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rsc.li/nanoscale

Nanoscalewww.rsc.org/nanoscale

ISSN 2040-3364

PAPERQian Wang et al.TiC2: a new two-dimensional sheet beyond MXenes

Volume 8 Number 1 7 January 2016 Pages 1–660

Nanoscale

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MOHAMMED, L. Velazquez, A. Chisenhall, D. Schiffels, D. Kuchnir Fygenson and R. Schulman, Nanoscale,

2016, DOI: 10.1039/C6NR06983E.

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DOI:10.1039/x0xx00000x

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Self-AssemblyofPreciselyDefinedDNANanotubeSuperstructuresUsingDNAOrigamiSeedsA.M.Mohammed,aL.Velazquez,bA.Chisenhall,aD.Schiffels,b,cD.K.Fygenson,b,dandR.Schulman*a,e

Wedemonstrate a versatile process for assemblingmicron-scalefilament architectures by controlling where DNA tile nanotubesnucleate on DNA origami assemblies. “Nunchucks,” potentialmechanical magnifiers of nanoscale dynamics consisting of twonanotubes connectedbyadsDNA linker format yields sufficientforapplicationandconsistentwithmodels.

A major goal of bottom-up synthesis is the self-assembly ofintegrated nanoscale devices.1, 2 One-dimensional structuresareimportantprimitivesforsuchstructures.Forexample,self-assembly of nanowires or carbon nanotubes into specificgeometriescouldformelectricalcircuits.3,4Nanoscaletubularfilamentscould likewiseassemble intomachinesandsensors,as demonstrated by the in vivo assembly of cytoskeletalstructuressuchasthecilium5andthemitoticspindle.6,7While the self-assemblyofDNA,8-12peptides,13, 14andother15types of filaments has been well-explored, relatively little isknown about how to organize these filaments intoarchitecturesfeaturingprecisenumbersofcomponents.16Theability to synthesize designed filament architectures couldmake it possible to construct molecular machines with newfunctions that can operate outside cells under a variety ofphysicalandchemicalconditions.Inthispaperweexploretheassembly of DNA tile nanotubes into an elemental multi-filamentarchitecture.Invivo,cytoskeletalfilamentsareoftenarrangedbycontrollingtheirnucleationusingproteincomplexessuchasArp2/317,18orthe centriole19 that template actin or microtubule growth.Analogously, formin20 and the γ-tubulin small complex21 can

promote filament nucleation in specific regions of a cell todirecttheformationoffilamentnetworksorbundles.We have previously developed structures that nucleate DNADAE-Etilenanotubes, termednanotubeseeds.22Hereweusethese structures to develop a biomimetic strategy for thecreation of nanotube architectures, in which these seedsorganize DNA tile nanotubes as they assemble. Wedemonstrate this strategy by self-assembling “DNA nanotubenunchucks”thatconsistoftwonanotubesjoinedattheirendsby a stretch of double-stranded DNA (dsDNA) of arbitrarylengthandsequence.

Fig.1Nanotubenunchuckdesignandassembly.(a)ADAE-EDNAnanotubetileconsistsoffiveDNAstrands.Eachstrandisshowninadifferentcolor.Single-strandedstickyendsonfourtileedgescanhybridizetocomplementarystickyendsonothertilestoformnanotubes.ADNAorigaminanotubeseedconsistsofalongscaffoldstrand(M13mp18)thatisassembledintoacylindricalshapeby72staplestrands.Hairpinsontheoutsideoftheseedensurethestructurecurlsinthecorrectdirectionduringfolding.Nanotubesgrowfromtileadapterstrandsononesideoftheseed(yellow).(b)Nunchuckseedsareassembledbyhybridizingtwonanotubeseedswithcomplementarylinkerstrands(greenandpurple).Nanotubescangrowfrombothgrowthsitesonanunchuckseed,producingnanotubenunchucks.

