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ELECTRONICSUPPLEMENTARYINFORMATION

miR-122directdetectioninhumanserumbytime-gatedfluorescenceimaging

EmilioGarcia-Fernandez,aM.CarmenGonzalez-Garcia,aSalvatorePernagallo,b,cMariaJ.Ruedas-Rama,aMarioA.Fara,bFranciscoJ.Lopez-Delgado,bJamesW.Dear,dHughIlyine,c

CristinaRess,eJuanJ.Díaz-Mochónb,f-gandAngelOrtea*

(a) DepartamentodeFisicoquimica.UnidaddeExcelenciadeQuímicaaplicadaaBiomedicinayMedioambiente,FacultaddeFarmacia,UniversidaddeGranada,CampusdeCartujas/n,18071–Granada,Spain.*E-mail:angelort@ugr.es

(b) DestiNAGenomicaS.L.,ParqueTecnológicoCienciasdelaSalud(PTS),Av.delaInnovación1,EdificioBIC,Armilla,Granada,Spain.

(c) DestiNAGenomicsLtd.,7-11MelvilleSt,EdinburghEH37PE,UnitedKingdom.(d) CentreforCardiovascularScience,UniversityofEdinburgh,TheQueen'sMedicalResearch

Institute,47LittleFranceCrescent,Edinburgh,EH164TJ,UK(e) OptoiMicroelectronics,ViaViennan°8,38121–Gardolo,Trento,Italy.(f) GENYO-CentreforGenomicsandOncologicalResearch:Pfizer/Universityof

Granada/AndalusianRegionalGovernment,Av.delaIlustración,114,18016–Granada,Spain.(g) DepartamentodeQuimicaFarmaceuticayOrganica,FacultaddeFarmacia,Universidadde

Granada,CampusdeCartujas/n,18071–Granada,Spain.

Tableofcontents

EXPERIMENTALPART 2

E1.MATERIALS 2E1.1.GENERALREAGENTSANDSTOCKSOLUTIONS 2E1.2.PNA122*ANDDGL122HYBRIDIZATIONPROBES 3E1.3.SYNTHESISOFSETAU425-LABELLEDALDEHYDECYTOSINE(SB-C*) 3E1.4.BLOODSERUMSAMPLES 4E2.METHODS 5E2.1.METHODOLOGYFORTHEDETECTIONOFMIR-122WITHLABELLEDPNA122*PROBE 5E2.2.DCL-TGIASSAYFORTHEDETECTIONOFMIR-122WITHSINGLE-BASESPECIFICITY 6E2.4.TGIIMAGINGINSTRUMENTATION 6E2.5.TIME-GATEDFILTERINGOFPHOTONS 7E2.6.TREATMENTOFHUMANSERUMSAMPLES 11

RESULTS 12

R1.SPECTRALCHARACTERIZATIONANDCHEMICALSTABILITYOFSETAU425ANDDERIVATIVES 12R1.1.SETAU425 12R1.2PNA122* 12R1.3.SB-C* 13R1.4.TIME-RESOLVEDFLUORESCENCESPECTROSCOPYOFSETAU425,PNA122*ANDSB-C* 14R2.PHOTOPHYSICALANDTGIIMAGINGCONTROLSWITHQ-SPHBEADS 15

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2019

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R2.1.Q-SPHBEADSAUTOFLUORESCENCE 15R2.2.UNSPECIFICADSORPTIONANDAUTOFLUORESCENCEFROMMIRS,DGL122,ANDPNA122* 15R2.3.UNSPECIFICADSORPTIONOFSB-C*ONQ-SPHBEADS 16R2.4.CONTROLSOFUNSPECIFICINTERACTIONSOFSB-C*WITHOTHERREAGENTS 18R2.5.NATUREOFTHEINTERACTIONBETWEENQ-SPHBEADSANDTHENUCLEICACIDCOMPLEXES 19R3.OPTIMIZATIONOFTHEDUPLEXADSORPTIONONTHEQ-SPHBEADS 20R4.OPTIMIZATIONOFTHEEXCITATIONLASERPOWERINPNA122*/MIR-122METHODOLOGY 21R5.OPTIMIZATIONOFTHETIME-GATINGWINDOWFORTHEDCL-TGIASSAY 23R6.DCL-TGIASSAYAPPLIEDTOTHEDETECTIONOFMIR-122INHUMANSERUMSAMPLES 24R6.1.CONTROLSWITHBOVINESERUMALBUMIN 24R6.2.SPIKE-INTESTONCONTROLHUMANSERUMSAMPLES 25R6.3.STATISTICALANALYSISOFTHERESULTSOFTHEDCL-TGIASSAYAPPLIEDTOHUMANSERUM 26

