1

QUADRUPLEXDETECTIONINHUMANCELLS

DavidMonchaud,Ph.D.

InstitutdeChimieMoléculairedel’UniversitédeBourgogne

ICMUB,CNRSUMR6302,UBFCDijon,France

david.monchaud@cnrs.fr

I.Introduction Page01

II.Four-strandedDNAstructuresandquadruplexligands,abitofhistory Page02

III.Immunodetectionofquadruplexesincells Page04

IV.Quadruplex-selectivefluorescentdyes Page06

IVa.Quadruplex-specificligandswithnon-optimizedspectroscopicproperties Page08

IVb.Fromfluorescentdyestoquadruplex-specificprobes Page09

IVb.1Fluorescentdyeswithnon-optimizedquadruplex-interactingproperties Page09

IVb.2Fine-tuningthequadruplex-interactingpropertiesoffluorescentprobes Page11

IVc.Fromquadruplexligandstoquadruplex-specificprobes Page14

IVc.1Optimizingthescaffoldofknownquadruplexligands Page14

IVc.2Addingtagstoknownquadruplexligands Page15

IVc.3Speciallydedicatedsmallmoleculeprobes Page17

IVc.4Usingmetallicprobestotrackquadruplexesincells Page18

V.Conclusion Page20

I.Introduction.

“Youwillneverbeabletohitatargetthatyoucannotsee”.1Thiswiseadvicesuitsperfectly

theexplorationoftheworldoftheinfinitelysmall,notablythewordofnucleicacids.Nearly

halfacenturyofresearchwasrequiredtounveilthecomplicatedprocessesthecanonicalDNA

duplex is involved in. These heroic decades, which saw the rise of cross-cutting scientific

disciplines (from molecular biology to chemical biology and epigenetics), have afforded

invaluableinsightsintothemanycellularprocessesinwhichtheduplexparticipates.2,3They

haveprovidedcriticalcluesaboutthecellularmechanismsunderlyingtheverynotionoflife,

and–becausetargetsweremadevisible–asmanynoveltherapeuticopportunities.

2

However,scarcelyhadtheduplexrevealed(notall)itssecretswhenalternativenucleicacid

structures appeared. They differ from the duplex in many ways, the most distinguishing

featurebeingtheirstrandness(fromtwotofourstrands),whichleadstothefoldingofhigher-

orderarchitecturessuchastriplexes,DNAjunctionsandquadruplexes.4-6Understandingthe

dynamicsofthesenewplayerswasanovel,ratherdauntingchallengeforthecommunityof

nucleic acids: how, where and when these structures fold in cells (if any)? From which

sequences?Withwhichstability?Etc.Anewburstofquestionsthatrequiredanewportfolio

ofmoleculartoolsaimedatcharacterizingfurtive,certainlylow-abundancestructuresinan

environment(thenucleus)thatwasalreadydifficulttocharacterize.

Effortswerethusinvestedtocombineanduseknowledgeandknow-howacquiredduring

thestudyofduplex-DNAtoassembleanarrayofmoleculartoolspoisedtoaddressnewand

challengingissues.Amongthem,thedetectionofquadruplexincells:dothesefour-stranded

DNA structures exist in cells? Can they represent a novel class of therapeutically relevant

targets?Etc.“Youwillneverbeabletohitatargetthatyoucannotsee”.Thisadviceprimarily

emphasizes theneed for tools able to cast lightonquadruplexes in cells. In the following

sectionswillbedemonstratedhowthecreativityofscientistshasbeeninstrumental inthe

designofexquisitemolecularflashlightsthathavebeendevelopedandarecurrentlyusedfor

understandinghow,whereandwhenquadruplexesfoldandunfoldincells.

II.Four-strandedDNAstructuresandquadruplexligands,abitofhistory.

ThestructureofthedoubleDNAhelixwasunraveledin1953byWatson,Crick,Wilkinsand

Franklin.7-9Theeventsthatswirlaroundtheelucidationofitsmolecularstructureareamong

themostfascinatingstoriesofmodernscience.10,11Thisdiscovery,whichmarkedthestartof

moderngenetics,drewonhard-wonresultsacquiredsincetheinitialattemptsbyMiecherin

1871tocharacterizethesubstancefoundinthenucleus,initiallyreferredtoas“nuclein”and

nowknownasnucleicacids.12Apartoftheseeffortswasdedicatedtothecharacterizationof

agivensubclassofnucleicacid,theguanylicacid,initiallyisolatedfrombovinepancreasby

Hammarsten in 1894. Levene (1909)13,14 and Bang (1910)15 noticed the tendency of

concentratedguanylicacidsolutionstoformhydrogels;theexactnatureofthestructuralunit

responsibleforthegelationprocess,theself-assemblyoffourguaninesinaG-quartet,16was

unveiled by Davies in 1962 by X-ray crystallography.17 Inferred as early as in 1982,18 the

formationofG-quartetsinbiologicallyrelevantguanine(G)-richsequences(immunoglobulin

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switchregions,telomeres)wasdemonstratedinvitrobyGilbert(1988)19andKlug(1989)20via

thestudyofsequencesinwhichcontinuousguaninetractsassembleintoG-quartetsthatself-

stacktoformhigher-orderG-quadruplexstructures.Furtherstructuralsupporttothistheory

weresoonprovidedinvitrobythefirstnuclearmagneticresonance(NMR,Feigon,21Patel22

andLilley,1992)23andX-raycrystallographyinvestigations(Rich,199224andLilley,1994).25As

discussedinthefollowingsections,thedemonstrationoftheformationofquadruplexesina

cellularcontexthasprovedmarkedlymorechallenging.