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We assess the assembly of both homogeneous nunchucks,consistingof twoconnectednanotubesof identical tile types,and heterogeneous nunchucks, in which two nanotubes ofdistincttiletypesareconnected.Our assembly strategy consists of linking DNA origami seedsend-wisewithasyntheticstretchofdsDNAandthengrowingnanotubes from the opposite ends of the seeds. Previousattempts to form nunchucks by directly linking pre-formedDNAnanotubesendwiseresultedinverylowyields(<1%,datanot shown).23 The seed-based assembly strategy developedhereproducesthesestructureswithdramaticallyhigheryields(>40%).DAE-Etilenanotubestypicallyself-assembleoverthecourseofa thermal anneal in two stages (Fig. 1a). First, DAE-E tilesassemble from five component strands at a relatively hightemperature (~45-65°C).8 Then, tile sticky ends hybridize toformnanotubesatadistinctly lower temperature (~40-30°C),which depends on tile concentration. A nanotube seed is aDNAorigamistructurewith12helicesarranged intoahollowcylinder. Adapter strands on one end of the seed presentstickyendspositionedsoastoresembleananotube.Nearthesticky-endmelting temperature, nanotubes grow from seedsmore rapidly than they nucleate spontaneously.22 For ourseededassemblystrategy, thethermalanneal involvesa longincubationnearthesticky-endmeltingtemperature,suchthatmostnanotubesgrowfromseeds.Because seeds are DNA origami structures, it is possible toexerciseexquisitecontroloverhowtheyattachtooneanotherusing DNA strands designed to hybridize to specific sites ontheir surface.Wedesigned a pair of partially complementarylinker strands that hybridize to the origami seed at locationsoppositethenanotubegrowthsite.Thedesignincorporatesanodd number of half-helical turns in order to maximize thedistancebetweentheseedcenters(Fig.1b,Fig.S3).Seedswithone linker strand were prepared separately from seeds withthesecondlinker.Thetwonanotubeseedswerethenmixedtoformnunchucksseedsvia linkerstrandhybridization(Fig.1b).We separated dimerized nunchuck seeds from monomericnanotubeseedsusingelectrophoresis throughanagarosegelstainedwithethidiumbromide(Fig.S7,ExperimentalSection).Relativeintensitiesofthedimerandmonomerbandsinthegel(Fig.S7)indicatedthatyieldofthehybridizationreactionwas~25%.We first used this strategy to construct homogeneousnanotubenunchuckseeds (SeeExperimentalSection).Atomicforce micrographs (Fig. 2a, Fig. S15) confirmed that theextracted nunchuck seeds consisted of two nanotube seedswith theexpected64by21nmdimensions22 connectedbyalinker of a length 27.9 ± 2.9 nm (n = 5), consistent with theexpected 90 base-pair (58 base-pair linker + 16 base-pairdouble stranded attachment region on either side) linker

lengthof29.7nm.Fornanotubenunchuckstoworkasatoolformeasuringthebendanglesofthe linkerembeddedwithinthem, it is essential that the nunchuck be able to explore allpossible angles that could frombetween the nanotubes. Therelativeorientationofthelinkednanotubesinnunchuckseedsranged from almost straight to roughly 0º (Fig. 2c). Apreference for unbent configurations is consistent with thefact that the length of the double-stranded DNA linkerbetween the nanotube seeds is less than its persistencelength.To create nanotube nunchucks, wemixed 40 pM of purifiednunchuck seedswith40nMofREd-SEdnanotube tiles22. ThetileswerelabeledwithCy3fluorescentdyemoleculesonthe5’end of their central strand, which is well removed from thesticky ends (Note S1). For contrast, nanotube seeds werelabeled by ATTO647N-conjugated strands hybridized to anunused region of the M13mp18 scaffold that emerges fromthe middle of the seed wall, well removed from both thelinker-presenting and the nanotube-nucleating facets (NoteS6). To prevent continued nanotube nucleation and growthduring room-temperature imaging, we added guard strandsthatremovetheshort,sticky-endbearingstrands(yellowandgreen, Fig. 1a) from freeDAE-E tiles andnanotubeends.22, 24Guard strands thus inhibit new nanotube growth withoutdestabilizing existing nanotubes during the imaging period(NoteS5).Fluorescence micrographs showed that two nanotubes grewfrommany of the nunchuck seeds (Fig. 3a-b,d). AFM imagesconfirmed that the two nanotubes in a nunchuck wereconnectedbynunchuckseeds(Fig.3c,Fig.S16).Asobservedinpreviousstudies,8,22DAE-EtileDNAnanotubesopenedonthemicasurfaceduetoelectrostaticinteractionbetweenthemicasurfaceandDNA,whileorigamiseedsremainedintact.