ESIREFERENCES 27

EXPERIMENTALPART

E1.Materials

E1.1.GeneralreagentsandstocksolutionsCommercially available chemicals were purchased from Sigma Aldrich and were used asreceived.Workingsolutionswerefreshlyprepared,using lowconductivitymilliQwater,priortoeachexperiment from stock solutionspreparedandkept refrigeratedat 4°C. The10mMphosphatebufferwaspreparedbymixingtheappropriateamountsofNa2HPO4andNaH2PO4with thepHadjusted to6.0usingHClorNaOH1M. For the lysisofplasma samples, itwasused the Stabiltech Buffer from DESTINA Genomics Ltd. The buffer is based on proprietarycombinationsofbufferingagents,milddetergents,proteinaseKandvarioussaltsthatenhancetheextractionofmiRsandmaintainthestabilityofthelysedmiR-122.Bovineserumalbumin(BSA)usedforcontrolexperimentswascommercialpowderfromSigma-Aldrich.Q-Sepharosebeads (Q-Sph) are crosslinked agarosematrix beads covered at the surfacewith quaternaryamines and were purchased from GE Healthcare Life Sciences. The diameter of the beadsrangedfrom45to165µm,asdetailedbythesupplier.Priortouse,Q-Sphwasequilibrated.Briefly, 1mL was centrifuged and the supernatant removed. The resin was subsequentlywashedcentrifugallywithH2O (1mL)onceand twicewith10mMphosphatebufferpH6 (1mL), before resuspending in the same buffer (0.5 mL). Immediately before use, the pre-equilibrated Q-Sph suspension was agitated to resuspend the resin beads. SeTau425 waspurchased as NHS ester from SETA BioMedicals (USA). As reducing agent in the dynamicchemistry reaction1, sodium cyanoborohydride (NaBH3CN) was employed. PNA probes(PNA122*andDGL122)aswellastheSMART-C-SETau425(SB-C*),weredesigned,synthesisedandcharacterizedbyDestiNAGenomicaS.L. (Spain), seebelow.All syntheticDNAoligomerswerepurchasedindesaltedformfromIntegratedDNATechnologies(IDT).

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E1.2.PNA122*andDGL122hybridizationprobesTwopeptidenucleicacid(PNA)probes(PNA122andDGL122)weresynthesizedusingstandardsolid phase chemistry on an INTAVIS MultiPep Synthesiser (IntavisAG GmH, Germany) (seeTableS1).TheN-terminal-SETau425-modifiedPNA122(PNA122*)wasdesignedtoallowanti-parallelhybridizationwithsyntheticmimicoligomerDNAmiR-122(seeTableS2). Incontrast,theDGL122sequencewasdesignedincludingandterminatedwithanacetyl-pegylatedgroupand an abasic “blank” site positioned at +13 from their C-terminal end so that post-hybridisation, with the mimic oligomer DNA miR-122 presents a guanine at that position(+13/+15fromthe5ʹ-terminusofmaturehsa-miR-122-5p)therebyallowingincorporationofacytosineintotheblanksiteorpocket (TableS2,nucleobasesunderinterrogationviadynamicchemistrylabelling,DCL,showninred).

TableS1.SequenceofPNA122*andDGL122probes.Reference Oligomersequence(Nʹ–Cʹ) Nʹ-modificationPNA122* AACACCATTGTCACACTC SETau425DGL122 AACAC--ATTGTCACACTC Acetyl-pegylatedgroup

'--':abasemonomer(blank).TableS2.Sequenceofsingle-strandedDNAoligonucleotides,mimicksoftargetmiRs.Reference Oligomersequence(5ʹ–3ʹ) LengthmiR-122 GAGTGTGACAATGGTGTT 18miR-122-22G TGGAGTGTGACAATGGTGTTTG 22miR-122-18A GAGTGTGACAATAGTGTT 18

'Inred':ThenucleobaseofmiR-122underinterrogationviadynamicchemistry.

E1.3.SynthesisofSETau425-labelledaldehydecytosine(SB-C*)Aldehyde-modified cytosine taggedwith the SETau425 reporter dyewas prepared followingthe synthetic routedescribedelsewhere2andpurifiedbyHPLCandcharacterizedbyMALDI-TOFmassspectrometry(seeFiguresS1-S2).

FigureS1.MolecularstructureofmodifiedcytosineSB-C*

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FigureS2.MADLI-TOFMassSpectraofSB-C*.Cation:ChemicalFormula:C32H36N7O5+;ExactMass:598.2772;

MolecularWeight:598.6835.Anion:ChemicalFormula:C32H36ClN7O5;ExactMass:633.2466;MolecularWeight:634.1340.

E1.4.BloodserumsamplesWe analysed samples coming from a single patient suffering from drug-induced liver injury(DILI). Theadultpatientwas recruited ina global study called theMarkersandParacetamolPoisoning (MAPP) study,3 fulfilling the study inclusion criteria: a history of paracetamoloverdose, resulting in treatment with intravenous acetylcysteine after evaluation of thetreating clinician. Full informed consent was obtained from the participant and ethicalapproval was given by the South East Scotland Research Ethics Committee and the East ofScotland Research Ethics Committee via the South East Scotland Human Bioresource. Thebloodsamplewastakenatthepresentationtohospitalanditwasstoredat−80°Casplasma.Thepatientwas affectedby anacute liver injury, pre-definedas apeakhospital stay serumalaninetransaminaseactivity(ALT)greaterthan100U/L4.

Human serum sample used as a negativemiR-122 control and for spiking experiments waspurchasedfromSigmaAldrich(H6914-HumanserumfrommaleABclottedwholeblood;USAorigin,sterile-filtered).

