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SupplementaryInformation

Single-MoleculeDNAOrigamiAptasensorsforReal-timeBiomarkerDetection

KeitelCervantes-Salguero,aMarkFreeley,aJorgeL.Chávez,b,*andMatteoPalmaa,*

a. SchoolofBiologicalandChemicalSciencesandMaterialsResearchInstitute,QueenMaryUniversityofLondon,London,UnitedKingdom.b. AirForceResearchLaboratory,711thHumanPerformanceWing,WrightPattersonAirForceBase,Dayton,Ohio,UnitedStates.Materials:AlloligonucleotidesandmodifiedoligonucleotideswerepurchasedfromIntegratedDNATechnologies(IDT)withtheexceptionoftheATTO655modifiedsequencewhichwaspurchasedfromEurofins.M13mp18viralDNAwaspurchasedfrom Tilibit. Neutravidin and DPBS and TAE buffers were purchased from ThermoFisher. BSA-biotin and cortisol werepurchasedfromMerck.MgCl2andNaClwerepurchasedfromFisherScientific.Time-dependentfluorescenceexperiments:ExperimentswereperformedonaCaryEclipseFluorescenceSpectrophotometer.FortheATTO488dyeandBHQ-1blackquencher,theexcitationandemissionweresetat503nmandat522nm,respectively.Slitsforexcitationandemissionweresetat5nm.Datawasobtainedevery10.2sec.ForcortisoldetectioninFigure1B,90nMATTO488-labelledaptamerin300μlof1xDPBSwasintroducedandallowedtoequilibrate.Then,thequencher(BHQ-1)wasaddedatafinalconcentrationof100nM(blackarrow),mixedandletequilibrate.Finally,1μlof3mMcortisolinethanolwasaddedto300μl1xDPBSsolution,whichwasaddedandmixedbypipetting.Fortheorangecurve:1μlethanolwasaddedandmixedbypipetting.Itisimportanttonotethattheduplexedaptamersystemisverysensitivetomechanicalperturbationsandsolutionchanges,i.e.thatevensimplesolutionmixingwithpipetteledtoduplexdehybridisationasshownbysmallchangesinintensityinFigure1Bat6min,36minand63min,althoughthesewouldquicklyre-equilibrate.Moreover,excessiveadditionsofethanolinthecontrolexperimentledtoasignificantintensitychangesimilartocortisoldetection;therefore,welimitedtheamountofethanolinsolutiontoamaximumof0.3%v/vinthetotalbuffersolution.Experimentsinsolutionatotherionicconcentrations:Inordertounderstandtheduplexedaptasensor’sdynamicnature,weperformedaseriesofexperimentsinsolutionatdifferentionicstrengthsat25°C.Weevaluatedtheeffectofionicstrengthatintermediateconcentrationsbetween1xDPBSand1xDPBS/1.5MNaClandat1×TAE/MgCl2(40mMTris,20mMaceticacid, 2 mM EDTA, 12.5 mMMgCl2) (see Figure S1). As 1x DPBS buffers contains ~150 mM NaCl, additional NaCl wassupplementedtoreachfinaltestingconcentrations.Forallcurves:theATTO488-labeledaptamerinbufferwasintroducedandallowedtoequilibrate.Then,thequencherwasaddedafter10min,mixedbypipettingandincubatedformorethan1hour.Finally,cortisol(10μM)wasadded(blackarrow)andmixedbypipetting,resultinginanaptamertocortisolratioof1:111. Experiments at all tested salt concentrations show formationof thedye/quencherduplex,despitepartial duplexdehybridisationbeforetheadditionofcortisol,asseenfromtheslowincreaseinfluorescenceoverlongperiods(seeschemeinFigure1Ainmaintext).At1×TAE/MgCl2,hybridisationiscompletewithin5minfollowedbyadehybridisationfasterthaninthecaseof1xDPBS/1.5MNaCl.In1xDPBS,afteraddingthequencher,hybridisationtakesatleast5minmorethantherestoftestedcasesbutdehybridisationhasasimilarrate.Weconcludedthateventhoughdetectionin1xDBPSispossible,thedehybridisationratealtersthesensingcapabilitiesofthesystem.DNA origami synthesis: The DNA origami is a triangular-shaped origami previously reported,1 based off a design byRothemund.2Itisasingle-layerDNAsheetwith120 nmsidelength.Itissynthesisedfrommorethan200shortsinglestrands(staples) and a 7249-nucleotide circular single-stranded DNA (scaffold). During origami preparation some staples wereextendedtoallowhybridisationoffunctionalcomponentsincludingtheaptamer,biotinanchors,andfluorophorelabels(seelistofsequencesatendofSI).TheaptamerandbiotinanchorsweredesignedtoextendfromoppositefacesoftheDNAorigami based on the number of turns in the DNA relative to each of their positions.3 The staples (Integrated DNATechnologies,100 μMin1 × TAEbuffer)andthe12modifiedstaplesforbiotinweremixedwitharatioof5:1withthescaffold(M13mp18viralDNA,Tibilit)atafinalconcentrationof1 × TAEbuffer/12.5 mMMg2