Fig.2(a)AFMimageofananotubeseednunchuck.Scalebar:50nm.(b)Nanotubeseednunchuckmodel.TheentangledsinglestrandedDNArepresentstheunusedM13mp18scaffoldnotfoldedintotheseed.Itisnotdrawntoscale.(c)Histogramshowingthedistributionofanglesformedbynunchuckseedstructures(n=175)afterdepositiononamicasurface.Errorbars,generatedviabootstrapping,representonestandarddeviation.

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Whilemostseedsintheresultingmixturegrewnunchucks,weobserved very few nunchucks overall. We suspectedadsorption to the walls of the test tubes in which thenunchucks were prepared was responsible for the low finalconcentration.Wefoundthatincludingbovineserumalbumin(BSA)inourreactionsolutionsincreasedthenumberofseedsobservedinnunchuckpreparations,presumablybecausenon-specificadsorptionofseedstothewallsofthereactiontubeswasreduced(NoteS7).Atthesametime,thepresenceofBSAalso increased the number of unseeded nanotubes weobserved, possibly because BSA altered the energetics ofnanotubenucleationandgrowth.Suchalterationofnanotubekinetics can be expected as the presence of BSA likely alsoprevented tiles from adsorbing to the walls of the reactiontubes, thereby increasing theoverall concentrationof tiles insolution. Increasing the temperature at which tiles wereincubatedwithnunchuckseeds from32ºCto34ºCeffectivelysuppressedunseedednucleationwhileallowingnanotubes togrowreliablyfromnunchuckseeds.

Analysis of fluorescence micrographs taken at randomlocations showed that 50 ± 5% of nunchuck seeds formednunchucks,45±5%hadonlyoneattachednanotubeand5±2%hadnoattachednanotubes(n=175).Assumingnanotubesgrow from each facet on a nunchuck seed with equalprobability,a50%proportionoftwo-tubenunchucksimpliesaprobability fornanotubegrowthona facetofp=sqrt(0.50±0.05) = 0.71 ± 0.03. This probability is consistent with theproportion of one nanotube arm observed (2·(0.71)(0.29) =0.41), as well as the proportion of nunchuck seeds with nonanotube arms (0.292 = 0.08). Single seeds nucleatenanotubesataslightlyhigherrate(78±2%).Asmall fractionofmonomer seeds that remained after gel purification couldaccountforthisslightdifference(NoteS12).We next synthesized heterogeneous nunchucks (SeeExperimentalSection), i.e.nunchuckswithtwodifferenttypesof nanotube arms (Fig. 3e-f, Fig. S19). Having two differentsets of DNA sequences that comprise the two arms of thenunchucks allows the arms to present either differentfluorescent labels, as we demonstrate, or to have distinct

Fig.3Nanotubesgrowfrombotharmsofnunchuckseeds.(a)Fluorescencemicrographofahomogeneous(REdSEd)nanotubenunchuck.NunchuckseedsarelabeledwithATTO647N(red)andREdandSEdnanotubetileswithCy3(green).Scalebar:2μm.(b)Interpretationofthenunchuckina.Greenandbluedotsonnanotubesandseedsrepresentthefluorescentdyesusedtovisualizethestructuresandarecoloredtodistinguishthedifferentdyes.Redloopsshowtheunfoldedscaffoldregionwithhybridizedfluorescentdyestrands.(c)AFMimageofahomogeneousnanotubenunchuck.Scalebar:100nm.(d)Assemblyyields,definedasthefractionofnunchuckormonomerseedstructuresfromwhichthedesignednumberandtypeofnanotubesgrew.Yieldsweremeasuredusingfluorescentmicrographstakenatrandomlocationsonmicroscopeslides.Errorbarsareonestandarddeviation.(e)Fluorescencemicrographofaheterogeneous(REsandSEs)nunchuck.REstilesarelabeledwithATTO488(blue),SEstileswithCy3(green)andnunchuckseedswithATTO647N(red).Scalebar:2μm.(f)Interpretationofthenunchuckine.

