A Modular, Dynamic, DNA-Based Platform for Regulating Cargo Distribution and Transport between Lipid Domains - Rubio-s et al. - 2021 - U

《A Modular, Dynamic, DNA-Based Platform for Regulating Cargo Distribution and Transport between Lipid Domains - Rubio-s et al. - 2021 - U》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

pubs.acs.org/NanoLettLetterAModular,Dynamic,DNA-BasedPlatformforRegulatingCargoDistributionandTransportbetweenLipidDomainsRogerRubio-Sánchez,SimoneEizagirreBarker,MichalWalczak,PietroCicuta,andLorenzoDiMichele*CiteThis:NanoLett.2021,21,2800−2808ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Cellmembranesregulatethedistributionofbiologicalmachinerybetweenphase-separatedlipiddomainstofacilitatekeyprocessesincludingsignalingandtransport,whichareamongthelife-likefunctionalitiesthatbottom-upsyntheticbiologyaimstoreplicateinartificial-cellularsystems.Here,weintroduceamodularapproachtoprogrampartitioningofamphiphilicDNAnanostructuresincoexistinglipiddomains.Exploitingthetendencyofdifferenthydrophobic“anchors”toenrichdifferentphases,wemodulatethelateraldistributionofourdevicesbyrationallycombininghydrophobesandbychangingnanostructuresizeandtopology.WedemonstratethefunctionalityofourstrategywithabioinspiredDNAarchitecture,whichdynamicallyundergoesligand-inducedreconfigurationtomediatecargotransportbetweendomainsvialateralredistribution.Ourfindingspavethewaytonext-generationbiomimeticplatformsforsensing,transduction,andcommunicationinsyntheticcellularsystems.KEYWORDS:DNAnanotechnology,lipidphaseseparation,lipiddomains,partitioning,artificialcells,syntheticmembranes,biomimicryiologicalmembranesarehighlyheterogeneous,containingfunctionalitiesofbiologicalinterfaces.AprecisecontroloverBupto20%oftheproteincontentofacellandfeaturingthelocalmolecularmakeupofsyntheticlipidbilayersis1,2thereforehighlydesirableandanecessarysteppingstoneforhundredsofdifferentlipidspecies.Suchadegreeofcomplexityevolvedalongsidethemyriadofbiologicalthedevelopmentofevermoresophisticatedlife-likeresponsesprocesseshostedandregulatedbymembranes,whichincludeinartificialcells.signaling,adhesion,trafficking,motility,anddivision.3ManyofDNAnanotechnologyhasdemonstratedgreatpotentialasathesefunctionalitiesrelyonlateralcolocalizationofmembranemeansofcreatingresponsivenanostructuresthatemulateproteins4requiredfortheemergenceofsignalinghubs,5,6focalbiologicalarchitecturesandfunctionalitiesandarebecomingDownloadedvia119.73.118.6onMay14,2021at06:39:03(UTC).adhesions,7andassembliesregulatingmembranearchitectureincreasinglypopularconstituentsofartificialcellularsys-89tems.33,34Inmanycases,biomimeticDNAnanostructures,topromoteendo-andexocytosisandcelldivision.Cellshaveevolvedavarietyofactiveandpassiverenderedamphiphilicbyhydrophobictags,havebeenmechanismstoregulatethelocalcompositionoftheirinterfacedtosyntheticlipidmembranestoreplicatetheSeehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.membranes,overcomingtheextrememolecularheterogene-responseofcell-membranemachinery.Examplesincludeity.10−12Amongthese,proteolipidphaseseparationisthoughtDNAarchitecturesmediatingartificialcelladhesionandtissue35−4142,43toplayanimportantroleinsignalingandsignaltransduction.13formation,regulatingtransport,enablingsignaltrans-4445,46duction,andtailoringmembranecurvature.Inthisprocess,nanoscaledomainsorraftsarebelievedtoImportantly,amphiphilicDNAnanostructureshavebeenemerge,richinsphingomyelinsandsterols,whichareabletodemonstratedtoundergopreferentialpartitioningwhenrecruitorexcludemembraneproteinsbasedontheirdifferent14,15anchoredtophase-separatedsyntheticbilayers,aneffectthataffinitiesforraftandnon-raftenvironments.47−49isreminiscentofmembrane-proteinpartitioninginrafts.Bottom-upsyntheticbiologyaimsatreplicatingfunction-ThepreferenceofDNAnanostructuresfordifferentlipidalitiestypicallyassociatedwithbiologicalcellsinmicrorobots16−18phasesandtheirdegreeofpartitioninghavebeenshowntodesigneddenovoor“artificialcells”.Justliketheirbiologicalcounterparts,manyartificial-celldesignsrelyonsemipermeablemembranesfortheircompartmentalizationReceived:December10,2020requirements,19−21whichcanbeconstructedfrompolymers22Revised:March3,2021andproteopolymersystems,23,24colloids25,26and,moreoften,Published:March18,202121syntheticlipidbilayers.However,withsomeremarkable27−31exceptions,reviewedinref32,themembranesofartificialcellsareoftenpassiveenclosures,lackingthecomplex©2021TheAuthors.PublishedbyAmericanChemicalSocietyhttps://doi.org/10.1021/acs.nanolett.0c048672800NanoLett.2021,21,2800−2808

1NanoLetterspubs.acs.org/NanoLettLetterdependonthechemicalidentityofthehydrophobicanchors,membranelipidcomposition,temperature,andsolvent40,50conditions.Forinstance,internarymodelmembranesof1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)/1,2-dipal-mitoyl-sn-glycero-3-phosphocholine(DPPC)/cholesteroldis-playingcoexistenceofliquidordered(Lo)andliquid1,51,52disordered(Ld)phases,DNAconstructsbearingasinglecholesteryl-tri(ethyleneglycol)(TEG)anchorshowaweakpreferenceforLo,whileduplexesend-functionalizedwithtwo53,54cholesteryl-TEGanchorspartitioninLomoreprominently.Conversely,oligonucleotidesfunctionalizedwithα-tocopherolpreferentiallylocalizewithintheLdphasesofternary1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/55,56sphingomyelin(SM)/cholesterolmembranes.Moreover,dynamiccontroloverthepartitioninghasalsobeenexemplifiedwithDNAnanodevicesthatredistributebetween57lipidphasesfollowingenzymaticcleavageorchangesinionic58strength.Theabilitytoinfluencepartitioningofmembrane-anchoredDNAconstructsmakesthempromisingtoolsforengineeringFigure1.Membrane-anchoredDNAnanostructuresdisplayprefer-themolecularmakeupandfunctionalityofartificialcellentialpartitioningbetweenthephasesofde-mixedgiantunilamellarmembranes.Yet,wehaveeffectivelyonlystartedtoscratchvesicles.(left)SchematicrepresentationofamulticomponentgiantthesurfaceofthemassivedesignspaceofamphiphilicDNAunilamellarvesicle(GUV),preparedwithsaturatedandunsaturatednanostructuresthat,alongsidethechemicalidentityofthelipidsmixedwithsterols,displayingliquid-ordered(Lo)andliquid-hydrophobes,canbefreelyengineeredwithrespecttotheirdisordered(Ld)phasecoexistence.TheouterleafletofthemembraneisfunctionalizedwithamphiphilicDNAnanostructures,exemplifiedsize,topology,andstimuliresponsiveness.herewithconstructsbearingtwocholesterolanchors,whichenrichHereweintroduceamodularplatformthatfullyexploitsthetheirpreferred(Lo)phase.(right)3Dviewofade-mixedGUVfromdesignversatilityofDNAnanotechnologytoconstruct59confocalz-stackasobtainedfromVolumeviewer(FIJI)usingnanostructureswhoseabilitytopartitioninthedomainsofmaximumprojection.DNAnanostructures(cyan)partitiontotheLophase-separatedsyntheticmembranescanbepreciselydomain,whiletheLdphase(red)islabeledwithTexasRed-DHPE.programmed.Besidesenablingstaticmembranepatterning,Scalebar=10μm.thenanodevicescanbedynamicallyreconfigureduponexposuretomolecularcues,triggeringredistributionbetweenpartitioningtendency.Försterresonanceenergytransferdomainsandunlockingsyntheticpathwaysforcargotransport,(FRET)betweenAlexa488ontheDNAandTexasRedonsignalingandmorphologicaladaptationinsyntheticcellularthelipids,alongsidefluorescent-signalcross-talk,couldinsystems.principlebiasfp,Lincertainpartitioningstates.ToruleoutthisoOurnanostructureswereconstructedfromsyntheticDNApossibility,weperformeddedicatedexperimentstodetermineoligonucleotides,someofwhichwerecovalentlylinkedtotheimpactofbothpotentialartifacts,aswellascontrolsonhydrophobicmoietiestomediateanchoringtothemembrane.GUVsthatlacktheTexasRedfluorophore,thusaltogetherFluorescenttags(Alexa488,unlessstatedotherwise)werealsoeliminatingthepossiblesourceofbias.DatainFigureS3,andincludedtomonitorthedistributionofthenanostructuresviatheassociatedSupplementaryDiscussion1,confirmthatFRETconfocalmicroscopy.AsdepictedinFigure1(left),weandfluorescencecross-talkcarryanegligibleimpactonfp,L.decoratedtheouterleafletofphase-separatedgiantunilamellarovesicles(GUVs)withtheDNAconstructs.UnlessspecifiedAssumingthattherecordedfluorescenceintensitiesareproportionaltonanostructureconcentrations,apartitioningotherwise,GUVswerepreparedfromternarylipidmixtures(DOPC/DPPC/cholestanol)todisplayLd−Locoexistence.TofreeenergycanbecalculatedasΔGp,Lo=−kBTlog(fp,Lo/fp,Ld).enablefluorescenceimaging,thevesiclesweredopedat0.8%ThelatterisdefinedasthefreeenergychangeassociatedwithmolarratiowithTexasRed-DHPE,whichpreferentiallylabelsrelocatingasingleconstructfromtheLdtotheLophase.theLdphase.Atroomtemperature,theGUVsreadilyde-mixedFirst,weappliedourdata-analysispipelineonsimpleDNAintotwomacroscopicLoandLddomains.TheresultingJanus-duplexesfeaturingwellcharacterizedsinglecholesterol-TEGlikemorphology,showninFigure1(right),enabledthefacile(sC)orsingletocopherol(sT)anchors,assummarizedinvisualizationandquantitationofthelateraldistributionoftheFigure2.Expectedly,whilesCanchorsledtoamarginalLo-preference(fsC≈0.6,ΔGsC≈−0.4kT),nanostructuresnanostructures.p,Lop,LoBTothisend,equatorialconfocalmicrographsoftheGUVsfeaturingsTstronglypartitionedinL(fsT≈0.14,ΔGsT≈dp,Lop,Lowerecollectedandanalyzedwithacustom-builtimage1.9kBT).processingpipeline,asdescribedindetailintheSI(seeImportantly,ashighlightedinFigureS4,thesizeMethods,andtheassociatedFiguresS1andS2),whichheterogeneityofelectroformedGUVsdoesnotaffectthedeterminedtheaveragefluorescenceintensitiesofthelateraldistributionofDNAanchoredspecies,asfp,Ldoesnotconstructsinthetwophases:IandI.Fromthesevalues,oLoLdcorrelatewithvesicleradius.fractionalintensitiesfp,Lo(Ld)=ILo(Ld)/(ILo+ILd)wereextracted.ThesubstantialdifferenceinthepartitioningbehaviorsThroughoutthisletter,werefertothefractionalfluorescenceinducedbysTandsCtracesaroutetoprogramlateralintensityofnanostructuresintheLophase,fp,Lo,togaugetheirdistributionbycombiningmultipleordifferenthydrophobic2801https://doi.org/10.1021/acs.nanolett.0c04867NanoLett.2021,21,2800−2808

2NanoLetterspubs.acs.org/NanoLettLetterFigure2.LateraldistributionofDNAnanostructuresdependsonanchorchemistryandcombination.(a)Fractionalintensity(fp,L)andfreeoenergyofpartitioning(ΔGp,Lo)ofDNAnanostructuresinLo,conveyedbybox-scatterplotsandlozengesrespectively,fortwoindividualrepeats(inblue/gray)ofDNA-decoratedGUVpopulationsfeaturingdifferentanchoringmotifs:singlecholesterol-TEG(sC),doublecholesterol-TEG(dC),singletocopherol(sT),oracombinationofsinglecholesterol-TEGandsingletocopherol(sC+sT).FordCandsC+sT,squaresindicatethepredictedfreeenergyvaluesforcombinedanchors,determinedfromeq1usingmeasuredvaluesofΔGsTandΔGsC.Thedottedlineindicatesnop,Lop,Lopartitioning(fp,Lo=0.5,ΔGp,Lo=0).(b)SchematicdepictionsofDNAduplexesbearingsC,sT,dC,orsC+sT.