+(annealingbuffer).ThemodifiedA40-staple,surfaceaptamerstrandandATTO655-strand,andbiotinstrandwereaddedina10:1,500:1,50:1,500:1ratiowithrespecttothescaffold.Themixturewasheatedto90 °Cfor5 minandannealedfrom90 °Ctoroomtemperatureattherateof0.2 °Cpermin,whichwasperformedinaPCRmachine(HybaidSprintPCRThermalCycler,ThermoScientific).TheDNAorigami was then purified by using 100 kDa MWCO spin filters (Amicon® Ultra, Ultracel-100 K, Millipore). The finalconcentrationoforigamiwas1.2nMin100μlannealingbuffer.Surface-basedaptasensorpreparationforsingle-moleculeexperiments:Glasscoverslipswerecleanedintensivelytoremoveall florescence impurity.Coverslipswereplaced inaTeflonrack,rinsedwithmilliQwater (mQ)andsonicated inpiranha

Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B.This journal is © The Royal Society of Chemistry 2020

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solution(3:1,sulfuricacidandhydrogenperoxide)for2hours.Afterthis,coverslipswererinsedwithmQandsonicatedinmQfor10min.Then,coverslipsweresonicatedinacetonefor10min,rinsedwithmQ,sonicatedinethanolfor10min,andfinallycoverslipswereblowndrywithArgon.Cloningcylindersweregluedtothecleanedmicroscopecoverslips,whereeachcloningcylindercouldbeusedasareactionchamberorwell.Coverslipswerestoredat-20°CinaFalcontubefilledwithArgon.Beforeusing,coverslipswereequilibratedatroomtemperature.Then,plasmacleanedfor30min.Coverslipswereallowedtocooltoroomtemperatureastheywereusuallyhot.Then,wellswerepassivatedwith1mg/mlBSA-biotinviaphysisorptionfor10min,rinsedwith1xDPBS,conjugatedwith1mg/mlneutravidinfor10min,rinsedwith1xDPBSandexchangedto1× TAEbuffer/12.5 mMMg2

+.Finally,theneutravidin-coatedsurfacewassparselyconjugatedwith240pMbiotinylatedDNAorigamiin1× TAEbuffer/12.5 mMMg2