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E2.METHODS

E2.1.MethodologyforthedetectionofmiR-122withlabelledPNA122*probeAs a first approach, designed to check whether the Q-Sph capturing and subsequent TGIimagingmaysucceed fordetectionofmiR-122,weemployeddirectlyacomplementaryPNAoligomer (PNA122*, Table S1) labelled with the fluorescent dye of long τ (SETau425). Bydefinition,thismethodologydoesnotreachsingle-nucleotidespecificity,anditonlyshouldbeconsideredasaproof-of-conceptfortheviabilityoftheDCL-TGIassay.

Theperformanceof thePNA122*/miR-122methodologywasconfirmedby thehybridizationofPNA122*withthecomplementarymiR-122strand(TableS2).Briefly,33μLofPNA122*(30μM),10μLofmiR-122(100μM)and57μLof10mMphosphatebufferpH6,weremixedina200μLeppendorftube.Hybridizationwasconductedat41°Cfor30minfollowedbycoolingdownslowlyatroomtemperature.Theresultingsolutionwithfinalduplexconcentrationof10μM was diluted to the desired concentration, and subsequently captured with the Q-SphbeadsandTG-imaged.

The separation of the PNA122*/miR-122 duplexeswas performed by adding 100 μL of pre-equilibratedQ-Sphbeadsto100μLofduplex-containingsolution.Solutionswerevortexedandincubated at room temperature for 20min at 1250 rpm.Afterward, solutionswerewashedthreetimeswith200μLofbufferandcentrifugedfor2minat2000rpm,andthesupernatantremoved.Finally,thebeadswereresuspendedin20μLofbufferbeforebeinganalysedinTGIimaging.

ChartS1depictsthewholeprocessforthismethodology.

Chart S1. PNA122*/miR-122methodology employed for detection ofmiR-122 in a single step hybridization andsubsequentpurificationwithQ-SphbeforeTGIimaging.

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E2.2.DCL-TGIassayforthedetectionofmiR-122withsingle-basespecificityThe labelling via DCL in the DCL-TGI assay was performed by incorporating the SB-C* baseuponthehybridizationofDGL122withtargetmiR-122sequences.Briefly,DGL122(50μM),SB-C*(45μM),NaBH3CN(2.5mM)andtargetmiRstrands(TableS2)togiveafinalconcentrationof1μMofduplexes,5μMofSB-C*and150mMofNaBH3CNwereaddedinasinglesteptoa200μLeppendorf,vortexedandincubated(41°Cfor30min,thermalcycler).Thesolutionwasallowedtoslowlycooldownatroomtemperature.

The capturing and separation of the hybridized and labelled DGL122/miR-122 duplexeswasperformedexactlyasdescribedinsectionE2.1forthePNA122*/miR-122methodology.

E2.4.TGIimaginginstrumentationFluorescence lifetime (FLIM) and TGI-filtered images were obtained in a MicroTime 200microscopesystem(PicoQuantGmbH).Theexcitationsourcewasa440-nmpulsedlaser(LDH-P-C-440, PicoQuant) controlled by a “Sepia” driver (PicoQuant) to adjust the power andrepetition rate to 5 MHz, giving a 200-ns total time-window. The laser powers typicallyemployedwere0.7,5.3and81.1µWatthephotodiode.Theexcitationpulsesweredirectedintothespecimenthrougha1.4NA,100×oilimmersionobjectiveofanIX-71invertedconfocalmicroscope (Olympus), after being reflected on a 440 DCXR dichroic mirror (Chroma). Thefluorescenceemissionwasfilteredbya460LP(AHF/Chroma)cut-offfilter,andfocusedontoa75-μm pinhole. The fluorescence emission was directed through a 550/50 bandpass filter(OmegaFilters)toasinglephotonavalanchediodeSPCM-AQR14(PerkinElmer)abletodetectindividualphotonsandsentthecorrespondingsignaltoaTimeHarp200singlephotontiming(SPT)module(PicoQuant).

The FLIM and TGI image acquisition was performed taking 10-20 μL of bead-containingsolutionandplacing itover themicroscopeslide.First, thebeadswere locatedandselectedwiththebrigthfieldattheocularsofthemicroscope,andthenaninitialpre-scan,atverylowlaserpowertoavoidphotobleachingeffects,ofan80×80μm2areawasperformedtotestthefluorescence intensityofthebead.Then,anotherscanofthesameareaatthesuitable laserintensitywitha256x256pixelresolutionwasrecorded.The laserwasfocusedontodifferentparticlesandthe imagesofdifferentparticlescollected.TheFLIMimageswereanalyzedandprocessedusingSymphoTime32software(PicoQuant).Theensembleimagedecaywasfittedtodetect thepresenceof the SeTau425 fluorescent probe lifetime. Time-gated filteringwasappliedbyselectingjustthephotonsofcertaintime-window,usually2filteringlevels:fullgateor from 55 ns onwards (time-gated). A previous spatial binning of 5x5 pixels andprehistogrammingof8temporalchannels(forafinalresolutionof464ps/channel)wereusedtoachievealargernumberofcountsineachpixel.Later,theintensityimageswereexportedtoImageJ1.52bsoftware5forprocessing.Inbrief,firstathresholdwasappliedtoremovethebackgroundcountsandlaterapseudocolourscaleforintensitywasapplied(Fig.S3).

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FigureS3.ProcessingofFLIMimagesintoTGintensityimages.Whitescalebarcorrespondsto20μm.