Despite the lack of firm evidences of the existence of quadruplexes in vivo, chemical

programsweresoonlaunchedtoidentifychemicalscapableofinteractingwithquadruplexes,

also referred to as G-quadruplex ligands. Neidle & Hurley reported in 1997 on the first

prototype of a small molecule specially dedicated to interact with quadruplexes for

therapeutic dividends,26 giving dramatic impetus to the field of quadruplex ligands. This

momentumhasneverslackenedsincethen,withdozensofnewligandsreportedeveryyear,

displayingeverbetterquadruplex-interactingproperties(affinity,selectivity).27,28Rapidly,the

ideaofusingligandsendowedwithspecificspectroscopicproperties(fluorescence)emerged

asawaytodetectquadruplexesincells.Theuseofsmall-moleculesisindeedapledgeofcell-

permeability,thepossibletoxicityissuebeingeasilytackledbyantiproliferativeinvestigations

implementedpriortoopticalcellularimaging.Firstattemptswerereportedassoonasin1999

withtheuseofmetallicporphyrinsonmetaphasespreadsfromhumancancercells.29These

firstimagesraisedmorequestionsthananswers,delineatingthelimitsofthisnewresearch

fieldtocome:arethesedyesquadruplex-specificenough(nottosayspecificfornucleicacids

overothercellularcomponentssuchasproteins)?Dotheirspectroscopicpropertiessuitedto

detect low-abundancetargets (mostly theamplitudeof the fluorescencemodulationupon

interactionwithquadruplexesonly)?Etc.Thesefirstimageswerenotconvincingenoughto

build a consensus across scientific fields (chemistry, biophysics, biology, genetics) on the

existenceofquadruplexes in cells, andbeyond this,on their relevanceasantiproliferative

targets.Thisskepticismcompelledscientist tofine-tunetheirstrategyandsharpenedtheir

moleculartoolstoprovideundisputableproofsoftheexistenceofquadruplexes inhuman

cells. This two-decade quest, further described below, has led to the design of highly

sophisticatedmoleculartoolsnowabletodeliverreliableevidencesofthebiologicalrelevance

of quadruplexes: antibodies on one side, a guarantee of high-affinity and specificity for

quadruplexes, and fluorescent ligands on the other side, which offer bioavailability and

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tunable spectroscopic properties. The respective advantages and drawbacks of these two

approaches(e.g.,poorbioavailabilityandtechnicalaccessfortheformer,lowquantumyields

andoff-targetlabelingforthelatter)makethemcomplementarytechniquesthatareoften

implementedinconcert.

III.Immunodetectionofquadruplexesincells.

Immunodetectionisaninvaluabletechniquefortaggingcellulartargets,30usuallyviaatwo-

stepprotocol comprising the target labelingper sewithaprimaryantibody followedbya

signal amplification with a secondary antibody conjugated to a fluorescent reporter. This

approachwassuccessfullyimplementedforthedetectionofquadruplexesincells;however,

duetotheirintracellularlocation,quadruplexeswerestainedinfixedandpermeabilizedcells

only,whichmightraisequestionsregardingtheirexistenceinfunctional,livingcells.

TheveryfirstantibodyreportedtoreactwithquadruplexeswasmenIIB4,describedin1998

byHardin.31Thisantibodywasnotusedfortheimmunofluorescentdetectionofquadruplexes

incellsbutwasassessedinvitroforitsabilitytodiscriminatequadruplexesfromotherDNA

structuresincludingduplexesandtriplexes.Itwasalsoshowntohaveahighaffinityforthe

quadruplexsequencesfoundintheciliatedprotozoanStylonychialemnaetelomeresd(TG4)

andd(T2G4).

Soonafter,Plückthun reportedonSty3 andSty49,32 also raisedagainst theStylonychia

lemnae telomeric sequences d(TG4T). Sty3 displayed better affinity for parallel versus

antiparallelquadruplexes,whileSty49wasfoundtointeractwithbothtopologies.OnlySty49

wasfoundefficienttolabelquadruplexesinisolatedStylonychiamacronuclei,afteratwo-step

protocolusingfluoresceinisothiocyanate(FITC)-labeledsecondaryantibody.Collectedimages

providedthefirstvisualconfirmationoftheexistenceofquadruplexesinnucleibutindicated

alsothatquadruplexesdoforminvegetativenucleionly,i.e.,inabsenceofreplication.This

points towards a possible protective roles of quadruplexes at telomeric levels (capping),

regulatedbytelomereend-bindingproteins(TEBPs)thatcontrolquadruplexformation,asit

wasconfirmedafterwards.33

Balasubramanianreportedonthehf2antibodythatwas,similarlytomenIIB4,described

foritsabilitytointeractwithquadruplexesinvitro.34hf2wasfoundtostronglydiscriminate

between various quadruplex topologies (with a preference for c-kit quadruplex) and was

subsequently used to pull-down quadruplex from genomic DNA, providing an unbiased,

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sequencing (seq)-based demonstration of the existence of quadruplexes in the double-

stranded genomic DNA of the human breast cancer MCF-7 cells.35 Regarding quadruplex

immunodetection,thesamegroupdevelopedBG4,anantibodyusedtovisualizebothDNA

andRNAquadruplexesinhumancells(Figure1a).Thethree-stepprotocol implementedto

amplify the fluorescence detection (BG4 incubation per se, then secondary and tertiary

labeledantibodies)allowedforvisualizingquadruplexesnotonlyinthenucleiofMCF-7and

osteosarcomaU2OScells36andboththenucleiandcytoplasmofSV40-transformedhuman

MRC5fibroblasts,37butalsoinmetaphasechromosomespreads(fromhumancervicalcancer

HeLa cells), suggesting that quadruplex formation occur during chromosome segregation.

Interestingly,thepretreatmentofcellswithDNAquadruplex-specific(pyridostatin,PDS)38and

RNA quadruplex-specific ligands (carboxyPDS, cPDS)39 prior to cell fixation and BG4

immunostainingincreasedthenumberofquadruplexfoci(ascomparedtountreatedcells),

lendingcredencetoboththeexistenceofquadruplexesinhumancellsandtheirdruggability

withcell-permeablesmall-molecules.BG4wasalsousedfortrackingquadruplexesinhuman

tissues:40theimmunohistochemicallabellingofstomachandlivercancertissuesrevealeda

higherdensityofquadruplexlandscapescomparedtonon-canceroustissues,supportingagain

the strategic relevance of quadruplex-directed anticancer strategy. Beyond quadruplex

detectionincellsandtissues,BG4wasalsoinstrumentalforthedevelopmentofG4ChIP-Seq,

a method that combines chromatin immunoprecipitation (ChIP) of quadruplexes and

sequencing.41 This protocol was implemented to map the genome-wide location of

quadruplexesinepidermalkeratinocyteHaCaTcells.G4ChIP-seqconfirmedtheprevalenceof

the quadruplex landscape in a chromatin context (with approximately 10,000 quadruplex

sites),provingbeyonddoubtstheirbiologicalrelevance.