+for2minandrinsedwith1× TAEbuffer/12.5 mMMg2+.Beforethe

TIRFMexperiments,thebufferinthewellswasexchangedto1xDPBS.TotalInternalReflectionFluorescence(TIRF)Microscopy:Imagingforthesingle-moleculedetectiononasurface(Figure3B)wasperformedusinganLSM710ELYRAPS.1inTIRFmodewith642nmlaserexcitationat30%power,LP655filterand100mscameraacquisitiontime.TIRFtimeseriesofthesameareawereperformedatthefollowingthreestates(SeeFigureS3forsnapshotsoftheacquiredtimeseriesandFigureS4forrepresentativesingle-moleculetraces):(1)recognitionofDNAorigamisviathesingle-moleculefluorescenceofATTO655whereweconfirmedsingle-moleculesbyphotobleachingevents(see representativeevent in FigureS4C); (2) additionofquencher (200nM final concentration) in1xDPBSwith10minincubationwherelossoffluorescenceindicateddye/quencherduplexformation–afterthe10minutesthisstatewasimagedforapproximately30secondstoconfirmthatthequencherhadbound;(3)additionofcortisolbiomarker(1μl;500μM-10μMfinalconcentration)in1xDPBStothechamberwithoutmixing-singlestepdetectionwasobserved14secaftercortisoladdition.Only2individualaptasensorsshowednodetectionafter1min47secofcortisoladdition.Analysis of TIRFMicroscopy:Using ImageJ,we analysed a TIRFmicroscopy time lapse (see Figure S3 for representativesnapshots).Initially,atimelapsewasloadedintoImageJasaStack.Then,brightspotswerevisuallyidentified.A2x2pixelsregionwasselectedonthebrightspotandshiftedaroundthespotinsuchawaythatthemaximumintensitywasobtainedusingtheImageJ’sPlotZ-axisProfiletool.Foreachbrightspotwealsoobtainedanearbybackgroundsignal.Inordertodetermineifafluorescentspotcamefromanindividualaptasensor,welookedatphotobleachingandblinkingofthedye,whereasingle-stepdecreaseinintensityequivalenttothemeasuredbackgroundwouldindicateasingle-moleculeaptasensor(exampleseeninfigureS4D).Thisbehaviourwasdistinctfromsinglemoleculebindingevents,wherethelossofintensitywouldnotdecreasetothebackgroundintensityandwasinsteaddimmed.Forsingle-moleculebindingeventstheobservedintensityfirstdecreasedinasinglestepafterthequencherwasaddedandhybridised.Ingeneral,allthebrightspotsinthetimelapsesdecreasetheirintensityafteraddingthequencherandwaitingthe10minutesincubation.Amongthosequenchedsingle-molecules,wechosethosewithsignaltonoiseratioofatleast1.44 (calculatedas signal2/noise2).Whencortisolwasdetected thesignal recovered,again ina single step, to the initialintensityrecordedpriortoaddingthequencher.Foradetectioncut-offafteraddingcortisol,weconsideredabindingeventtooccurwhenthe intensityrecoveredtothe initial intensityrecordedbeforethequencherwasadded.Weconfirmed1single-moleculebrightspotper36.4μm2afterevaluating68brightspotsinanareaof1310.5μm2.AtomicForceMicroscopy(AFM):AFMcharacterisationinliquidwascarriedoutonaBrukerDimensionFastScaninPeakForceTappingmodewithFastScanDtipsfromBrukerandwasperformedonafreshlycleavedmicasubstrate.Scanningbufferwastheorigamiannealingbuffer(1× TAEbuffer/12.5 mMMg2

+).Theoreticalmeltingtemperaturecalculations:ThemeltingtemperatureswerecalculatedusingOligoAnalyzersoftwarefromIDTwheretheDNAwassetat1μMwithvariedconcentrationsofNaClandMgCl2.Calculatedmeltingtemperatureforthetrigger(highlightedingreeninthesectionsrelatedtoDNAsequencesforthein-solutionaptasensorandsingle-moleculesurface-basedaptasensor:1μM150mM(1xDPBS)35.7°C,1μM300mMNaCl38.2°C.Calculatedmeltingtemperatureforthequencherduplex(highlightedinlightblueintheDNAsequencessection):1μM150mMNaCl(1xDPBS)56.7°C,1μM300mM NaCl 59.6°C. Other DNA segments such as the “ATTO 655-strand” and the surface aptamer capturing strand(highlighted in purple in the DNA sequences section) were designed using simulation packages such as NUPACK andOligoAnalyzerfromIDTforthetested150mMNaClinsuchawaythat100%hybridisationratewaspredictedat25°C.DNAsequencesforthein-solutionaptasensor:in-solutionaptamer:/5ATTO488N/ctctcgggacgacGCCCGCATGTTCCATGGATAGTCTTGACTAgtcgtcccquencher-BHQ1:gtcgtcccgagag/3BHQ_1/blocker:gggacgacTAGTCAAGACTATCCATGGAACATGCGGGDNAsequencesforthesingle-moleculesurface-basedaptasensor:Surfaceaptamer:CACGCTGTTGAATGTCTGACctctcgggacgacGCCCGCATGTTCCATGGATAGTCTTGACTAgtcgtcccmodifiedA40-staple:CGTGATTAGGTAAGGCCTTAGTCAGACATTCAACAGCGTGTTTTTTTAGTATCGCCAACGCTCAACAGTCGGCTGTCATTO655-strand:/ATTO655/TAAGGCCTTACCTAATCACGquencher-iowa:gtcgtcccgagag/3IAbRQSp/