E2.5.Time-gatedfilteringofphotonsFLIMmicroscopy takesadvantageof time-resolved fluorescencemethods since they registerbothintensityandlifetimeofthesampleforeachinterrogatedpixel,buildingupafinalimagecontaining this informationwith spatial resolution. Single photon timing (SPT) permits us todiscriminate individual photonsdependingon the arrival time to thedetector. Thedecayofthe fluorescence emission is a random phenomenon and the lifetime of fluorescence, τ, isdefinedasthetimewhen(100/e)%ofthetotalexcitedmoleculeshasrelaxedtothegroundstate.Foreachpixelandfortheensembleimageweobtainahistogramof intensity(counts)vs. arrival time. This is illustrated in figure S4. In SPTmode,we candifferentiate short- andlong-lived emitting molecules by selecting an appropriate time window (time-gating)subsequentlytoacquisition.ReconstructingtheimageonlywiththephotonsdetectedwithinthistimewindowproducestheTG-filteredimage.

Toillustratethis,wehavemodelledahypotheticalsituationinwhichwehaveafluorescencedecaytracethatisthesumofthreedifferentcontributions:aninstrumentresponsefunction(IRF), with an apparent lifetime of 0.5 ns; a short-lived fluorescence emission from thebackground,withanapparent lifetimeof5ns;andthe fluorescenceof theSeTau425probe,witha25nsfluorescencelifetime(Fig.S4).Applyingatimewindowstartinginlinea(from5nsonwards), the photons selected will come from both the 5-ns and the 25-ns sources, butdiscardingIRFphotons.Ifweclosethetime-windowstartingnowinlineb,theonlysourceofphotonswillbethespeciesthatemitswithalifetimeof25ns.

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Figure S4. Simulated fluorescencedecay tracesof threedifferent emitterswith average lifetimevaluesof 0.5ns(red), 5 ns (blue), and 25 ns (orange), and the triexponential sum of equal contributions of the three of them(green).Thedashedlines(a)and(b)representstime-windowsstartingin10nsand70ns,respectively.Inourparticularcase,wehaveafulltime-windowof200ns(5MHzpulserepetitionrate).Infigure S5, we show the application of different filtering time-windows for the duplexPNA122*/miR-122adsorbedoverQ-Sphbeads,andacontrolwiththeQ-Sphbeadsonly.Themajority of fluorescence fromQ-Sph is detected in times shorter than 55 ns. Therefore,wemay exclusively detect fluorescence photons from SeTau425 by filtering out all photonsarrivingbefore55ns.Thisfilteringcausesabiglossof intensity(89%),butwecansafelyandunequivocally attribute the sourceof the remaining counts. It is clear that thenarrower thedetection time-window is, the fewer thenumberofdetectedphotons is,but in contrast thesmallerthecontributionfromQ-Sphis.TheTGimagesresultingfromthisfilteringprocessareshowninFigureS6.

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FigureS5. Fluorescencedecay traces fromQ-Sph (blue) andPNA122*/miR-122duplexadsorbedonQ-Sphbeads(red).Differenttime-windowsareappliedtothehistograms.

Figure S6. TG intensity images from (top) a single Q-Sph bead and (bottom) a Q-Sph bead in the presence ofPNA122*/miR-122 duplex. Four different detection time-windows were applied: 35 ns, 55 ns, 72 ns and 90 ns.Imagessizeis80x80µm2.

In order to accurately estimate the best time-window choice, we have also performed asimulation for a DCL-TGI assay comparing the decay curves for a solution of target miR-122/DGL122/SB-C*duplex(solution1)andablankQ-Sphsolution(solution0).Signal-to-noise(S/N)functionwascalculateddividingsmoothedintensityofsolution1oversolution0foreachtime,figureS7.ResultsshowthatthisS/Nratio ismaximizedfortimes40-90nswhendecay

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curveof solution0 starts todrop to residual counts.According to the firstderivativeof S/Nfunction,figureS7,themaximumsignal-to-noiseratioisachievedfortimeca.55ns,whichisinagreementwithdataoffiguresS5andS6.

FigureS7.(top)Signal-to-NoiseratioofintensitydecaysoffigureS5solutionsatdifferenttimes(blackpoints),redlines is the smoothed S/N function after fitting it to an asymmetric double sigmoidal function. (bottom) Firstderivativeofsignal-to-noiseratioshowingthepointofmaximumS/Nratio.

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E2.6.TreatmentofhumanserumsamplesSerumsampleswerestudiedusingtheDCL-TGIassay,asreferredinsectionE2.2andChart1inthemaintextofthemanuscript,butwithsomemodificationsduetothenatureofrealhumanserumsamples.Inafirststep(a),10μLofserumsampleweretreatedwith25μLoflysisbuffer,7.5μLofDGL122(13μM)andwater fora finalvolumeof45μL.Thiswaskeptreacting for60minutesat30°Candagitationat1200r.p.m.Inasecondstep(b),thissamplewasmixedduring30minutesat41°Cwith1μLofSB-C*(500μM),3μLofNaBH3CN(5mM)andphosphatebufferpH6 fora final volumeof100μL. Later, solutionwas separatedwith theminimumpossibleamountofQ-Sph(15-20beads)andledtothemicroscope.FormiR-122spike-inexperiments,desired concentrationsofmiR-122were added to step (a) insteadofwater and followed tostep(b)aspreviouslydescribed.