Lansdorpreportedonaseriesofimmunofluorescencestudiesperformedwiththeantibody

1H6 (Figure 1b). This antibody was found efficient to label both nuclei and metaphase

chromosome spreads from HeLa cells,42 and the macronuclei of Stylonychia lemnae.43

Similarly to BG4, the pretreatment of cells with quadruplex ligands, TMPyP444 and

telomestatin,45increasedthenumberofquadruplexfoci,furthersupportingthecapabilityof

1H6todetectquadruplexesincells.However,itwassubsequentlyreportedthat1H6cross-

reacts with non-quadruplex targets, primarily single-stranded thymidines, highlighting the

needtoexercisecautionwheninterpreting1H6sub-cellularbindingpatterns.46

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Finally,D1antibodywasalsoreportedtobeeffectiveinimmunostainingquadruplexesin

humancervicalcancerSiHacells (Figure1c),47andassessingthemodulationofquadruplex

landscapesuponligandtreatments,includingN-methylmesoporphyrinIX(NMM).48

Figure1.ImmunodetectionofDNAquadruplexesby(a)BG4inU2OScells(redfoci,thenucleibeing

counterstainedwithDAPI,inblue),36(b)1H6inHeLacells,42and(c)D1inSiHacells(greenfoci,co-incubated

withanantibodyraisedagainstTRF2(redfoci),thenucleibeingcounterstainedwithDAPI,inblue).47

Altogether,theseinvestigationshaveestablishedthatantibodiesareexquisitemoleculartools

tovisualizequadruplexesincellsbyopticalimaging.Theonlysournoteofimmunostainingis

thatinvestigationshavetobeperformedwithfixedandpermeabilizedcells,thatis,withcells

whose morphology is chemically altered, thus giving rise to doubts as to the biological

relevanceofobservedstainingpatterns.Thisissuewasalleviatedthankstotheversatilityof

antibodies, which could be used in ChIP-seq protocols to gain reliable insights into the

prevalenceofthequadruplex landscapes inthehumangenome.Thecombinationofthese

twoapproachesthusallowedtheinitial,bonafidedoubtsabouttheexistenceofquadruplexes

incellstobereliablydispelled.Asfurtherdiscussedbelow,asimilarstrategywasdevisedwith

small-molecules,combiningchemicallabellingandchemicalaffinitycaptureandsequencing

(chem-seq)49toobtaincomplementarycluesontherelevanceofquadruplexesinfunctional,

livingcells.

IV.Quadruplex-selectivefluorescentdyes.

Quadruplexligandsareusuallysmall-molecules(molecularweight<2000g.mol-1)thatdisplay

water-solubilityandcellpermeabilitypropertiessuitedtobeusedinlivingcells.Thediversity

ofscaffoldsthatcanbedesignedandsynthesizedthroughorganicsynthesishasofferedaccess

toabroadportfolioofmoleculartoolsendowedwithevermoreaccuratepropertiesinafully

controllablemanner.Thisexplainsthewiderangeofligandsnowreported,27,28,50,51andmade

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available for cell-based investigations. When displaying fluorescence properties, ligands

becomequadruplex-specificprobes,52-54pronetobeusedinbothfixedandlivingcells,the

laterallowingtotrackanddetectquadruplexesinafunctionalcontext.Thesedyescouldbe

dividedinseveralcategoriesasafunctionoftheirchemicalnature(organometalliccomplexes

versusorganicmolecules),cell-permeability(fixedcellsversuslive-cellimaging)andselectivity

(DNAversusRNAquadruplexes,orboth).

Itisnocoincidencethat,historically,thefirstsmall-moleculesdescribedasquadruplex-

interacting compounds, i.e., a cyanine (DODC)55 and a porphyrin (NMM)56 in 1996, an

anthraquinonein1997,26aperylenetetracarboxylicdiimide(PIPER)57in1998andaseriesof

metal complexes of tetra(N-methyl-4-pyridyl) and tetra(N-methyl-3-quinolyl) porphines

(TMPyP4andQP3,respectively)29in1999,werealsoendowedwithspectroscopicproperties

thatmakethemsuitedtobeusedasquadruplexprobes.Theselaidthefoundationforanew

area of research focused on the use of condensed polyaromatic molecules and metal

complexestotrackquadruplexesincells.Severalstrategieswereimplementedoverthepast

years,fromthefine-tuningofthechemicalstructureofknownquadruplexligands(tomake

themfluorescent)tothatofknowndyes(tomakethemquadruplex-specific).

Most of the early examples of quadruplex ligands displayed spectroscopic

(fluorescence)propertiespotentiallysuitedtobeingusedfortrackingquadruplexesincells.

However, their polyaromatic nature along with that of their binding sites within the

quadruplex structure (external G-quartets)makes them sensitive to the presence of their

nucleic acid targets, which usually results in a “turn-off” (or “light-off”)-type interactions,

mostlyviaphotoinducedelectrontransfermechanisms.58While“turn-off”probesturnedout

tobeusefulforinvitroinvestigations,52theyhaveonlypoorapplicabilityforthedetectionof

quadruplexinhumancells.Astutestrategieshavebeendevisedtocounteractfluorescence

quenching.52,54,59,60Thefluorescenceofproperlydesignedaromaticmoleculescanbe“turned-

on”uponinteractionwithquadruplexesthroughapanelofpossiblemechanismsthatinclude

i-therestrictionofinternalrotation(RIR),whichoccurswithcyanine(e.g.,DODC,55thiazole

orange(TO)61andThioflavinT (ThT)),62carbazole (e.g.,BMVC)63or triphenylmethanedyes

(e.g.,malachitegreen (MG),64crystalviolet (CV));65 ii- theprotectionprovidedbytheDNA

matrixagainst solvent-mediatednon-radiativedeexcitation (e.g., porphyrins (e.g.,NMM)66

and metal complexes);67 iii- the quadruplex-mediated disaggregation of non-emitting

aggregates (e.g.,NDI)68 or conversely, iv- the aggregation-induced emission (AIE,e.g., the

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tetraphenyletheneTTAPE);69v-thequadruplex-mediatedmolecularrearrangementofsmart

probes;70Etc.Whilehundredsofquadruplex-specificprobeshavebeenstudiedandreported,

we will focus in the next sections on quadruplex-specific probes whose spectroscopic

propertieshavebeenusedtoassesstheexistenceofquadruplexlandscapesincells.