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Modifiedstaplesforthebiotinanchorandbiotinstrand:A52-staple:CCCATCCTCGCCAACATGTAATTTAATAAGGCCTGATGATTGATACCG A44-staple:TCAATAATAGGGCTTAATTGAGAATCATAATTCTGATGATTGATACCG A07-staple:AAAGACAACATTTTCGGTCATAGCCAAAATCACTGATGATTGATACCG A15-staple:GGAGGGAATTTAGCGTCAGACTGTCCGCCTCCCTGATGATTGATACCG B52-staple:GTACAACGAGCAACGGCTACAGAGGATACCGACTGATGATTGATACCG B44-staple:ATTGTGTCTCAGCAGCGAAAGACACCATCGCCCTGATGATTGATACCG B07-staple:AACCAGACGTTTAGCTATATTTTCTTCTACTACTGATGATTGATACCG B15-staple:GATTAGAGATTAGATACATTTCGCAAATCATACTGATGATTGATACCG C52-staple:CGCGCGGGCCTGTGTGAAATTGTTGGCGATTACTGATGATTGATACCG C44-staple:CCAGGGTGGCTCGAATTCGTAATCCAGTCACGCTGATGATTGATACCG C07-staple:GGACATTCACCTCAAATATCAAACACAGTTGACTGATGATTGATACCG C15-staple:TGACCTGACAAATGAAAAATCTAAAATATCTTCTGATGATTGATACCG Biotinstrand:/5Biosg/CGGTATCAATCATCAGStaplestrandsforthetriangularDNAorigami:A01:CGGGGTTTCCTCAAGAGAAGGATTTTGAATTAA02:AGCGTCATGTCTCTGAATTTACCGACTACCTTA03:TTCATAATCCCCTTATTAGCGTTTTTCTTACCA04:ATGGTTTATGTCACAATCAATAGATATTAAACA05:TTTGATGATTAAGAGGCTGAGACTTGCTCAGTACCAGGCGA06:CCGGAACCCAGAATGGAAAGCGCAACATGGCTA07:AAAGACAACATTTTCGGTCATAGCCAAAATCAA08:GACGGGAGAATTAACTCGGAATAAGTTTATTTCCAGCGCCA09:GATAAGTGCCGTCGAGCTGAAACATGAAAGTATACAGGAGA10:TGTACTGGAAATCCTCATTAAAGCAGAGCCACA11:CACCGGAAAGCGCGTTTTCATCGGAAGGGCGAA12:CATTCAACAAACGCAAAGACACCAGAACACCCTGAACAAAA13:TTTAACGGTTCGGAACCTATTATTAGGGTTGATATAAGTAA14:CTCAGAGCATATTCACAAACAAATTAATAAGTA15:GGAGGGAATTTAGCGTCAGACTGTCCGCCTCCA16:GTCAGAGGGTAATTGATGGCAACATATAAAAGCGATTGAGA17:TAGCCCGGAATAGGTGAATGCCCCCTGCCTATGGTCAGTGA18:CCTTGAGTCAGACGATTGGCCTTGCGCCACCCA19:TCAGAACCCAGAATCAAGTTTGCCGGTAAATAA20:TTGACGGAAATACATACATAAAGGGCGCTAATATCAGAGAA21:CAGAGCCAGGAGGTTGAGGCAGGTAACAGTGCCCGA22:ATTAAAGGCCGTAATCAGTAGCGAGCCACCCTA23:GATAACCCACAAGAATGTTAGCAAACGTAGAAAATTATTCA24:GCCGCCAGCATTGACACCACCCTCA25:AGAGCCGCACCATCGATAGCAGCATGAATTATA26:CACCGTCACCTTATTACGCAGTATTGAGTTAAGCCCAATAA27:AGCCATTTAAACGTCACCAATGAACACCAGAACCAA28:ATAAGAGCAAGAAACATGGCATGATTAAGACTCCGACTTGA29:CCATTAGCAAGGCCGGGGGAATTAA30:GAGCCAGCGAATACCCAAAAGAACATGAAATAGCAATAGCA31:TATCTTACCGAAGCCCAAACGCAATAATAACGAAAATCACCAGA32:CAGAAGGAAACCGAGGTTTTTAAGAAAAGTAAGCAGATAGCCGA33:CCTTTTTTCATTTAACAATTTCATAGGATTAGA34:TTTAACCTATCATAGGTCTGAGAGTTCCAGTAA35:AGTATAAAATATGCGTTATACAAAGCCATCTTA36:CAAGTACCTCATTCCAAGAACGGGAAATTCATA37:AGAGAATAACATAAAAACAGGGAAGCGCATTAA38:AAAACAAAATTAATTAAATGGAAACAGTACATTAGTGAATA39:TTATCAAACCGGCTTAGGTTGGGTAAGCCTGTA40:TTAGTATCGCCAACGCTCAACAGTCGGCTGTCA41:TTTCCTTAGCACTCATCGAGAACAATAGCAGCCTTTACAGA42:AGAGTCAAAAATCAATATATGTGATGAAACAAACATCAAGA43:ACTAGAAATATATAACTATATGTACGCTGAGAA44:TCAATAATAGGGCTTAATTGAGAATCATAATTA45:AACGTCAAAAATGAAAAGCAAGCCGTTTTTATGAAACCAAA46:GAGCAAAAGAAGATGAGTGAATAACCTTGCTTATAGCTTAA47:GATTAAGAAATGCTGATGCAAATCAGAATAAA