(c)RepresentativeconfocalmicrographsoftheDNA-functionalizedGUVs.TheLdphasewaslabeledwithTexasRed(red),andDNAconstructs(cyan)werelabeledwithAlexa488.Scalebars=10μm.moietieswithinthesamenanostructure.Withthismodularcase,anchorco-operativityandothernonadditivecontribu-“mixandmatch”approach,wecanindeedthinkofreinforcingtionsnegligiblyaffectpartitioning.partitioningineitherlipidphasebyincreasingthenumberofWhentesting“chimeric”duplexes,bearingatocopherolandcholesterolortocopherolmoietiesonournanodevices,ortoacholesterolanchor(sC+sT),weobservedapronouncedaccessintermediatepartitioningstateswithnanostructurespreferenceforLd,consistentwiththeexpectationthatfeaturingbothmoieties.tocopherolshoulddominateinviewofthestrongerfreeIntheabsenceof(anti)co-operativeeffectsandatenergyshiftassociatedwithitspartitioning(Figure2,fsC+sT≈p,Losufficientlylowconstructconcentrations,thepartitioningfree0.3).Here,thefreeenergypredictionfromeq1slightlyenergyofananostructurefeaturingmultiplehydrophobicoverestimatedthemeasuredvalueofΔGsC+sT≈0.9kTmoietiesshouldbeadditiveinthecontributionsfromp,LoB(statisticallysignificantdifference,p=8.5×10−11,usingtheindividualanchors:nonparametricMann−WhitneyWilconsonTest).Sincethis∑inonadditivebehaviorwasnotobservedforthedCconstruct,ΔGGp,Loo=Δp,Li(1)wearguethatitmayresultfromthedistinctchemicalnatureoftheanchorsinsC+sTandtheconsequentdifferencesintheirwheretheindexirunsoveralltheanchorsintheconstruct.interactionswiththesurroundinglipids.Thesimplerelationshipineq1canguidethedesignofTofurtherchallengeourmodulardesignapproach,wemultianchormotifs,andinFigure2,wetesteditonaduplexstudiedthepartitioningbehavioroftheconstructsinFigure2featuringtwocholesterol-TEGanchors(dC).Asexpected,theinaquaternarylipidmixture(DOPC/DPPC/cholestanol/dCmotifdisplayedanenhancedpreferenceforLodomainscardiolipin).Whilethismorecomplexmixturestilldisplayed40,53dCcomparedtosC,withfp,L≈0.7.ThemeasuredoLo−Ldphasecoexistence,theincorporationofthehighlypartitioningfreeenergywasΔGdC≈−0.8kT,nearlyidenticalp,LoBunsaturatedcardiolipinhasbeenshowntoenhanceLo-totwicethatofsCnanostructures.Thisquantitativeagreementpartitioning,owingtotheincreasedlateralstressintheLd40withthepredictionofeq1suggeststhat,atleastinthisspecificphase.Thedata,summarizedinFigureS5,largelyconfirmed2802https://doi.org/10.1021/acs.nanolett.0c04867NanoLett.2021,21,2800−2808

3NanoLetterspubs.acs.org/NanoLettLetterFigure3.AnchorcouplingandconstructtopologymodulateDNAnanostructurepartitioning.(a,top)TunablelateralsegregationofDNAarchitecturesattainedbycouplingtwosetsofanchors:dC+dCandsT+dC.Thepartitioningbehaviorisdemonstratedbythefractionalintensity(fp,Lo,box-scatterplots)andfreeenergyofpartitioning(ΔGp,Lo,lozenges)inLo.Squaresindicatethepredictedfreeenergyvaluesforcombinedanchors,determinedfromeq1usingmeasuredvaluesofΔGsTandΔGsC(Figure2).(a,bottom)Schematicrepresentationsofthenanostructuresp,Lop,LoandrepresentativeconfocalmicrographsofdecoratedGUVs.(b,top)Effectofnanostructuresizeonpartitioning,shownviafp,Lbox-scatterplotsoofDNAnanostarsbearingsingletocopherol(sT)ordouble-cholesterol(dC)anchors,comparedagainstthemeanfp,Lachievedbytheirduplexocounterparts(dashedlines,redfordC,blueforsT).(b,bottom)DepictionofDNAnanostarsalongsiderepresentativeconfocalmicrographsofDNA-decoratedGUVs.Inplotsinbothpanelsaandb,thedottedlineindicatesnopartitioning(fp,Lo=0.5,ΔGp,Lo=0).TheLdphasewaslabeledwithTexasRed(red),andconstructs(cyan)werelabeledwithAlexa488(DNA−DNAcomplexes)orfluorescein(nanostars).Scalebars=10μm.thepredictivepowerofeq1,butsomenonadditivedeviations+dCconstruct,asexpected,displaysaverystrongLoarehighlighted.Specifically,weobservedthatapplyingtherulepreference,withΔGdC+dC≈−1.4kTandfdC+dC≈0.8.p,LoBp,Loineq1ledtoanoverestimationofthenanostructures’SimilarlytothecaseofthesC+sTconstructs,thepartitioningtendencytolocalizewithintheLoforbothdCandsC+sTofsT+dCnanostructureswasdominatedbythestrongLdmotifs.Thedifferenceinmagnitudebetweennonadditivefreepreferenceofthetocopherol,withΔGsT+dC≈1kTandfsT+dCp,LoBp,Loenergytermsobservedforternaryandquaternarylipid≈0.26.compositionsisonlypartiallysurprising,asonewouldexpectDespitetheremarkableaccuracyofeq1inpredicting(anti)co-operativeeffectstobehighlysensitivetothelipidpartitioningstatesinmultianchorconstructs,wehighlightedmicroenvironmentoftheanchors.Forinstance,onemaynonadditivedeviations.Whilesomeoftheseappeartospeculatethatthecardiolipin-richLdphaseinthequaternarycorrelatewiththedetailsofanchorandlipidchemistry(sC-mixturemaybebetterabletoaccommodatelargerinclusionssTinFigure2andFigureS5)andarethusdifficulttocontrol,likethosegeneratedbytwo-anchormotifs(dC,sC+sT),whichothernonadditivecontributionscanpotentiallybeexploitedto40mayinturnhelptorelaxthebuilt-inlateralstress.Thisfine-tunepartitioningstatesaroundthebaselinedefinedbyphenomenonmayleadtoalesspronouncedLopreferenceforanchorcombination.Forinstance,theuseoflargerandmoretwo-anchorcomparedtosingle-anchorconstructs.complexnanostructuresmayinfluencelateralsegregationThankstothedesignfreedomofDNAnanotechnology,ourowingtostericorelectrostaticinteractionsbetweenthemotifs.modularstrategyisnotrestrictedtosimplemotifswithoneorOnemayindeedexpectthat,forlargernanostructures,twoanchors.WecanindeedregardtheduplexconstructsinexcludedvolumeeffectsmayhinderaccumulationinoneFigure2as“anchoringmodules”andfurthercombinetheminspecificphase,thussuppressingpartitioning.Figure3blargernanostructurestoexpandtherangeofaccessiblesummarizesthepartitioningbehaviorofthree-pointedDNApartitioningstates.Forinstance,asschematicallydepictedinnanostarsanchoredtothebilayersusingdCandsTmodules.Figure3,twoanchoringmoduleswerecoupledbysimplyThesemotifshadroughly4×themolecularweightoftheconnectingthemwithatransversal,fluorescentlylabeledlinkersmallerduplexarchitecturesinFigure2and,indeed,duplex.Foraddedconformationalflexibility,3-ntsingle-systematicallydisplayedareducedpartitioningtendency.stranded(ss)DNAdomains(αinFigure3)wereincludedMoreover,FigureS6provesthattheeffectisnotuniquetobetweenthehydrophobicallymodifiedandlinkerduplexes.thebranchednanostructures,aslinearduplexesanchoredviaTwomodularcombinationswereinvestigated:dC+dCanddCalsoshowedageneralweakeninginpartitioningwithsT+dC.Notably,forbothdesigns,themeasuredpartitioningincreasinglength.Infurthersupporttothehypothesisthatfreeenergiescouldbequantitativelypredictedbyaddingupstericnanostructure−nanostructureinteractionsmayhaveanthecontributionsoftheindividualanchormodules.ThedCeffectonpartitioning,weperformedmeasurementsforsmaller2803https://doi.org/10.1021/acs.nanolett.0c04867NanoLett.2021,21,2800−2808

4NanoLetterspubs.acs.org/NanoLettLetterFigure4.Toehold-mediatedstranddisplacementenablescargotransportbetweenlipiddomainsvialateralredistributiontoprogrammedpartitionedstates:(a)SchematicdepictionofthemechanismunderpinningDNAnanostructuredimerizationandcargoredistributioninducedbytheadditionofFuel/Antifuelstrands,whichexploitdomainsδ1,α1,δ2,andα2astoeholds.Thecorrectresponseofthemolecularcircuitwastestedwithagarosegelelectrophoresis,assummarizedinFigureS8.(b)EvolutionofthepartitioningtendencyofthefluorescentDNAelement(panela),conveyedbyfp,Lbox-scatterplots,afteradelaytimeforequilibrationupontheadditionofFuel/Antifuelstrands.Thelightbluedashedlinemarksothemeanfp,LofthecargohybridizedtothesT-bearingmoduleandintheabsenceofthedC-anchoringmodule,whilethereddash-dotlinemarksothatofthecargohybridizedtothedC-bearingmoduleandintheabsenceofthesTanchoringmodule.Thedottedlineindicatesnopartitioning(fp,Lo=0.5,ΔGp,Lo=0).