Figure2.Imagesof(a)metaphasespreadsofMCF7cellslive-treatedbyQP3.In,29andA459cellslive-treated

withRHPS4(b)71andCX-3543(c).72

IVa.Quadruplex-specificligandswithnon-optimizedspectroscopicproperties.

Thefirstimmediatestrategytotrackquadruplexeswithincellsistoexploitthefluorescence

propertiesofestablishedquadruplexligands,evenifthesepropertiesarenon-optimized.As

statedabove,thefirstreportedexamplereported(in1999)reliedonthelive-incubationof

MCF7 cellswith a sub-toxic dose (40µM, 3 days) themetal complexQP3.In prior to the

preparationofmetaphasespreads(Figure2a).29Thecollectedimageswereratherill-defined

andallowedfordemonstratingtheaccumulationofthecomplexinthenucleiofcancercells

only. Soon after, Stevens reported on the synthesis and studies of a series of acridinium

salts,71,73 including RHPS4 whose complex with a quadruplex was the very first to be

elucidatedbyNMR.74Theyalsoexploitedthe intrinsicfluorescencepropertiesofRHPS4 to

gaininsightsintothedruglocalizationaftershortlive-cellincubations(30µM,15min)inboth

MCF7andhumanlungcarcinomaA459cellsbyconfocalmicroscopy(Figure2b).Again,the

low-resolution imagesdidnotallow for identifyingprecisely thequadruplex sitesbut they

enlightenanaccumulation in thenucleoli ofA459 cells.Ofnote,higher-resolution images

werecollectedrecentlyinmouseembryonicfibroblast(MEF)cellsusingahigh-speedconfocal

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microscope, and showed an accumulation of RHPS4 in mitochondria.75 Later on, the

fluorescenceofthefluoroquinoloneCX-3543(orquarfloxin)wasexploitedforamoreaccurate

analysis of the biodistribution of quadruplex ligands (5µM, 1 h), confirming a privileged

accumulation of quadruplex ligands in the nucleoli of A459 cells after live-cell incubation

(Figure2c).72Thisobservationopenedthewaytowardsaredefinitionofthein-celltargetsof

quadruplex ligands, which challenge the ribosomal biogenesis efficiently. However, these

imagesfailedinprovidedirrefutableargumentsabouttheexistenceofquadruplexesincells,

andhighlighttheneedofdesigningspeciallydedicatedmoleculartoolstothisend.

IVb.Fromfluorescentdyestoquadruplex-specificprobes

Anotherapproachwasthusdevisedwiththeaimofprovidingundebatableevidenceabout

therelevanceofquadruplexesincellularcontexts.Thisstrategywasbasedontheuseof–and

then, the design of new systems from–commercially available nucleic acid stains. This

approachwasyetthoroughlyfollowedbut,asfurtherdetailedbelow,didnotaddressallthe

aforementionedpitfalls.

IVb.1.Fluorescentdyeswithnon-optimizedquadruplex-interactingproperties.

TheillustrativeexampleofafirmlyestablisheddyethatfoundnumerousapplicationsasDNA

stainingagentisthiazoleorange(TO).Itsabilitytointeractwith(owingtoitspositivecharge)

and label nucleic acids in a “turn-on” manner (RIR mechanism) whatever its secondary

structurewasthoroughlyexploited,notablyforthedevelopmentoffluorescentintercalator

displacement(FID)assaysusedtoassessandquantifytheapparentaffinityofsmallmolecules

toduplexes76 and/orquadruplexes.77However,TO doesnotdiscriminatebetweennucleic

acidstructures,makingitofpoorinterestforthedetectionofquadruplexesincells.Thioflavin

T(ThT)isacloselyrelateddyewithbetterquadruplex-versus-duplexdiscriminatingability.78

Itwasusedforvisualizingquadruplexesinpolyacrylamidegels79andalsofoundtobesuited

for cellular imaging (Figure 3a).80 Collected images indicated a privileged accumulation of

quadruplex-specific probes in the nucleoli of cancer cells (MCF7), both for post-fixation

labelling (5µM, 15min) or via live-cell incubation protocols (5µM, 24 h). These results

indicatedthatThTmightinteractwitheitherribosomalDNA(rDNA)orribosomalRNA(rRNA),

orboth.TheRNA-interactingpropertiesofbothThTandthecloselyrelatedanalogThT-NE

werefurtherinvestigatedusingthetwoprobesforenlighteningviralRNA(vRNA)inhuman

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hepatoma cells that express the full-length RNA genome of the hepatitis C virus (HCV).81

Obtainedimages(Figure3b)demonstratedaclearaccumulationofthefluorescentfociwithin

thecytoplasmoflivingcells(1µM,10min),makingthisvRNA“turn-on”systemapromising

strategy for viral infection diagnosis. Another ThT analog, N-Isopropyl-2-(4-N,N-

dimethylanilino)-6-methylbenzothiazoleorIMT,wasusedforlabellingquadruplexesinboth

fixedandlivinghumancervicaladenocarcinoma(HeLa)cells(Figure3c).82IMTturnedoutto

beparticularlysuitedforlive-cellinvestigations,allowingformonitoringthemodificationsof

theDNAquadruplexlandscapesreal-time(4µM,0-90min),notablyupontreatmentwiththe

quadruplexligandsPDS,RHPS4andTMPyP4.83

Figure3.Imagesof(a)livingMCF7cellstreatedbyThT(yellowfoci,thenucleibeingcounterstainedwith

SYTO59,inred),80(b)livinghepatomacellsinfectedbythehepatitisCvirustreatedbyThT-NE(greenfoci,the

nucleibeingcounterstainedwithDAPI,inblue),81and(c)livingHeLacellstreatedbyIMT(redfoci,thenuclei

beingcounterstainedwithDAPI,inblue).82

Theseresultswereinterestinginthattheyshowedthattheuseofcommercialdyes(or

closelyrelatedanalogues)indeedenlightenwhatmightcorrespondtoquadruplexlandscapes

incellsinastraightforwardmanner.However,onlyadditionalmanipulations(DNase/RNase

treatments, co-incubation with firmly established quadruplex ligands, etc.) provide more

circumstantial evidence of the bona fide relevance of the observed patterns. Also, using

commercial dyes (or closely related analogues) leads to an a posteriori analysis of the

fluorescentpatternratherthananaprioricontroloverthedyestrajectory.Therefore,inspired

by this wealth of promising data, numerous chemical programs have been launched to

improvetheperformancesofTO/ThT-basedquadruplexprobes.