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A48:CACCGGAATCGCCATATTTAACAAAATTTACGA49:AGCATGTATTTCATCGTAGGAATCAAACGATTTTTTGTTTA50:ACATAGCGCTGTAAATCGTCGCTATTCATTTCAATTACCTA51:GTTAAATACAATCGCAAGACAAAGCCTTGAAAA52:CCCATCCTCGCCAACATGTAATTTAATAAGGCA53:TCCCAATCCAAATAAGATTACCGCGCCCAATAAATAATATA54:TCCCTTAGAATAACGCGAGAAAACTTTTACCGACCA55:GTGTGATAAGGCAGAGGCATTTTCAGTCCTGAA56:ACAAGAAAGCAAGCAAATCAGATAACAGCCATATTATTTAA57:GTTTGAAATTCAAATATATTTTAGA58:AATAGATAGAGCCAGTAATAAGAGATTTAATGA59:GCCAGTTACAAAATAATAGAAGGCTTATCCGGTTATCAACA60:TTCTGACCTAAAATATAAAGTACCGACTGCAGAACA61:GCGCCTGTTATTCTAAGAACGCGATTCCAGAGCCTAATTTA62:TCAGCTAAAAAAGGTAAAGTAATTA63:ACGCTAACGAGCGTCTGGCGTTTTAGCGAACCCAACATGTA64:ACGACAATAAATCCCGACTTGCGGGAGATCCTGAATCTTACCAA65:TGCTATTTTGCACCCAGCTACAATTTTGTTTTGAAGCCTTAAAB01:TCATATGTGTAATCGTAAAACTAGTCATTTTCB02:GTGAGAAAATGTGTAGGTAAAGATACAACTTTB03:GGCATCAAATTTGGGGCGCGAGCTAGTTAAAGB04:TTCGAGCTAAGACTTCAAATATCGGGAACGAGB05:ACAGTCAAAGAGAATCGATGAACGACCCCGGTTGATAATCB06:ATAGTAGTATGCAATGCCTGAGTAGGCCGGAGB07:AACCAGACGTTTAGCTATATTTTCTTCTACTAB08:GAATACCACATTCAACTTAAGAGGAAGCCCGATCAAAGCGB09:AGAAAAGCCCCAAAAAGAGTCTGGAGCAAACAATCACCATB10:CAATATGACCCTCATATATTTTAAAGCATTAAB11:CATCCAATAAATGGTCAATAACCTCGGAAGCAB12:AACTCCAAGATTGCATCAAAAAGATAATGCAGATACATAAB13:CGTTCTAGTCAGGTCATTGCCTGACAGGAAGATTGTATAAB14:CAGGCAAGATAAAAATTTTTAGAATATTCAACB15:GATTAGAGATTAGATACATTTCGCAAATCATAB16:CGCCAAAAGGAATTACAGTCAGAAGCAAAGCGCAGGTCAGB17:GCAAATATTTAAATTGAGATCTACAAAGGCTACTGATAAAB18:TTAATGCCTTATTTCAACGCAAGGGCAAAGAAB19:TTAGCAAATAGATTTAGTTTGACCAGTACCTTB20:TAATTGCTTTACCCTGACTATTATGAGGCATAGTAAGAGCB21:ATAAAGCCTTTGCGGGAGAAGCCTGGAGAGGGTAGB22:TAAGAGGTCAATTCTGCGAACGAGATTAAGCAB23:AACACTATCATAACCCATCAAAAATCAGGTCTCCTTTTGAB24:ATGACCCTGTAATACTTCAGAGCAB25:TAAAGCTATATAACAGTTGATTCCCATTTTTGB26:CGGATGGCACGAGAATGACCATAATCGTTTACCAGACGACB27:TAATTGCTTGGAAGTTTCATTCCAAATCGGTTGTAB28:GATAAAAACCAAAATATTAAACAGTTCAGAAATTAGAGCTB29:ACTAAAGTACGGTGTCGAATATAAB30:TGCTGTAGATCCCCCTCAAATGCTGCGAGAGGCTTTTGCAB31:AAAGAAGTTTTGCCAGCATAAATATTCATTGACTCAACATGTTB32:AATACTGCGGAATCGTAGGGGGTAATAGTAAAATGTTTAGACTB33:AGGGATAGCTCAGAGCCACCACCCCATGTCAAB34:CAACAGTTTATGGGATTTTGCTAATCAAAAGGB35:GCCGCTTTGCTGAGGCTTGCAGGGGAAAAGGTB36:GCGCAGACTCCATGTTACTTAGCCCGTTTTAAB37:ACAGGTAGAAAGATTCATCAGTTGAGATTTAGB38:CCTCAGAACCGCCACCCAAGCCCAATAGGAACGTAAATGAB39:ATTTTCTGTCAGCGGAGTGAGAATACCGATATB40:ATTCGGTCTGCGGGATCGTCACCCGAAATCCGB41:CGACCTGCGGTCAATCATAAGGGAACGGAACAACATTATTB42:AGACGTTACCATGTACCGTAACACCCCTCAGAACCGCCACB43:CACGCATAAGAAAGGAACAACTAAGTCTTTCCB44:ATTGTGTCTCAGCAGCGAAAGACACCATCGCCB45:TTAATAAAACGAACTAACCGAACTGACCAACTCCTGATAA