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RESULTS

R1.SpectralcharacterizationandchemicalstabilityofSeTau425andderivatives

R1.1.SeTau425Absorption,excitationandemissionspectraofSeTau425areshowninfigureS8.SeTau425ischaracterizedbyalongStokesshift(ca.125nm)withmaximainitsUV-Visabsorptionspectraat340nmand425nmandasingleemissionbandat542nminwater.Thiscompoundhasanaveragefluorescencelifetimeofca.25ns(Fig.S12andTableS3)andafluorescencequantumyield around 0.39 (free dye). This dye shows good chemical stability and its excitation andemissionspectraandlifetimesdonotshowsignificantchangeswithpHbetween2and7.Thedyeisalsostableatconditionsemployedinthiswork,includingtheincubationat41°Cinthepresence of the reducing agent NaBH3CN. The addition of miR and PNA/miR duplexes toSeTAu425 did not lead to important changes, neither in its absorption, emission spectra orfluorescence lifetime. This suggests that there is no direct interaction, such as intercalation,between the SETau425 dye and the nucleic acids used in this work, at the experimentalworkingconditions.

FigureS8.(left)Absorption,and(right)excitation(λem=550nm)andemissionspectra(λexc=425nm)ofSeTau425inphosphatebuffer,pH6.0.

R1.2PNA122*WhentheSeTau425dyewas incorporate to thePNA122*probe,viacovalentbond,andthecorrespondinghybridizedPNA122*/miR-122duplex, itshowedsimilarphotophysical featuresthanthefreeSeTau425insolution.AslightdecreaseintheStokesshiftwasdetectedduetoabathochromiceffectontheabsorptionandahypsochromiceffectontheemissionspectra(Fig.S9).Thefluorescencelifetimeofthedyespannedbetween22-25ns,afterfunctionalizationofthePNA122andhybridizationwithmiR-122(Fig.S12andTableS3).

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FigureS9.Normalizedfluorescence(a)excitationspectra(λem=550nm)and(b)emissionspectra(λexc=425nm)offree SETau425 (blue line), PNA122* (red), PNA122*/miR-122 duplex (green), and PNA122*/miR-122-18A duplex(purple).Arrowsindicatetheshiftoftheband.

R1.3.SB-C*AbsorptionspectraofSB-C*canbeconsideredas thesumofbands fromtwocontributions:theSMARTBase(SB)coreandtheSeTau425moiety(figureS10).Thus,excitationofSB-C*atwavelengths above the SB absorption (350nm) showed strictly the emission profile of theSeTau425dye.Likewise,theexcitationspectracollectedattheemissionwavelengthof550nmonly showed excitation of the SeTau425 dye. This confirms that there is no energy transferprocessfromthebasecoretothefluorescentdye.

Figure S10. Absorption (blue line), excitation (λem = 550nm) (green line) and emission (λexc = 425nm) (red line)spectraofSB-C*.Forcomparison,theabsorptionspectraoftheSB(purpleline)isalsoshown.

A smalldecrease in the fluorescence intensitywasobserved in theSeTau425dye labelafterperforming the hybridization, dynamic incorporation of the SB-C* base, and subsequentcovalent linkagebyreductionwithNaBH3CN(Fig.S11).However, thereducedSB-C*wasstillfluorescent enough, keeping the same spectral properties of SB-C* and the free SETau425.Furthermore, the lifetimeof SeTau425 in the incorporatedSB-C* remainedwithin the22ns

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range.Therefore,evenafterthisreaction,thedyestillkeepsgoodspectralandphotophysicalcharacteristicstoactaslong-lifetimefluorescentreporter.

FigureS11.Emissionspectra(λexc=425nm)ofSB-C*inphosphatebuffer,pH6.0.(a)SB-C*before(blue)andafter(red)reductiontreatmentwithNaBH3CN.(b)SB-C*treatedwithNaBH3CN(blue)andmiR-122-18G/DGL122(red).

R1.4.Time-resolvedfluorescencespectroscopyofSeTau425,PNA122*andSB-C*

Figure S12. Fluorescence decay traces of free SETau425 (blue line), PNA122* (red), PNA122*/miR-122 duplex(green),andPNA122*/miR-122-18Aduplex(cyan),withλexc=405nmandλem=550nm.

TableS3.Averagefluorescencelifetimes,,ofSeTau425freeinsolutionandlabellingthenucleicacidstrandsusedinthiswork.Sample /nsaSeTau425-NHS 25.64PNA122* 21.25PNA122*/miR-122 25.61PNA122*/miR-122-18A 25.42

aTheaveragelifetimeswereobtainedfromglobalanalysesofthreedifferentdecaytraces,collectedat540,550,and560nm,afterpulsedexcitationat405nm.

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R2.PhotophysicalandTGIimagingcontrolswithQ-Sphbeads

R2.1.Q-SphbeadsautofluorescenceWe carried out the characterization of the autofluorescence emission of Q-Sph beads withFLIM imaging, to measure both the emission intensity and lifetime. We observed weaklyfluorescent beads, of tens ofmicrons of diameter (Fig. S13). The fluorescence decay tracesfromtheseimagesfollowedmulti-exponentialdecayfunctions,withanaveragelifetimeofca.4ns.This fluorescencemaycomefromscatteringofexcitation lightandautofluorescenceofthematerialsthatconstitutetheQ-Sphbeads.