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IVb.2.Fine-tuningthequadruplex-interactingpropertiesoffluorescentprobes

The two chemical motifs found in the TO scaffold, i.e., theN-methylquinolinium andN-

methylbenzothiazoliummoieties(thelatterbeingalsofoundintheThTscaffold),weretruly

inspirationalandsubsequentlythoroughlyexploitedwiththehopeofobtainingquadruplex-

specificprobeswithanalternativecellularactivity.Forinstance,thep-surfaceoftheTOwas

extendedwithabenzofurantoobtainthebenzothiazole-fusedbenzofuroquinoliniumdye1,84

whichproveduseful for labelling livingMCF7cells (2µM,15min),highlightingnuclei and

nucleolionly(Figure4a).Higher-resolutionimageswerecollectedwithastyryl-substitutedTO

referredtoas4a,85whichwasusefulforlabellingbothliveandfixedhumanprostatecancer

PC3cells(5µM,15min),thelatterprovidingaclearvisualizationofanucleolaraccumulation

ofthedye(Figure4b).Thep-conjugationwasfurtherexpandedintroducing3-vinylindolearms

buttheimagesoflivePC3cellsincubatedwithcompound3c(5µM,5min)demonstrated,

again,thatthe localizationoffoci remainedgloballycenteredonthenucleoli (Figure4c),86

withoutprovidingdeeperinsightsintothelocalizationofquadruplexesincells.

Figure4.Nuclearlabellingof(a)livingMCF7cellstreatedbythebenzofuroquinoliniumdye1,84andofliving

PC3cellstreatedbythestyryl-substitutedthiazoleorange4a(b)85andthevinylindole-substituedN-

methylquinolinium3c(c).86

AhigherlevelofprecisionwasreachedwithN-methylbenzothiazolium-basedprobes.

Forinstance,thecyanineCyTwasfoundtobeparticularlysensitivetoRNAquadruplexesand

suitedtothedetectionofRNAquadruplexesinlivingA459cells(1.25µM,24h),thefocibeing

exclusivelylocalizedinthecytoplasmofthecells(Figure5a).87Anotherbenzofuroquinolinium

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dyenamedCYTOwasimplementedwithbothliveandfixedPC3cells(1µM,30min)andleads

toaninterestingnuclearpatternfromwhichnucleoliwerefoundastheprivilegedlabelling

sites along with some–unfortunately unexploited–minor nuclear foci (Figure 5b).88 The

squaraine-basedcyanineCSTSwasfoundlivecell-compatible(2µM,30min)andaccumulated

in the lysosomesofMCF7 cells.89 Similarly, another cyaninenamedDMOTYwas found to

accumulate in lysosomesof livingHeLa andMCF7 cells (10µM,4h) andbe fluorescently

responsive to the presence of quadruplexes (Figure 5c),making it a promising autophagy

probes.90

Figure5.Labellingof(a)RNAquadruplexesbyCyTinlivingA459cells(redfoci,thenucleibeingcounterstained

withHoechst33258,inblue),87(b)thenucleoliofPC3cellsbyCYTO(redfoci,thenucleibeingcounterstained

withDAPI,inblue),88and(c)thelysosomesoflivingHeLacellsbyDMOTY(greenfoci,co-incubatedwith

LysoTracker,inred).90

N-methylquinolinium-basedprobeswerealsofurtherdevelopedandstudied.Twored-

emittingdyes(withemissionwavelength(lem)>650nm)werenotablydeveloped: ISCH-1,

whosestructureresultsinmergedisaindigotoneandcoumarinescaffolds,wasusedwithboth

fixedandlivingA459andHeLacells(5µM,30min)andleadtoanaccumulationoffoci in

nucleoli (Figure 6a), presumably targeting rDNAquadruplexes as further demonstratedby

ChIPassay;91NCT,whosestructure results inconjugatedTO andcoumarinescaffolds,was

used with living HeLa cells (1 µM, 15 min) and found to accumulate exclusively in

mitochondrialDNAquadruplexes,92asdemonstratedbyco-localizationwithMitoTrackerTM

(Figure6b).Averycompletestudywasrecentlyreportedusingthecoumarine-basedQUMA-

1forlabellingquadruplexesinbothfixedandlivingHeLacells.93Thefixedconditions(1µM,

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15 min) were useful not only to show the very high selectivity of QUMA-1 for RNA

quadruplexes(cytoplasmicfocialongwithsomeisolatednucleolarsites,Figure6c)butalsoto

performcompetitiveexperimentswithRNAquadruplex-interacting ligands suchascPDS,39

whichresultsinthelossofQUMA-1foci.Live-cellconditions(0.5µM,3h)uniquelyprovided

insightsintothedynamicsofRNAfoldingandunfolding,notablyuponadditionofthehelicase

DHX36,whichunfoldsefficientlyRNAquadruplexesresultinginthecompletedisappearance

ofthecytoplasmicfoci.

Figure6.ConfocalimagesoffixedHeLacellstreatedby(a)ISCH-1,91(b)NCT(redfoci,co-incubatedwith

MitoTracker,inred,resultinginyellowfoci),92and(c)QUMA-1(redfoci,thenucleibeingcounterstainedwith

DAPI,inblue).93

Tremendous results have thusbeen collected through thesenovel strategies. Fine-

tuning thechemical structuresofestablished fluorophoreshas indeed lead toquadruplex-

specific systems that illuminated the versatility of the quadruplex landscapes in cells.