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B46:AGGTTTAGTACCGCCATGAGTTTCGTCACCAGGATCTAAAB47:GTTTTGTCAGGAATTGCGAATAATCCGACAATB48:GACAACAAGCATCGGAACGAGGGTGAGATTTGB49:TATCATCGTTGAAAGAGGACAGATGGAAGAAAAATCTACGB50:AGCGTAACTACAAACTACAACGCCTATCACCGTACTCAGGB51:TAGTTGCGAATTTTTTCACGTTGATCATAGTTB52:GTACAACGAGCAACGGCTACAGAGGATACCGAB53:ACCAGTCAGGACGTTGGAACGGTGTACAGACCGAAACAAAB54:ACAGACAGCCCAAATCTCCAAAAAAAAATTTCTTAB55:AACAGCTTGCTTTGAGGACTAAAGCGATTATAB56:CCAAGCGCAGGCGCATAGGCTGGCAGAACTGGCTCATTATB57:CGAGGTGAGGCTCCAAAAGGAGCCB58:ACCCCCAGACTTTTTCATGAGGAACTTGCTTTB59:ACCTTATGCGATTTTATGACCTTCATCAAGAGCATCTTTGB60:CGGTTTATCAGGTTTCCATTAAACGGGAATACACTB61:AAAACACTTAATCTTGACAAGAACTTAATCATTGTGAATTB62:GGCAAAAGTAAAATACGTAATGCCB63:TGGTTTAATTTCAACTCGGATATTCATTACCCACGAAAGAB64:ACCAACCTAAAAAATCAACGTAACAAATAAATTGGGCTTGAGAB65:CCTGACGAGAAACACCAGAACGAGTAGGCTGCTCATTCAGTGAC01:TCGGGAGATATACAGTAACAGTACAAATAATTC02:CCTGATTAAAGGAGCGGAATTATCTCGGCCTCC03:GCAAATCACCTCAATCAATATCTGCAGGTCGAC04:CGACCAGTACATTGGCAGATTCACCTGATTGCC05:TGGCAATTTTTAACGTCAGATGAAAACAATAACGGATTCGC06:AAGGAATTACAAAGAAACCACCAGTCAGATGAC07:GGACATTCACCTCAAATATCAAACACAGTTGAC08:TTGACGAGCACGTATACTGAAATGGATTATTTAATAAAAGC09:CCTGATTGCTTTGAATTGCGTAGATTTTCAGGCATCAATAC10:TAATCCTGATTATCATTTTGCGGAGAGGAAGGC11:TTATCTAAAGCATCACCTTGCTGATGGCCAACC12:AGAGATAGTTTGACGCTCAATCGTACGTGCTTTCCTCGTTC13:GATTATACACAGAAATAAAGAAATACCAAGTTACAAAATCC14:TAGGAGCATAAAAGTTTGAGTAACATTGTTTGC15:TGACCTGACAAATGAAAAATCTAAAATATCTTC16:AGAATCAGAGCGGGAGATGGAAATACCTACATAACCCTTCC17:GCGCAGAGGCGAATTAATTATTTGCACGTAAATTCTGAATC18:AATGGAAGCGAACGTTATTAATTTCTAACAACC19:TAATAGATCGCTGAGAGCCAGCAGAAGCGTAAC20:GAATACGTAACAGGAAAAACGCTCCTAAACAGGAGGCCGAC21:TCAATAGATATTAAATCCTTTGCCGGTTAGAACCTC22:CAATATTTGCCTGCAACAGTGCCATAGAGCCGC23:TTAAAGGGATTTTAGATACCGCCAGCCATTGCGGCACAGAC24:ACAATTCGACAACTCGTAATACATC25:TTGAGGATGGTCAGTATTAACACCTTGAATGGC26:CTATTAGTATATCCAGAACAATATCAGGAACGGTACGCCAC27:CGCGAACTAAAACAGAGGTGAGGCTTAGAAGTATTC28:GAATCCTGAGAAGTGTATCGGCCTTGCTGGTACTTTAATGC29:ACCACCAGCAGAAGATGATAGCCCC30:TAAAACATTAGAAGAACTCAAACTTTTTATAATCAGTGAGC31:GCCACCGAGTAAAAGAACATCACTTGCCTGAGCGCCATTAAAAC32:TCTTTGATTAGTAATAGTCTGTCCATCACGCAAATTAACCGTTC33:CGCGTCTGATAGGAACGCCATCAACTTTTACAC34:AGGAAGATGGGGACGACGACAGTAATCATATTC35:CTCTAGAGCAAGCTTGCATGCCTGGTCAGTTGC36:CCTTCACCGTGAGACGGGCAACAGCAGTCACAC37:CGAGAAAGGAAGGGAAGCGTACTATGGTTGCTC38:GCTCATTTTTTAACCAGCCTTCCTGTAGCCAGGCATCTGCC39:CAGTTTGACGCACTCCAGCCAGCTAAACGACGC40:GCCAGTGCGATCCCCGGGTACCGAGTTTTTCTC41:TTTCACCAGCCTGGCCCTGAGAGAAAGCCGGCGAACGTGGC42:GTAACCGTCTTTCATCAACATTAAAATTTTTGTTAAATCAC43:ACGTTGTATTCCGGCACCGCTTCTGGCGCATC