FigureS13.80x80µm2FLIMimageofaQ-Sphbeadsuspendedinphosphatebuffer,pH6.0;andthecorrespondingoverallfluorescencedecaytraceobtainedfromtheimage.

R2.2.UnspecificadsorptionandautofluorescencefrommiRs,DGL122,andPNA122*We have also checked other eventual autofluorescence sources, which might causeinterferences inthedetectionprotocol.Wehave investigatedtheemissionfromthe labelledPNA122*probe,theunlabelledmiR-122andthemiR-122/DGL122duplexadsorbedonQ-Sphbeadsatcommonworkingconcentrations(Fig.S14).FLIMimagesoftheQ-Sphbeadsexhibitedlifetimecomponentsofca.1,4,and12ns,similartothoseobtainedforbareQ-Sphparticles.NosignificantcontributionfrommiR-122orDGL122totheTGIimagesappearedundertheseconditions, as the TGI filtering removed most of the fluorescence from the particle, asexpected.SimilarresultswereobtainedfromasolutionofPNA122*,meaningthatthisprobedoesnotinteractwithQ-Sphbeadsattheworkingconcentrations.

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Figure S14. Intensity images of Q-Sph beads in a blank solution, and in the presence of 1 μM miR-122, 1 μMPNA122*or1μMoftheDGL122/miR-122duplex.Fullwindow(a)andTGI-filteredimages(b,55nstimewindow)areshown.Colourscalesindicateemissionintensity.

R2.3.UnspecificadsorptionofSB-C*onQ-SphbeadsApotentialsourceofinterferenceinourDCL-TGIassaywouldbetheunspecificadsorptionofthelabelledSB-C*baseonthebead.Thiswouldpreventtheactualidentificationoftheduplex,sincefree,unreactedSB-C*mayremainonthebead,evenafterthewashingsteps.Hence,asacontrol,wecheckedthepotentialadsorptionofSB-C*onQ-Sphbeadswithbothsteady-statefluorescencemeasurementsandFLIMimaging.

Should this interaction happens, the addition of Q-Sph beads to a solution of SB-C* woulddecreasethefluorescenceintensity,sinceSB-C*moleculeswouldberetiredfrombulksolutionto the bottom of the cuvette upon deposition of the beads. Importantly, we did find anegligible decrease of the intensity after a first treatment with Q-Sph (Fig. S15). Excitationspectra showed similar results. Therefore, we can confirm that Q-Sph beads are notsignificantlyadsorbingSB-C*molecules,thusthedyeremainsinthebulksolution.

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FigureS15.Emissionspectra(λexc=425nm)ofSB-C*insolutionbefore(blue)andafter(red)theadditionofQ-Sphbeads.Theemissionspectrumofablanksolution,onlycontainingQ-Sphbeads,isalsoshown(greenline).

WehavealsoincubatedtwodifferentconcentrationsofSB-C*(50nMand5μM)withQ-Sphbeads,andimagedthemintheFLIMandTGIimagingwithoutfurtherseparation(Fig.S16).Atlow SB-C* concentration, the TGI image was similar to a blank solution. No significantfluorescencefromthedyeislocalizedovertheparticleinthefull-gatedimage,norintheTGIimage,meaningthatthelongcomponentofSeTau425intheSB-C*wasnotpresent.Forhighconcentrations of the SB-C* base, 5 μM (results not shown), the full-gated image showedhomogeneity outside the Q-Sph bead, from SB-C* in the bulk solution, but even loweremissionintensityintheareaoftheparticle, inagreementwithitsmoreinaccessiblenature.Consistently,theTGI imagealsoshowedless intensity intheareaoftheparticle,andwedidnotobserveahigherconcentrationofSB-C*overthebeadsinrelationtothebulk.

AlltheseexperimentsallowedtodrawtheconclusionthatfreeSB-C*basesdonotrepresentasourceofpotentialinterferencesintheDCL-TGIassay,duetounspecificadsorptionontheQ-Sphbeads.

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FigureS16.IntensityimagesofQ-Sph(left)andQ-Sph+SB-C*50nM(right)inphosphatebuffer,pH6.0,atdifferentdetectiontime-windows:(a)Full-gated,and(b)55-nsTGI-filteredimages.Imagessizeis80x80µm2.

R2.4.ControlsofunspecificinteractionsofSB-C*withotherreagentsWehavecheckedseparatelythepotentialinteractionoftheSB-C*basewitheachcomponentoftheDCL-TGIassaytoruleoutotherpossiblesourcesofinterferences.Wehaveinvestigatedthe interaction of SB-C* with the individual miR-122 and DGL122 strands, not forming theduplex,andwiththemiR-122/DGL122duplexbutwithoutthepresenceofthereducingagentthat promotes the covalent binding of the base within the interrogation pocket. Results inFigure S17 evidence that significant amounts of long-lived photons from SeTau425 are notdetectedinanycase.TheTGIimagesshowedsomeintensitywhentheSB-C*baseispresent,due to transient diffusion of the labelled base, but homogeneously found in solution andsurrounding the bead. It is worth noting that even in the presence of themiR-122/DGL122duplex,undernon-reducingconditions,theadsorptionofSB-C*wasnotdetected.Thismeansthat the reducing agent, NaBH3CN, is necessary for a permanent insertion of SB-C* to theduplex. In this case,a slightlyhigher intensitywas found in the surroundingsof theparticle,possibly due to the transient dynamic binding/unbinding events of SB-C*within the duplexpocket. Importantly,emission fromtheSB-C*base isunequivocallyandexclusivelydetectedwhen it is incorporated according to the full protocol. These negative controls are in clearcontrastwiththepositiveresultsshowninthemaintext.