However–and again–, additional manipulations (enzymatic digestions, co-incubation with

ligands,helicasesorspecificantibodies,etc.)werestillrequiredtodemonstratetherelevance

of these approaches, and some pitfalls still exist, notably the influence of local

microenvironments(pH,viscosity,etc.)thatmighttriggerthefluorescenceofthedyes ina

quadruplex-independentmanner.Smarter–andevenmoresophisticated–molecularsystems

were thus required to provide ever more convincing evidence about the existence of

quadruplexesincells.

14

IVc.Fromquadruplexligandstoquadruplex-specificprobes.

Anotherstrategywasthusimplementedinthehopeofprovidingmoleculartoolswithfirst,a

high affinity and selectivity for quadruplexes and then, the capacity of delivering readily

detectableoutputsonceininteractionwiththeirtargets.Thisstrategywasmostlybasedon

themodificationsofthechemicalstructureofwell-establishedquadruplexligands.

IVc.1.Optimizingthescaffoldofknowquadruplexligands

As indicated above, fusing known quadruplex-interacting and TO scaffolds was soon

envisionedasawaytodesignquadruplexprobessuitedtocellular investigations.Thiswas

pioneeredfusingtheTOscaffoldwiththepyridodicarboxamide(PDC)coreof360Atoprovide

PDC-M-TO,94 with the acridine core of BRACO-19 to provide a dual, pH sensitive and

quadruplex-specificprobe,95andwithanisaindigotonecoretoprovideISCH-1(videsupra),91

whichwastheonlydyefromthatseriesthatwasattemptedincells.Somewell-knownnucleic

acid stacking motifs were also revisited with successes, such as the quinacridone motif,

notablywithQABthatwasmadewatersolubleandbioavailableviatheadditionofsolubilizing

amine appendages,96 the anthracene motif, notably with 9CI that was used for labelling

exogenous quadruplexes,97 or the carbazolemotif, whose quadruplex labelling properties

havebeenthoroughlystudied,spurredonbythecompellingresultsobtainedwithBMVC63

anditscloselyrelatedanalogo-BMVC.Thesetwocarbazoleswerethefirstprobesusedfor

thedetectionofquadruplexinlivinghumanlungcancerCL1-0cells(5µM,2h),beingsuited

to fluorescence lifetime imaging microscopy (FLIM) that enhanced the resolution of the

collectedimages(Figure7a).98Theylocalizeddifferently,BMVCbeinglocatedinthenucleus

witho-BMVCinthecytoplasm,likelyinmitochondria.ThecarbazolesthatbelongtotheBMVC

familyfoundnumerousapplications:theyhavebeenusedtotrackexogeneousquadruplexes

inCL1-0 cells (BMVC),99 to illuminatemitochondrialDNAquadruplexes in livingHeLa cells

(BMVC-12C-P,5µM,24h;o-BMVC-12C-P,5µM,4h),100toimplementabiosensingassayfor

thedetectionofcancerversushealthycells(o-BMVC,5µM,10min,includingfixedcancerous

MCF-7andCL1-0cellsversusnon-cancerousfibroblastMRC5andBJcells,applicableintissue

biopsiesaswell),101andtoassessthemodificationsofthequadruplexlandscapesinHeLaand

MRC-5cellslive-treatedwiththequadruplexligandsBRACO-19andTMPyP4(10µM,16h)

afterfixation(o-BMVC,5µM,10min).102Manyotherderivativeshavebeensynthesizedand

studied for live-cell imaging, suchBPBC and BTC, found to localize in the cytoplasm and

15

nucleoliofMCF7cells(BPBC,10µM,2h),103andinthenucleoliofhumanlivercancerHepG2

cells(BTC,5µM,4h,Figure7b).104Finally,anotherwell-knownnucleicacidstackingmotifthat

wasimplementedforopticalimagingpurposeswasthenaphthalenediimide(NDI)unit.This

motifwasthoroughlyexploitedfortherecognitionofquadruplexesinvitro,68andexamples

werealsoreportedfortrackingthemincells.ThespectroscopicpropertiesofNDI,notablyof

core-extendedNDI (c-exNDI), arebasedonaquadruplex-mediateddisaggregationofnon-

emittingaggregates.LiveincubationofhumanembryonickidneyHEK293Tcellswithc-exNDI

(1µM,30min)allowedforanaccumulationoffociwithinthenuclei(Figure7c),whichpartially

overlappedwith1H6immunostaining,andmorepreciselyinthenucleoli,asdemonstratedby

nucleolindisplacementinvestigations.105

Figure7.(a)Time-gatedFLIMimagesoffixedHeLacellstreatedwitho-BMVC(redfocifordecaytimes≥2.4ns,

correspondingtoquadruplexes,greenfocifordecaytimes<2.4ns);102(b)nucleolilabellingofHepG2cellsby

BTC;104(c)nuclearlabellingofHEK293Tcellsbyc-exNDI.105

IVc.2.Addingtagstoknownquadruplexligands

Attemptshavebeenalsomadetocovalentlylinkafluorescenttagdirectlytoaquadruplex

ligandscaffold.Forinstance,aBODIPYhasbeenlinkedtoamacrocyclicheptaoxazole(L1BOD-

7OTD),106aTOandaBODIPYtothePDCcore(PDC-L-TO94andPDC-BODIPY,107respectively),

aCy5toaPDSderivative(PDP-Cy5)108andafluoresceintoatriarylimidazole(IZFL-2).109This

approachturnedouttobehighlyvaluableforinvitroquadruplexdetection:L1BOD-7OTDand

PDP-Cy5wereusedtovisualizequadruplexesbygelelectrophoresis,whilePDC-L-TO,PDC-

BODIPYandIZFL-2wereusedtodetectquadruplexesviafluorescencetitrations.Doingsoin

16

cellsrepresentedanotherlevelofdifficulty,giventhatthestructureofthequadruplexligand

was drastically modified by the fluorescent appendage, diverting its intrinsic properties

