesirablecompo-nentsfromthenanotubebufferbyexchangingtheannealingbufferforpurewater.Weexperimentedwiththreemethodsofbufferexchange:dialysis,spinfiltration,andultracen-trifugation(seetheSupportingInformation).Unligatednanotubessurviveallthreemanipulationswhensolutionconditionsarekeptconstant,butfallapartimmediatelyandcompletelywhenthebufferisexchangedforpurewater,regardlessofthemethodused.WethereforeconcludethatitisthechangeinsolutioncompositionratherthanthemechanicalmanipulationinvolvedinbufferexchangethatdestroysunligatedDNAnanotubes.Four-cornerligatednanotubessurviveallthreebufferexchangeproceduresandarestableinpurewaterforatleastonemonth.Hereweperformedligationafterself-assemblytofortifyatile-basedDNAlattice.Ligationmayalsobeofuseduringself-assembly.Forexample,ligationrestrictsthetorsionalflexibilityofnickeddsDNA.Ifastickyendjunctionwereligatedimmediatelyfollowinghybridization,itmightusefullyconstraintheglobalgeometryofagrowingtilelattice.Therelativekineticsoflatticeformationandligationwillbecrucialindeterminingtheeffectsligasehasduringself-assembly.Thechallengeinsuchanapproachwillbeinavoidingligationsofimproperhybridizationsthatcouldinhibitassemblyofthedesiredstructure.Insummary,wehaveshownthatT4DNAligasesealsnicksbetweentilesinDNAnanotubes,makingthemmorerobusttopracticalmanipulations.Theextentofligationatindividualcornernicksrangedfrom46to85%withanaverageof6877%whenthreeofthefourcornernickswerephosphorylated.Theseresultssuggestthattile-basedDNAsuperstructuresaregenerallyviablesubstratesforefficientligation.Similarexperimentsonothertile-basedlatticeswouldhelpdeterminethegeometricalconstraintsimportantforligationandestablishdesignprinciplesformorefullyligatableDNAlattices.LigationalreadyplaysacriticalroleincharacterizingDNAnanostructures.Inthefuture,itmaybeevenmoreimportantasameansoffortifyingthesestructuresfortechnologicalapplications.WethankErikWinfreeandKyleChipmanforinsightfuldiscussions.ThisworkwassupportedbyanNERAwardNo.0404440fromtheNationalScienceSupportingInformationAvailable:Gelpreparation,bandanalysis,fluorescencemicroscopy,AFM,andpersis-tencelengthmeasurements.ThismaterialisavailablefreeofchargeviatheInternetathttp://pubs.acs.org.(1)Liu,D.;Park,S.H.;Reif,J.H.;LaBean,T.H.Proc.Natl.Acad.Sci.U.S.A.,717.(2)Mitchell,J.C.;Harris,J.R.;Malo,J.;Bath,J.;Turberfield,A.J.Am.Chem.Soc.,16342.(3)Rothemund,P.W.;Ekani-Nkodo,A.;Papadakis,N.;Kumar,A.;Fygenson,D.K.;Winfree,E.J.Am.Chem.Soc.,16344.(4)Braun,E.;Keren,K..Phys.,441.(5)Liu,Y.;Lin,C.X.;Li,H.Y.;Yan,H.Angew.Chem.,Int.Ed.,4333.(6)Yan,H.;Park,S.H.;Finkelstein,G.;Reif,J.H.;LaBean,T.H.,1882. Figure5.Averagesquaredend-to-enddistanceplottedagainstcontourlength(seeinset)forligated()andunligated(nanotubes.Linesareleast-squaresfitstothedatausingthe2DPorodmodel.Theresultingmeasurementofpersistencelengthforligated(28.6m)andunligated(26.2nanotubesisthesamewithinerror. NanoLett.,Vol.6,No.7, (7)Sharma,J.;Chhabra,R.;Liu,Y.;Ke,Y.G.;Yan,H.Angew.Chem.,Int.Ed.,730.(8)Le,J.D.;Pinto,Y.;Seeman,N.C.;Musier-Forsyth,K.;Taton,T.A.;Kiehl,R.A.NanoLett.,2343.(9)Noji,H.;Yasuda,R.;Yoshida,M.;Kinosita,K.(10)Petrillo,M.L.;Newton,C.J.;Cunningham,R.P.;Ma,R.I.;Kallenbach,N.R.;Seeman,N.C.,1337.(11)Yang,X.P.;Wenzler,L.A.;Qi,J.;Li,X.J.;Seeman,N.C.J.Am.Chem.Soc.,9779.(12)LaBean,T.H.;Yan,H.;Kopatsch,J.;Liu,F.R.;Winfree,E.;Reif,J.H.;Seeman,N.C.J.Am.Chem.Soc.,1848.(13)Sa-Ardyen,P.;Vologodskii,A.V.;Seeman,N.C.Biophys.J.,3829.(14)Winfree,E.;Liu,F.R.;Wenzler,L.A.;Seeman,N.C.,539.(15)Johnson,A.;O'Donnell,M.Curr.Biol.,R90.(16)Fu,T.J.;Seeman,N.C.,3211.(17)Weusedcombinationsofchemicallyphosphorylatedandunphos-phorylatedoligomerssynthesizedbyIntegratedDNATechnologies(Skokie,IL).http://www.idtdna.com.(18)Incontrast,othertiledesignsexistthatyieldlongªreporterºstrandsuponligationasinref10.(19)Thiscommerciallyavailablebuffer(FisherT4DNAligationbuffer)wasusedforconvenience.ATPandDTTarenotnecessaryatthisstageandthermallydegradeduringtheanneal.(20)Definingthedetectionlimitbythelightestbandinthegel,theabsenceofaslowmovingbandcorrespondingtoacyclizedno.3strandmeanstheextentofligationofthenickinthetilecoreis(21)Tomakesurethatstrandno.3was5phosphorylated,acontrolexperimentwasperformedinwhichstrandno.3andashort(16base)strandwerehybridizedtostrandno.1.Afterexposuretoligase,therewasaclearlyvisiblebandcorrespondingtotheligationoftheendofstrandno.3totheshortstrand.(22)Tuma,R.S.;Beaudet,M.P.;Jin,X.K.;Jones,L.J.;Cheung,C.Y.;Yue,S.;Singer,V.L.Anal.Biochem.,278.(23)SecondarystructuresweredeterminedusingtheMfoldwebserverwith0.1MNaCl,20C.http://www.bioinfo.rpi.edu/applications/(24)Zuker,M.NucleicAcidsRes.,3406.(25)Doi,M.;Edwards,S.F.TheTheoryofPolymerDynamics;ClarendonPress:Oxford,1986.(26)Zhang,Y.L.;Crothers,D.M.Abstr.PapAm.Chem.Soc.,U520.NanoLett.,Vol.6,No.7,