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Figure S17. Intensity images of Q-Sph beads in a blank solution, and in the presence ofmiR-122 1μM + SB-C*,DGL122 1μM + SB-C*, and DGL122/miR-122 duplex 1μM + SB-C*, at different detection time-windows: (a) Fullwindow,and(b)TG-filtered(55ns)images.Colourscalesindicateemissionintensity.

R2.5.NatureoftheinteractionbetweenQ-SphbeadsandthenucleicacidcomplexesTheadditionofNaCl1MtotheQ-SphbeadscontainingthefullSB-C*/DGL122/miR-122duplexexhibited a clear release of some of the fluorescence to the bulk solution (Fig. S18). ThereleaseoflabelledduplexdemonstratestheelectrostaticnatureoftheinteractionwiththeQ-Sphbeads.

FigureS18.Emissionspectra(λexc=425nm)ofSB-C*/DGL122/miR-122duplexsolution(afterDCL-TGIassay)before(redline)andafter(blueline)theadditionofNaCl1M.

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R3.OptimizationoftheduplexadsorptionontheQ-SphbeadsDuplexperbeadratioandincubationtimearetwokeypointsforabetterdetectionoftargetmiR.Weimprovedthedetectionreducingthenumberofavailablebeadsbydirectlyselectingjust one single bead (see Chart S2) and performing a kinetic study of the adsorption ofPNA122*/miR-122 over the Q-Sph beads (Fig. S19). For this purpose, we placed a dilutedsolution of Q-Sph on a microscope slide. Visualization of the beads through the opticalmicroscopehelped topickasinglebeadusingaspirationwithamicropipette.Thisbeadwasledtotheconfocalmicroscopeanddepositedovertheslidewhichcontained10μLofa10nMsolutionofPNA122*/miR-122duplex. Immediatelyafter,weregisteredFLIMandTGI imagescontinuouslyduring90min.ResultsareshowninFigureS19.Strikingly,thisfigureshowshowthebeadsurroundingsunderwentanenhancementinthefluorescenceemissionintensity(andaverage lifetimes, not shown) with time. This is especially noticeable in the TGI images,indicating thepresenceof theSeTau425dye,corresponding to further loadingof theduplexPNA122*/miR-122.Accordingtotheseresults,weestablishedanincubationtimeofatleast50min for the adsorption of the formed duplexes on the bead surface when single-beadapproachisperformed.

ChartS2.Procedure for theselectionof justonesinglebead,subsequent incubationwithPNA122*/miR-122andacquisitionattheconfocalmicroscope.

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FigureS19.Full-gatedandTGI(55nswindow) intensity imagesofaQ-Sphbead inthepresenceofPNA122*/miR-12210nMatdifferentincubationtimes.Colourscalesindicateemissionintensity.

R4.OptimizationoftheexcitationlaserpowerinPNA122*/miR-122methodologyWehaveperformedseveralexperimentstodetectdifferentconcentrationsoflabelledduplex,ranging over 5 order of magnitude, under the PNA122*/miR-122methodology. One of theinconveniencies found in working with such broad range was the saturation of the singlephoton detectorswhen the emission intensitywas too high. Figures S20 and S21 show full-gated and TGI intensity images, respectively, from PNA122*/miR-122 duplex samples, withconcentrations ranging from 1 μM to 100 pM. As expected, for samples with the lowestconcentrationsweneededto increasetheexcitationpowerofthe laser.However,high laserpowerscausedsaturationonthedetectorwithsamplesofhigherconcentrations.Hence,theexcitationpowerisanotherkeypointfordetectionoflowamountsofduplexes.Underthesehighpowerconditions,wecouldclearlydistinguish1nMduplexsamplesfromblanksolutions.As shown in figure 1 of the main text, further improvement in the limit of detection wasachievedbyincreasingtheacquisitiontime-per-pixel.Althoughthismethodincreasesthetimefortheanalysis,onecanunequivocallydetectconcentrationsaslowas100pMofduplex(Fig.1inmaintext).

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Figure S20. Full-gated intensity images of Q-Sph beads with PNA122*/miR-122 duplex solutions at differentconcentrations (100 pM – 1 μM), and different excitation laser powers, comparedwith a blank solution. Colourscalesindicateemissionintensity.

FigureS21.TGI intensity images (55-ns timewindow)ofQ-SphbeadswithPNA122*/miR-122duplexsolutionsatdifferentconcentrations (100pM–1μM)anddifferentexcitation laserpowers,comparedwithablanksolution.Colourscalesindicateemissionintensity.