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C44:CCAGGGTGGCTCGAATTCGTAATCCAGTCACGC45:TAGAGCTTGACGGGGAGTTGCAGCAAGCGGTCATTGGGCGC46:GTTAAAATTCGCATTAATGTGAGCGAGTAACACACGTTGGC47:TGTAGATGGGTGCCGGAAACCAGGAACGCCAGC48:GGTTTTCCATGGTCATAGCTGTTTGAGAGGCGC49:GTTTGCGTCACGCTGGTTTGCCCCAAGGGAGCCCCCGATTC50:GGATAGGTACCCGTCGGATTCTCCTAAACGTTAATATTTTC51:AGTTGGGTCAAAGCGCCATTCGCCCCGTAATGC52:CGCGCGGGCCTGTGTGAAATTGTTGGCGATTAC53:CTAAATCGGAACCCTAAGCAGGCGAAAATCCTTCGGCCAAC54:CGGCGGATTGAATTCAGGCTGCGCAACGGGGGATGC55:TGCTGCAAATCCGCTCACAATTCCCAGCTGCAC56:TTAATGAAGTTTGATGGTGGTTCCGAGGTGCCGTAAAGCAC57:TGGCGAAATGTTGGGAAGGGCGATC58:TGTCGTGCACACAACATACGAGCCACGCCAGCC59:CAAGTTTTTTGGGGTCGAAATCGGCAAAATCCGGGAAACCC60:TCTTCGCTATTGGAAGCATAAAGTGTATGCCCGCTC61:TTCCAGTCCTTATAAATCAAAAGAGAACCATCACCCAAATC62:GCGCTCACAAGCCTGGGGTGCCTAC63:CGATGGCCCACTACGTATAGCCCGAGATAGGGATTGCGTTC64:AACTCACATTATTGAGTGTTGTTCCAGAAACCGTCTATCAGGGC65:ACGTGGACTCCAACGTCAAAGGGCGAATTTGGAACAAGAGTCCLink-A1C:TTAATTAATTTTTTACCATATCAAALink-A2C:TTAATTTCATCTTAGACTTTACAALink-A3C:CTGTCCAGACGTATACCGAACGALink-A4C:TCAAGATTAGTGTAGCAATACTLink-B1A:TGTAGCATTCCTTTTATAAACAGTTLink-B2A:TTTAATTGTATTTCCACCAGAGCCLink-B3A:ACTACGAAGGCTTAGCACCATTALink-B4A:ATAAGGCTTGCAACAAAGTTACLink-C1B:GTGGGAACAAATTTCTATTTTTGAGLink-C2B:CGGTGCGGGCCTTCCAAAAACATTLink-C3B:ATGAGTGAGCTTTTAAATATGCALink-C4B:ACTATTAAAGAGGATAGCGTCCLoop:GCGCTTAATGCGCCGCTACAGGGC