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functionalities.Heterogeneousnunchuckseedsconsistedofalinkedpairoforigamiseedsthatwere identicaltothoseusedinconstructinghomogeneousnunchucks,exceptoneseedwasmade with adapter sequences compatible with nanotubesconstructedfromonetypeoftile,termedREs,whiletheotherseed was made with adapter sequences compatible with asecondtiletype,termedSEs(SeeNoteS1forREsandSEstilesequences). As before, the two seeds were preparedseparatelyand thenmixed to formnunchuck seedsvia linkerhybridizationandpurifiedbyagarosegelelectrophoresis.REsandSEstilesweredesignedpreviously8tohavestickyendswith minimal crosstalk. Accordingly, when REs and SEs tileswereannealed together,nonanotubesconsistingofboth tiletypeswereobserved(NoteS10).In experiments involving only REs tiles and seeds, 87±3%seedsgrewnanotubes,and inexperiments involvingonlySEstilesandseeds,90±2%ofseedsgrewnanotubes.Giventhesenucleation rates, the yield of heterogeneous nanotubenunchucks (30±4%) was significantly less than expected.However, we also found that the yields of REs and SEsnanotubes from heterogeneous nunchuck seeds were lowerthanfrommonomerseeds.WhenonlyREstileswerepresent,67±6% of heterogeneous nunchuck seeds grew nanotubesand when only SEs tiles were present, 60±5% ofheterogeneous nunchuck seeds grew nanotubes. Theserespective yields were consistent with observed yield ofheterogeneousnunchucks(NoteS12).We hypothesized that heterogeneous nunchucks grew withlower thanexpectedyieldsbecausewhen the seeds forbothtypes of nanotube are present, the adapter tiles thatdistinguish the nucleating facets for the two types ofnanotubesmaydynamicallyrearrange.Asaresult,somefacetsmay present a mixture of binding sites for the two types ofnanotubes(Fig.S12).Neithertypeofnanotubewouldnucleateas easily from such mixed templates. This hypothesis wassupportedbythepresenceofnunchuckswithtwoarmsofthesamenanotubetype(4%oftheobservedstructures).To directly test whether dynamic “switching” of adapterstrandswasresponsibleforthelowyield,wecharacterizedtheproportionofREsnanotubesthatgrewfrommonomericseedsmade with REs adapters when free SEs adapters were alsopresent. SEs adapters lowered the yield of REs nanotubes to70±4%. A similar reduction in nanotube yield for SEsnanotubes(80±2%)wasobservedinthepresenceoffreeREsadapters (Note S9). Adapter switching therefore likelyaccounts for some, but not all, of the reduced yield ofheterogeneous nunchucks over homogeneous ones. Otherpossible reasons for reduced yield include incomplete linkerhybridizationduringtheannealingprocess,whichwouldmakenunchuckseedssusceptibletodissociationandrecombination.A recent study25 suggests that longer incubation timesof theindividualnanotubeseedspriortoco-incubationforassemblyofthenunchuckseedstructurecouldaddressthisproblemby

minimizingkinetictrapsthatpreventhybridizationfromgoingto completion.Minor interference between the two types oftiles,REsandSEs,couldalsointerferewithnucleationkineticsand result in lower yields. While such routes to increasingnunchuckyieldsmeritexploration,yieldsof30%aresufficientfor use of individual nunchucks as mechanical magnifiers ofthebendingdynamicsoftheembeddedDNAstrand.Forotherapplications, improved yields of heterogeneous nunchucksmightbeachievedbyusingdistinctorigamiseeds foreachofthedifferentnanotubes inadesiredsuperstructure,althoughsuch an approach would increase the complexity of thenunchuck design and the cost of purchasing the componentDNAsequences.

ConclusionsThis paper demonstrates a bio-mimetic strategy forhierarchically self-assembling nanotube architectures. Theresulting structures have micron-scale arms that are readilyvisible using standard epi-fluorescence techniques. Thestrategywe used to assemble these structures could also beextended in order to form more complex arrangements ofnanotubes, such as by increasing the number of arms in thestructure. Rigid nanostructures with multiple growth sitescouldbeusedtocreatejointswithwell-definedangles.Finally,the capacity to organize nanotubes both by nucleating themandbyattachingstructuresattheirgrowthendscouldmakeitpossible to assemble qualitatively more elaboratearchitectures. To assemble such higher-order structures withhighyields, itwillbe importanttounderstandandcorrectforthe causes of decreased nucleation of nanotubes from seedcomplexesvs.nucleationfromsingleseeds.