(c)RepresentativeconfocalmicrographsofDNA-functionalizedGUVsovertime,showcasingthedistinctpartitionedstatesattainedbythesystem.TheLdphasewaslabeledwithTexasRed(red),andDNAconstructswerelabeledwithAlexa488(cyan).Scalebars=10μm.dCandsTduplexesoverawiderangeof(nominal)DNA-to-largerconcentrationofsolubilizedconstructs,effectivelylipidmolarratiosandthusofsurfacedensitiesofthemotifs,asreducingthesurfacedensityatfixedDNA/lipidratios.summarizedinFigureS7.Weobservedanearstationaryfp,LOurabilitytoprogramthedomainpartitioningofDNAoconstructsbylinkingdifferentanchoringmodules,asoverabroadrangeofDNA/lipidratiosaroundthevalueuseddemonstratedinFigure3,canbecombinedwiththedynamicforallotherexperimentsthroughoutthiswork(∼4×10−4).reconfigurabilityofDNAnanostructurestoreversiblytriggerForbothdCandsTconstructs,however,fp,LostronglydeviatedredistributionofafluorescentcargowithintheGUVs’surfacesathighDNA/lipidratios,approaching∼0.5.Suchadeviationuponexposuretomolecularcues.WedemonstratethiseffecthintsatasaturationoftheLoandLdphasesandthewithananostructurefeaturingbothdCandsTanchoringmodules,similartothatshowninFigure3butinwhichtheconsequentimpossibilityforthenanostructurestofurtherfluorescentdsDNAlinkermodule(cargo)connectingtheaccumulateinthosedomains.SaturationoccurredathigheranchorduplexescanreversiblybindtoordetachfromeitherDNA/lipidratiosforsTcomparedtodC,andwearguethat61viatoehold-mediatedstranddisplacement,amechanismthatthisdifferencemayarisefromdifferencesintheoverallisreminiscentoftwo-componentbiologicalreceptorsunder-membraneaffinityoftheanchors.Indeed,whiledCmembranegoingligand-induceddimerization.5,6AssketchedinFigure4a,60insertioniseffectivelyirreversible,anchoringviasTmaybeweinitiatedoursystemfromaconfigurationinwhichtheweakersothatmembrane-anchoredsTduplexescoexistwithafluorescentcargowasattachedtothesTanchorandthus2804https://doi.org/10.1021/acs.nanolett.0c04867NanoLett.2021,21,2800−2808

5NanoLetterspubs.acs.org/NanoLettLetterFigure5.Staticanddynamicengineeringofpartitioningstatesacrossthefree-energylandscapebyprescribinganchorcombinationandnanostructuremorphology.(a)Summarygraphdemonstratingthemeanfp,L±standarddeviationsofseveralmembrane-associatedDNAonanostructuresasafunctionoftheirmeasuredfreeenergyofpartitioning(ΔGp,L).Theeffectofanchorcoupling,size,andgeometryisshowcased.oArrowsconnectprogrammedpartitionedstatesachievedbytheresponsivenanodevicediscussedinFigure4.(b)PartitioningfreeenergyofDNAnanostructuresistoagoodapproximationadditiveincontributionsfromindividualanchoringmotifs,asshownforseveralmultianchornanostructureswiththemean±standarddeviationsofmeasuredΔGp,Lvsthosepredictedwitheq1.olocalizedintheLdphase(State1).Here,thefluorescentlinkerlikelylimitedbythediffusionofthefuelstrandthroughthemodulewaspreventedfromconnectingtothedCanchoringsample,giventhat,inordertopreventGUVdriftandmoduleasitsdomainγ*1,complementarytoγ1onthedCdisruption,thefuelsolutionisgentlyaddedfromthesamplemodule,wasprotectedbyanAntifuel1strandofdomainsurfacewithoutanyactivemixing.Theratesoftoehold-sequenceδ1γ1α*1.Notethatthefp,Lovaluerecordedinthismediatedstranddisplacementanddiffusionofthemembrane-configurationmatchedexactlytheonemeasuredifthedCanchoredconstructsareexpectedtobecomparativelyfastermodulewasabsentfromthebilayer,markedbyadashedline(seediscussioninthecaptionofFigureS9).TheredistributioninFigure4b,thusconfirmingtheabsenceofbindingtothedCrecordedforournanodevicesiscomparabletothoseofmodule.Antifuel1couldbedisplaceduponadditionofFuel1,biologicalmembrane-anchoredagentsinvolved,forexample,in63leadingtotheexposureofγ*1onthelinkermoduleanditstherecruitmentofreceptorsuponT-cellactivationor8,64bindingtothedCmodule.Uponacquiringthisconfiguration,clathrin-mediatedendocytosis,bothofwhichrangebetweentensandhundredsofseconds.Finally,notethatState2,thenanostructuresshiftedthecargotowardLo.AdditionofAntifuel2,withsequenceα*2γ2δ2,triggeredtheStates1and3displayedalowertendencytopartitioninLddisplacementofthelinkerfromthesTmodulefollowingaandLo(respectively)comparedtotheirsTanddCduplexanalogues(Figure2).Theshiftislikelyaconsequenceofthetoeholdingreactioninitiatedatdomainα2,leadingtothegreaterstericencumbranceoftheresponsivemotifs,discussedemergenceofState3andafurthercargoredistributiontowardabovewithrespecttoFigure3andFigureS6.theLophase.Alsointhisconfiguration,thefp,LovaluealignedInsummary,weintroducedamodularapproachtoengineertothatmeasuredintheabsenceofsTanchors,indicatingathelateraldistributionofamphiphilicDNAnanostructuresnear-completeprogressionofthetoeholdingreaction(dot-betweencoexistingphasesofsyntheticmembranes.WedashlineinFigure4b).Finally,sequentialadditionofFuel2exploitedtheabilityofindividualcholesterolandtocopherolandAntifuel1couldreversethesystems’configurationtoStatesanchorstoinducepartitioninginLoandLd,respectively,and2andthen1.Thelasttwostepspushedthefluorescentcargocombinedthemtoproduceanarrayofmultianchornano-backtowarditsinitialLdpreference,butthefp,Lovaluesdevicesthatspanabroadrangeofpartitioningbehaviorsfromremainedslightlyhigherthanthoserecordedatfirst.This∼80%preferenceforLoto∼85%partitioninginLd,asincompletereversibilitymaybeduetopartialinefficienciesinsummarizedinFigure5a.Thecomparisonbetweenmeasured62thereversetoeholdingreactionsortoasmallthermodynamicpartitioningfreeenergiesandthoseextractedfromeq1,shownunbalancethatfavorsState3overState1.NotethatthesysteminFigure5b,provesthattoagoodapproximationΔGp,Lisowasallowedtofullyequilibrateaftertheadditionoffuel/additiveinthecontributionsfromindividualanchors,thusantifuelstrandspriortocollectingthedatainFigure4,asofferingapredictivedesigncriterion.Small,nonadditiveeffectsdemonstratedbythemeasurementsacquiredatintermediatecontributetoadifferentextentdependingonanchortimepointsandshowninFigureS9a.Inturn,FigureS9bcombinationsandlipid-membranecomposition,hintingatshowsthetimeevolutionoffp,LoforanindividualGUV,in(anti)co-operativeeffectsdependentonsystem-specificwhichthenanostructurestransitionfromState1toState2chemistry,whileexcluded-volumeeffectsemergedforbulkieruponfueladdition.Theequilibrationtimescalesof∼5minaremotifsandhighernanostructureconcentrations.2805https://doi.org/10.1021/acs.nanolett.0c04867NanoLett.2021,21,2800−2808

6NanoLetterspubs.acs.org/NanoLettLetterThemodularityofourdesignstrategyenablesdynamicW120BZ,UnitedKingdom;orcid.org/0000-0002-1458-controloverthepartitioningstatebyalteringtheanchor9747;Email:l.di-michele@imperial.ac.ukmakeupofthenanostructures,asweshowedwithaproof-of-conceptexperimentinwhichfluorescentcargoeswereAuthorsreversiblytransportedacrossvesiclesurfacesuponexposureRogerRubio-Sánchez−BiologicalandSoftSystems,tomolecularcues.ThisstrategyevokesthatusedbycellstoCavendishLaboratory,UniversityofCambridge,Cambridgecontrolspatiotemporallocalizationofmembranepro-CB30HE,UnitedKingdom;orcid.org/0000-0001-5574-teins47−49,65andcanbefurtherextendedtorespondtoother5809physico-chemicaltriggersbyincludingstimuli-sensitivemotifsSimoneEizagirreBarker−BiologicalandSoftSystems,suchasaptamers,66DNAzymes,67G-quadruplexes,68pH-CavendishLaboratory,UniversityofCambridge,Cambridgeresponsivemotifs,69andotherfunctionalDNAarchitec-CB30HE,UnitedKingdom;orcid.org/0000-0001-8920-tures.70,710428OurapproachcouldbeeasilyextendedtoincludemoietiesMichalWalczak−BiologicalandSoftSystems,Cavendishotherthancholesterolandtocopherol,suchasalkylchains,40Laboratory,UniversityofCambridge,CambridgeCB30HE,porphyrin,72andazobenzene,73whichbesidesunlockingaUnitedKingdombroaderrangeofpartitioningstatesmayalsoenablePietroCicuta−BiologicalandSoftSystems,Cavendishresponsivenesstoawiderspectrumofstimuli.SimilardesignLaboratory,UniversityofCambridge,CambridgeCB30HE,principlescouldevenbeappliedtomembrane-associatedUnitedKingdom;orcid.org/0000-0002-9193-8496entitiesdifferentfrom(amphiphilic)DNAnanostructures,Completecontactinformationisavailableat:suchasperipheralandintegralproteins,toprogramtheirhttps://pubs.acs.org/10.1021/acs.nanolett.0c0486774,75colocalizationinmembranedomains.Ourplatformpavesthewayforthedevelopmentofnext-NotesgenerationbiomimeticDNAdevicesforthebottom-upTheauthorsdeclarenocompetingfinancialinterest.engineeringoflife-likebehaviorsinsyntheticcellularsystems,whichcouldinthelongtermfindapplicationinhigh-tech■ACKNOWLEDGMENTStherapeuticsanddiagnosticsolutions.Forinstance,thestimuli-R.R.S.acknowledgessupportfromtheMexicanNationaltriggeredreshufflingofmembrane-boundobjectsbetweenlipidCouncilforScienceandTechnology(CONACYT,GrantNo.phasescanenablehighlysoughtbehaviorssuchassignal472427)andtheCambridgeTrust.R.R.S.andS.E.B.also45transduction,communication,andlocalmembranesculpting.acknowledgefundingfromtheEPSRCCDTinNanoscienceExamplesincludestimuli-inducedcolocalizationofsyntheticandNanotechnology(NanoDTC,GrantNo.EP/L015978/144receptorsinitiatingartificialsignalingcascadesandherdingofandEP/S022953/1,respectively).L.D.M.acknowledgesobjectscapableofinfluencinglocalmembranecurvature,thusfundingfromaRoyalSocietyUniversityResearchFellowship76directingmorphologicalrestructuringsuchastubulargrowth(UF160152)andfromtheEuropeanResearchCouncil(ERC)46,77,78andexosome/endosomebudding.undertheHorizon2020ResearchandInnovationProgramme(ERC-STGNo851667NANOCELL).TheauthorsthankB.■ASSOCIATEDCONTENTM.Mognettifordiscussionsontheproject,andD.Morzyfor*sıSupportingInformationcommentsonthemanuscript.AlldatainsupportofthisworkTheSupportingInformationisavailablefreeofchargeatcanbeaccessedfreeofchargeathttps://doi.org/10.17863/https://pubs.acs.org/doi/10.1021/acs.nanolett.0c04867.CAM.64579.Experimentalmethods,discussionofimpactoffluo-■rescencecross-talkandFRET,imageanalysispipelineREFERENCESformeasuringDNA-constructfluorescenceintensityin(1)Heberle,F.A.;Feigenson,G.W.PhaseSeparationinLipidequatorialconfocalmicrographs,circlefittingroutinetoMembranes.ColdSpringHarborPerspectivesinBiology2011,3,No.a004630.correctforimperfectsegmentationoflowintensity(2)Lorent,J.H.;Levental,K.R.;Ganesan,L.;Rivera-Longsworth,equatorialmembranedomains,impactoffluorescentG.;Sezgin,E.;Doktorova,M.;Lyman,E.;Levental,I.PlasmalipidmarkerandGUVpolydisperityonpartitioningmembranesareasymmetricinlipidunsaturation,packingandproteintendency,lateraldistributionofduplexDNAnanostruc-shape.Nat.Chem.Biol.2020,16,644−652.turesinquaternary(DOPC/DPPC/cardiolipin/chol)(3)Alberts,B.Molecularbiologyofthecell,6thed.;GarlandyScience:GUVs,lateraldistributionofDNAnanostructuresofNewYork,2017.increasingmolecularweight,suppressionofpartitioning(4)Sezgin,E.;Levental,I.;Mayor,S.;Eggeling,C.ThemysteryofbyhighDNA/lipidratioduetomembranesaturation,membraneorganization:composition,regulationandrolesoflipidDNAnanostructurereconfigurabilityviatoeholdingrafts.Nat.Rev.Mol.CellBiol.2017,18,361−374.reactions,DNAnanostructurereconfigurationand(5)Akira,S.;Takeda,K.Toll-likereceptorsignalling.Nat.Rev.Immunol.2004,4,499.redistribution,andDNAsequencesofthenanostruc-(6)Schlessinger,J.ReceptorTyrosineKinases:LegacyoftheFirstturesusedthroughoutthiswork(PDF)TwoDecades.ColdSpringHarborPerspect.Biol.2014,6,No.a008912.■(7)Acebrón,I.;etal.StructuralbasisofFocalAdhesionKinaseAUTHORINFORMATIONactivationonlipidmembranes.EMBOJournal2020,39,No.e104743.CorrespondingAuthor(8)Haucke,V.;Kozlov,M.M.Membraneremodelinginclathrin-LorenzoDiMichele−Biologicalan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