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R5.Optimizationofthetime-gatingwindowfortheDCL-TGIassayUsingtheDCL-TGIassay,wecandifferentiatesinglepointmutationsinthemiR-122sequence,asshowninfigure2inthemaintext.TheadvantageofusingacompleteFLIManalysisisthatdifferenttimewindowscanbetestedandoptimized.FigureS22showsthefull-gatedandTGIimages, using different gatingwindows, for the sequencesmiR-122,miR-122-22G, andmiR-122-18A, analysed using theDCL-TGI assay; this is the hybridizationwith theDGL122 probeandtheincorporationofthelabelledSB-C*base.FigureS22clearlyshowsthatthebestsignal-to-noiseratioisachievedwitha55-nstimewindow,whichisinagreementwithpreviousdataoffigureS7.

FigureS22.TGIintensityimagesofQ-SphbeadsinthepresenceofmiR-122,miR-122-22G,andmiR-122-18A(1μM),analysedwiththeDCL-TGIassay(withDGL122andSB-C*).Colourscaleindicatesemissionintensity.

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R6.DCL-TGIassayappliedtothedetectionofmiR-122inhumanserumsamples

R6.1.ControlswithbovineserumalbuminWecarriedoutsomecontrolexperimentswiththebovineanalogueofhumanserumalbumin(HSA)todeterminepossiblesourcesof interference inthehybridizationreactionof theDCL-TGI assay, since blood serum contains a huge variety of molecules with different chemicalbinding abilities, e. g., sugars, substrates, electrolytes and proteins (emphasizing serumalbumin),justtomentionsomeofthem.Thesecomponentsfeaturedifferentchemicalbindingabilities andmay cause some kind of disruption in proper functioning of themethodologiesproposedhereduetounspecificbinding.

Forexample,we tested thehybridization reaction in thepresenceofBovineSerumalbumin(BSA)at0.4%(w/V),findinganegligibleincreaseintheintensityofQ-SphbeadsthatvanishesafterapplyingTGIfiltering.Therefore,norelevantdifferencesintheperformanceofthemiR-122detectionwerefoundinpresenceofBSA(FigureS23).

Figure S23. Effect of BSA in theperformanceof theDCL-TGI assay. Fullwindowand TGI-filtered (55ns) intensityimagesofQ-Sphbeadsin(A)ablanksolution,andinthepresenceof(B)SB-C*5μM,(C)SB-C*5μM+DGL1221μM,(D)SB-C*5μM+miR-1221μM,(E)fullreaction:SB-C*5μM,DGL122/miR-122duplex1μM.Allthesamplescontain0.4%(w/V)ofBSA.Colourscalesindicateemissionintensity.

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R6.2.Spike-IntestoncontrolhumanserumsamplesTo test thedetectioncapabilities towardsmiR-122and the limitofdetectionof theDCL-TGIassay in serum samples, we employed a control, commercial human serum and a totalconcentration of 100 pM of miR-122 was spiked in (2 femtomoles of miR-122 in 20 µL ofhuman serum).Wecompared theobtained signalwith thebackgroundnoiseobtained fromtheuntreatedserumsolution(blank),SB-C*inserumsolution,andDGL122andSB-C*inserumsolution as controls. The latter experiment would be representative of a complete DCL-TGIassayappliedtoaseruminwhichthemiR-122levelsarenegligible.Indeed,ourresultsshowedsomebackgroundsignalinthosecontrolexperimentsthatareattenuatedwhenTGIfilteringisapplied. In any case, significantly distinguishable signal is obtained between the samplecontaining100pMofspiked-inmiR-122andthatintheabsenceofmiR-122(Fig.S24).

FigureS24.IntensityperareaofQ-Sphbeadsobtainedfromintensityimagesofcontrolserumsolution(blank),andinpresenceofSB-C*5μM,SB-C*5μM+DGL1221μM,andthefullDCL-TGIassay (SB-C*5μM,DGL1221μM+miR-122 100 pM). The different detection time-windows are shown: (blue) Full window, and (red) TGI-filtered(55ns)images.

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R6.3.StatisticalanalysisoftheresultsoftheDCL-TGIassayappliedtohumanserumWe performed DCL-TGI assays of control human serum and serum from a DILI patient(triplicatedmeasurementsof fourdifferent aliquotseach, for aN=12 ineach sample). Theresults of average intensity per area unit were related to theminimum value for a controlserummeasurement (Fig.S25).Statistical,non-parametric test for two independent samples(NPH-TS, Mann-Whitney test) was performed to validate the hypothesis that the twopopulations,controlandDILIpatient,aresignificantlydifferent.Ourresultsshowthatthetwopopulationsarestatisticallydifferent,withaconfidencelevelof96%,usingtheDCL-TGIassay.Usingafull-gateanalysis,whichwouldbecomparabletoconventionalmicroscopytechniques,resulted in very similar results, although the confidence level for validating the hypothesisdecreaseddownto93%.

FigureS25.BoxplotoftherelativeaveragefluorescenceintensityinDCL-TGIassays,usingfull-gate(dashedboxes)orTGI-filtering(solidboxes),ofacontrolhumanserumsamples(N=12,green)andaDILIpatientserumsamples(N=12,red).Boxesandwhiskersrepresentthestandarderrorandstandarddeviation,respectively.Squaresymbolsandhorizontallinerepresenttheaverageandmedian,respectively.Non-parametricstatisticalMann-Whitneytestof the data confirmed the two populations are different at 0.93 confidence level (full-gate, p = 0.068) or 0.97confidencelevel(TGI-filtered,p=0.033).

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