ExperimentalsectionDNAstrandsandReagents:DNAtile,staple,linkerandadapterstrandsequencesareinthesupportinginformation.AllstrandsweresynthesizedbyIntegratedDNATechnologies,Inc.exceptM13mp18(P-107,BayouBiolabs).Tile,linkerandadapterstrandswerepurchasedPAGEpurified.ReactionswereperformedinTAEbuffer(40mMTris-Acetate,1mMEDTA,pH8.3)with12.5mMmagnesiumacetate(TAE/Mg2+buffer)unlessmentionedotherwise.TopreventadsorptionofDNAtoPCRtubes,0.15mg/mlBSAbiotin(A8549,Sigma-AldrichCo.)wasaddedduringnanotubenunchuckgrowth(SupplementaryNoteS7).

Nunchuckseedstructuresynthesis:TwosolutionseachcontainingM13mp18(10nM),staplestrands(100nM),adapterstrands(100nM)andoneofthelinkerstrands(10nM)wereseparatelyannealedfrom90°Cto20°Cat-1°C/min.Toformnunchuckseeds,thesesolutionswerecombinedinasinglethermocyclertubeat32°Cfor12hours(homogeneous)or4days(heterogeneous).Themixturewasthenrunthrougha1%agarosegelwith1.25μg/mlEthidiumBromidestainat100Vfor1hourandthedimerbandwasextractedusingaFreezeN’SqueezeDNAGelExtractionSpinColumn(Biorad).

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Growingnanotubenunchucks:TileandseedlabelingstrandsweremixedinTAE/Mg2+buffercontaining0.15mg/mlBSA-BiotintominimizeadsorptionofDNAtothePCRtubewalls.26Sampleswereheldat90°Cfor5mins,thencooledfrom90°Cto45°Cat-1°C/min,heldat45°Cfor60mins,thencooledfrom45°Cto34°Cat-0.1°C/min.Gel-purifiednunchuckseedswereaddedat40°C.Aftertheanneal,thesampleswereheldat34°Cfor15hourstoallowfornanotubegrowth.Fluorescencemicroscopy:2μlof4μMguardstrandswereaddedto20μlofannealednanotubemixtureat34°Candincubated1minutejustbeforeimaging(Fig.S8).Samplesweretransferredtoglassslides.AcooledCCDcamera(iXON3,Andor)attachedtoaninvertedmicroscope(OlympusIX71)usinga60x/1.45NAoilimmersionobjectivecapturedepi-fluorescenceimages.MultipleimagesatthesamelocationwereacquiredusingfiltersforCy3,ATTO647NandATTO488dyes,processedtocorrectforunevenilluminationandsuperimposedtoproducemulticolorimages.Toestimateyieldsusingfluorescenceimages,onaverageatleast2independentexperimentsandatotalof10imagescapturedatrandomlocationswereusedtomeasuretheassemblyyieldsdescribedinthepaper.Errorbarsinthemeasurementsrepresentonestandarddeviationoftheyieldsobservedbetweendifferentcapturedimages.AtomicForceMicroscopy:Guardstrandsweremixedwithsamplesasinfluorescencemicroscopyexperiments.SeedlabelingstrandswerenotaddedtothenanotubeseednunchucksamplesusedforAFMimaging.10μlofthesamplewasthentransferredtoafreshlycleavedmicapuckonwhichwhich100μlofTAE/Mg2+bufferhadbeenaddedandincubatedfor2mins.ThemicawasthenwashedwithTAE/Mg2+buffertoremoveexcessDNAtiles.ImageacquisitionwasperformedonaDimensionIcon(Bruker)usingScanasystmodeandSharpNitridelever(SNL10-C,Bruker)probes.ImageswereflattenedbysubtractingalinearfunctionfromeachscanlineusingNanoscopeanalysissoftware.

AcknowledgementsTheauthorswouldliketothankD.AgrawalandE.Francoforvaluablediscussionsandadviceonthemanuscript.ThisresearchhasbeensupportedbyNSFCAREERaward125387,DOEgrantDE-SC0010595(reagents,supplies),UCSBstartupfundsandprivatedonations.

AuthorcontributionsAbdulM.Mohammed,LourdesVelazquez,DeborahK.FygensonandRebeccaSchulmandesignedtheexperimentsanddidtheexperimentalanalysis.AbdulM.Mohammed,LourdesVelazquezandAllisonChisenhallconductedtheexperiments.DanielSchiffelswasinvolvedwiththeconceptionofthesystem.Alltheauthorsdiscussedtheresultsandwrotethemanuscript.

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