(pharmacodynamics,bioavailability)andtargetengagement.Despitethesepitfalls,PDP-Cy5

wassuccessfullyusedforthedetectionofexogenousquadruplexesinHeLacells(10µM,2h,

Figure8a).108

Figure8.Detectionof(a)exogenouslyaddedfluorescein-labelledquadruplexRNAinHeLacellsbyPDP-Cy5

(redfoci,flurosceiningreen,resultinginyellowfoci,thenucleibeingcounterstainedwithDAPI,inblue),108(b)

DNAquadruplexesinfixedU2OScellsthankstoPDS-aafterCuAACwithAF-594(redfoci,co-localizationwith

GFP-hPif1ingreen,thenucleusbeingdelineatedbyadottedwhiteline),110and(c)DNAquadruplexesinfixed

HT29,afterlive-treatmentwithPhenDC-azandSPAACwithCy5-DBCO.111

An elegant way to tackle these issues is to exploit the tremendous potential of

bioorthogonalchemistry.112Thiswasdemonstratedusinganalkyne-containingPDSderivative

PDS-a,110whichisminimallystructurallymodifiedascomparedtotheparentPDS,alteringits

biodistribution only marginally. The chemical labelling of PDS-a (1µM, 12 h) once in its

genomic sites within fixed human osteosarcoma U2OS cells using click chemistry with

AlexaFluor594-azideallowed for adirect visualizationofDNAquadruplexes (Figure8b), as

confirmed by co-localization experiments performed with the green fluorescent protein

(GFP)-labelledquadruplex-specifichelicasePif1. This approachwasextendedwithanother

17

well-established ligand,PhenDC,113usingboth copper-catalyzedazide-alkyne cycloaddition

(CuAAC) betweenPhenDC-alk andCy5-az, and strain-promoted azide-alkyne cycloaddition

(SPAAC)betweenPhenDC-azandCy5-DBCO(Figure8c).111Experimentswereperformedlive

incubatinghumancolonadenocarcinomaHT29cellswithclickablePhenDCderivatives(5 µM,

24h)priortofixationandcatalyzedornon-catalyzedAAC.Thecomparisonofthetwomethods

showedthatcautionmustbeexercisedwheninterpretingtheinsituclickchemistrylabelling

results,duetopossiblerelocationofintermediatemetalcomplexeswithinthenucleoli.

IVc.3.Speciallydedicatedsmallmoleculeprobes

Theleastdisruptivewaytolabelasmallmoleculeisisotopiclabelling.Thiswasdemonstrated

asearlyas in2005via studiesperformedwith thePDC360A.Thetritium-labeled 3H-360A

accumulated in the nuclei of living glioblastoma T98G cells (0.3 µM, 24 h) and the

autoradiographyofmetaphasespreadsof3H-360A-treatedcellsconfirmedthatgenomicDNA

was the target of the ligand (Figure 9a).114 Similar experiments performed with T-

lymphoblasticCEM1301cells(characterizedbylongertelomerethanT98Gcells)andnormal

peripheralblood lymphocyte(PBL)cellsclearlyshowedthepreferential (butnotexclusive)

binding of 3H-360A to the terminal region of the chromosomes, lending credence to the

relevanceoftelomericquadruplexesasoneoftheprivilegedtargetsforligands.Anotherway

nottodisruptthemolecularorganizationofaquadruplexligandtomakeitafluorescentprobe

istobuildthedyeexnihilo.ThiswasdemonstratedwithNaphthoTASQ(orN-TASQ)whose

designwasbasedonabiomimetic interactionwithquadruplexes.N-TASQ comprised four

guaninearmsthatself-assembleintoanintramolecularG-quartetuponinteractionwiththeir

quadruplextargets.Thesearmsarearrangedaroundafluorescenttemplate(anaphthalene

unit) whose fluorescence is “turned-on” by the formation of the template-assembled G-

quartet(TASQ),makingN-TASQatwice-as-smartquadruplexligand(bothasmartligandand

asmartprobe).N-TASQwasusedwithbothfixedandlivingcells, interactingpreferentially

with RNA quadruplexes and accumulating in sub-cytoplasmic sites (stress granules and P-

bodies)oflivingU2OSandMCF7cells(5µM,48h,Figure9b),115andlabellingeitherDNAor

RNAquadruplexesinfixedMCF7cells,dependingonthecellpreparationprotocol.116N-TASQ

wasalso founduseful toassess the themodulationofquadruplex landscapesupon ligand

treatments,includingTMPyP4andBRACO-19.117Anotherexampleofdedicatedquadruplex

probeisthetrianguleniumDAOTA-M2.118Thisprobeturnedouttobecompatibleforlive-cell

18

imaging performed with U2OS cells (20µM, 24h) in which it bound to nuclear DNA (co-

localizationwithHoechst33342andDRAQ-5)andmitochondrialDNA (co-localizationwith

rhodamine-123)butallowedforidentifyingquadruplexesviaFLIMimages(Figure9c),given

thatitsemissivelifetimeswasstronglydependentonthetopologyofthenucleicacidtargets

(higherlifetimes(t2values)uponinteractionwithquadruplexes(upto16ns)versusduplex-

DNA(<10ns)).

Figure9.(a)AutoradiographofmetaphasespreadsofT98Gcellslive-treatedwith3H-360A(blackarrows

indicatechromosomeends,redarrowsintrachromosomalsites);114(b)detectionofRNAquadruplexesinthe

cytoplasmofMCF7cellslive-treatedwithN-TASQ;116(c)generalstainingobtainedwithU2OScellstreatedwith

DAOTA-M2.118

IVc.4.Usingmetallicprobestotrackquadruplexesincells

AftertheinitialimpetusgivenbyQP3.In,manymetalcomplexeshavebeeninvestigatedas

quadruplex-specificreporterprobes.Thequadruplexfieldwasindeedafertileplaygroundfor

organic and inorganic chemists, owing to the manipulation of metal chelating moieties

(sometimes referred to as ligands, avoided here for disambiguation)with broad aromatic

surfaces (e.g., phen, dppn, dppz, dpq, etc.),119,120 prone to interact efficiently with the

accessiblequartetofquadruplexes.Whiledozensofmetalcomplexeshavebeenstudied in

vitro,67 far less complexeshavebeenused for cellular imaging,which canoriginate in the

globallylowerresolutionoftheobtainedimagesascomparedtothoseobtainedwithorganic

probes.Thedinuclearrutheniumcomplex[(phen)2Ru(tpphz)Ru(phen)2]wasforinstanceused

19

to labelthegenomicDNAofbothhumanMCF7cells (500µM,1h)andmouse lymphoma

L5178Y-R cells (characterized by longer telomere thanMCF7 cells; 200µM, 30min), but

allowedforidentifyingquadruplexesvialambdastackingexperiments(Figure10a),giventhat

itsemissivemaximaweredependentonthetopologyofthenucleicacidtargets(630-640nm

forquadruplexesversus670-700nmforduplex).121Thetetrakis-(diisopropylguanidinio)zinc

phthalocyanineZn-DIGPwasusedtolabelquadruplexesinlivingmelanomaSK-Mel-28cells

(3µM,2h)122ormouseembryofibroblastNIH3T3cells(2µM,24h,Figure10b),123revealing

foci localized inperinuclear regions,while themetal-free counterpart labellednucleionly.

Interestingly,the intrinsicchiralityofthemetalcomplexeswasfoundtoplayan important

roleinthebiodistributionoftheprobes.Forinstance,thetwoenantiomersoftheruthenium

complex[Ru(bpy)2(p-BEPIP)],referredtoasRM0627werefoundtoenterrapidlylivingbreast

cancerMDA-MB-231 cells (5 µM, 2 h) and accumulate either in perinuclear regions (the

dextrorotaryD-RM0627)orinthenucleiexclusively(thelevorotaryL-RM0627).124

Figure10.(a)Mutliple-emissionimagesofL5178Y-Rcellstreatedwith[(phen)2Ru(tpphz)Ru(phen)2](L=

phen),highlightingthegenomicDNAinred(emission:670-700nm)andthequadruplexfoci(emission630-640

nm)inyellow;confocalimagesof(b)NIN3T3cellstreatedwithZn-DIGP,123and(c)fixedMCF7cellsstainedby

Pt-SCC.125

20

Morerecently,theplatinumcomplexPt-SCC(forsupramolecularcoordinationcomplex)was

usedinfixedMCF7cells(50µM,30min,Figure10c)andleadtoanintensenuclearlabelling

andtheinteractionwithquadruplexeswasconfirmedviacompetitionwithquadruplexligands

(e.g., PDS, TMPyP4) and co-localization with quadruplex-specific antibody (BG4).125

Interestingly,Pt-SCCwasfoundtoprovideaduallabeling,fluorescentlystainingthegenomic

DNAononehandandincreasingthecontrastofnucleoliindirecttransmissionmode(DIC,for

differential interference contrast) on the other hand, thus providing anothermodality (so

called‘darkstaining’,relyingontheabsorptionoflightbythePt-SCC/quadruplexcomplex)for

theobservationofquadruplexincells.

Globallyspeaking, theoptical imagescollectedsofarwithmetalcomplexesdidnot

reachanexquisitelevelofresolution,whichcanbeattributedtothefactthattheirstructures,

for most of them, relied on planar metal chelating moieties that failed in creating

discriminatinginteractionswithquadruplexes,assimpleorganicintercalatorsdid.However,

thechemicalandstructuraldiversitythatuniquelyoffersthisfieldofresearch,alongwiththe

variousmodalitiesof imagesthatcanbe implementedwithmetalcomplexes,arethebest

promisesforfutureadvancesandsuccesses.

V.Conclusion.

Thedetectionofquadruplexinhumancellswasandremainsachallengingtask.Inspiteofthe

massiveeffortsthathavebeeninvestedforalmost20yearsnow,noturnkeysolutionshave

been delivered and the examples discussed above highlighted that only multipronged

approaches(co-localizationofsmallmoleculeprobesandantibodies;competitionbetween

establishedligandsandprobes;etc.)canprovidesolidevidenceabouttheirexistenceincells.

The visualizationof quadruplexes involves probes (antibodies, chemicals) suited to optical

imagingbuttheverynotionofdetectionofquadruplexesisboarder:italsoencompassesthe

sequencing-based techniques that have been developed recently to gain insights into the

relevance and prevalence of the quadruplex landscapes.126-128 Once again, the role of

quadruplex-specific antibodies and chemicalswas instrumental in these investigations: for

instance,theantibodyBG4wasthecentralmoleculartoolforthedevelopmentofG4ChIP-

seq(videsupra),41whichassessedtheformationofDNAquadruplexesinthehumangenome;

thesmallmoleculePDSwasusedinbothG4-seq129andrG4-seq130topromoteand/orstabilize

21

DNAandRNAquadruplexes, respectively, tomakethemdetectableduringthesequencing

analysissteps;andbiotinylatedPDS131andTASQ-basedBioTASQ132wasusefultopurify(pull-

down)andidentifyquadruplexes(i.e.,G4RP-seq).133Theseexamplesdemonstrateagainthat

(bio)molecular tools created to detect quadruplexes have to be versatile to be used in

complementaryfields,sinceonlycross-disciplinaryinvestigationscanprovidereliableinsights

intothecellularrelevanceandfunctionsofquadruplexes.Theseexampleshavealsocasteda

brightlightonantibodiesandsmallmoleculesbutscientistshaveotherstringsintheirbow,

such as fluorescently labelled proteins (e.g., the helicase GFP-hPif1, vide supra) or short

oligonucleotides (e.g., the oligonucleotide-templated reactions (OTR)134 or G-quadruplex-

triggered fluorogenichybridization (GTFH)135 approaches). This highlights theneed for the

quadruplexcommunitytobothkeepgrowingandpulltogethertodeveloptransdisciplinary

researchprogramsaimedatprovidingevermoreconvincingevidencetosupporttherapeutic

strategiesbasedonthequadruplextargetingwithadhocdrugs.

“Youwillneverbeabletohitatargetthatyoucannotsee”.Wearenowabletohitthis

target.Theexamplespresentedaboveshowedthatscientistshavenowapanelofmolecular

lightstoenlightenquadruplexesinourgenomeandtranscriptome,atargetthatcannolonger

hide–andwillbehit–evenintheremotestpartsofthehumancells.

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