FigureS1.Experimentsinsolutionatdifferentionicconcentrationconditions

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FigureS2.RepresentativeAFMimagesinliquidofthetriangularDNAorigami

FigureS3.TIRFsnapshotsofthesamearea,wherethebrightspotsindicatethepresenceofDNAorigamiwithaptasensor.Theseareimagedinthreedifferentstages:A.“beforedetection”,whereTIRFisusedtolocatetheDNAorigamiwiththeaptasensor;B.“quencher”,wheretheorigamiisimaged10minutesafteradding200nMquencher;andC.“aftercortisol”,where10μMcortisolisaddedandtheDNAorigamiisimagedapproximately14secondslater.

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FigureS4.Representativesingle-moleculetraces.A)Representativesingle-moleculedetectioneventfor10μMcortisolbyTIRFMatthreedifferentstates:(1)beforeaddingthequencher,(2)afteraddingexcessamountofquencherinsolution,and(3)afteradditionof10μMcortisol.Thestates1,2and3correspondtothestatesintheschemeinFigure3Cinthemaintext.Detectionwasachieved26.5secaftercortisoladdition.B)Detectionofcortisolafter37.9secaftercortisoladdition.C)Detectionofcortisolafter81.8secaftercortisoladdition;italsoshowsasinglestepphotobleachingeventafter94.4sec.D)Detectionofcortisolwithin14secaftercortisoladdition;italsoshowsasinglestepphotobleachingeventafter35sec.Redtraces indicatethesingle-moleculeaptasensor,whileblacktraces indicatethereferencebackground.Cameraacquisitiontimewassetat100ms.

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