amplified catalytic detection of nucleic acids- …...amplified catalytic detection of nucleic acids...

AmplifiedCatalyticDetectionofNucleicAcids RijksuniversiteitGroningen

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AmplifiedCatalyticDetectionofNucleicAcids

JaspervanderVelde,Prof.A.HermannZernikeInstituteofAdvancedMaterials,UniversityofGroningen,TheNetherlands

18‐06‐2011

AbstractTemplatedcatalyticreactionshaveemergedasapowerfultoolforthedetectionanddiagnosticsforDNAandRNA. Detection schemes that allow the amplification of the signal are of high interest in the search ofpolymerase chain reaction free nucleic acid diagnostics. The basis of templated catalytic reactions is thecatalyticactivityof templatewhichcantriggernumerousreactionfor thegenerationofsignallingmolecules.Becauseofthecatalyticactivityofthetemplate,asingletemplatecangivemultipleturnovers.However,therearethreeimportantpointstoconsiderinthedesignofreactiveprobes:thereactivityofthefunctionalgroups,affinityforthetemplateandthereadoutsystem.Recently,fluorogenicreactionshavebeenexploitedforthedetectionofnucleicacids.Fluorogenicreactionsareparticularlypromisingbecausetheysimplifyandshortenthe reaction protocol. The different reactions exploited for the generation of a fluorogenic signal can beclassifiedintotwoclasses.Thefirstclassisbasedontheactivationofafluoresceinwhereasinthesecondclassaquencheriscleavedwithaccompaniedfluorescenceenhancement.Bothclassesofferthepossibilityforthedetectionofnucleicacidmoleculesinlivingcells.Additionally,theresearchoftemplatereactionsopensadoortopositionspecificdrugrelease.

ListofContents1. Introduction 2

1.1. BackgroundandPerspectives 22. BasicPrinciplesofNucleicAcidTemplateReactions 4 2.1. BasicPrinciple 4 2.1.2. SelectivityofNucleicAcidTemplatereactions 4 2.2.2. ParametersinNucleicAcidDetection 5 2.2. TriggeredChemicalReaction 73. FluorogenicTemplateChemicalReactions 7 3.1. ActivationofaFluorescein 7 3.1.1. StaudingerReaction 8 3.1.2. MetalMediatedActivationofaFluorescein 9 3.2. FluorescentEnhancement 10 3.2.1. TransferReactions 10 3.2.2. QuenchedAutoligation 12 3.3. NonetheredReagentsinNucleicAcidTemplateReactions 134. Discussion 14

4.1. NucleicAcidDetectioninLivingCells 144.2. SequenceSpecificFluorogenicDetectionofDoubleStrandedDNA 15

5. FutureProspects 166. Conclusion 17


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1. Introduction

Afundamentalbiochemicalfeaturethatiscommontoallcellularorganismistheuseofnucleicacidsequencesforthestorageofgenetic information,calledDNAandRNA.1Alongwith the discovery that nucleic acid sequences – mostly RNA – control critical cellularfunctionsandareunique foreach cellularorganism, thedevelopmentofmethods for thedetection and identification of nucleic acid sequences has gained an increase interest. Todate, the detection of specific DNA and RNA sequences is used to explore genes andgenomes inorganisms, in forensicapplicationsandmost importantly in thehealthcare forthe identificationof infectiousorganisms anddiagnosis and/orprognosesof diseases, likecancer. To combat these diseases effectively, accurate detection in an early stage of thedisease is of great importance. In combinationwith the focus ofmedicine on the geneticcauseofdiseases,which is linkedto thedevelopmentofmorepersonalizedmedicine, thisleads to thedemandofultrasensitivesequencespecificnucleicaciddetection. Inmodern‐dayresearch,PolymeraseChainReaction(PCR)isthestandardmethodforthedetectionofnucleicacidmolecules.2However,PCRhas somesignificantdrawbacksand researchesarecontinuouslylookingfornewmethods.Templatedcatalyticchemicalreactionshaveturnedout to be promising candidates for ultrasensitive and selective detection of nucleic acidsequences.Particularlyappealingarefluorescentapproachesthatsignificantlyshortenandsimplifythedetectionschemebecauseofthelackofseveralpurificationsteps.3Additionally,templatedfluorescentreactionshavethepotentialtobeusedinintactcells.1.1. BackgroundandPerspectives

The standardmethod for thedetectionofDNAandRNA–nucleic acidmolecules – inpresent day biology, diagnostics and genomics is based on the enzymatic amplification ofnucleicacidmoleculesbyamethodcalledPolymeraseChainReaction(PCR).2PCRrepresentthe ultimate limit of nucleic acid sensitivity. In PCR single ormultiple copies of a specificnucleic acid sequence are amplified several orders ofmagnitude before being indentifiedandisolatedafterwards.2However,PCRhassomesignificantdrawbacks.

Enzymatic methods like PCR require optimized conditions for the enzymes to achievehighly specific and selective amplification. As a result it is a complex and costlymethod.Besides, PCR is highly sensitive to contamination causing amplification of spurioussequences.Toavoidtheamplificationofspurioussequences,variousreagentscanbeaddedorthecatalyticpropertyoftheenzymehastobeimprovedmakingPCRevenmorecomplex.If multiple targets need to be detected, multiplexing, PCR can not be used. That is thedetection of multiple distinct target sequences in a single assay is not possible. Finally,because PCR is an enzymaticmethods it can not be used in intact ells because it is verydifficulttogettheenzymesthroughthemembrane.4Moreover,PCRonlyamplifiesatargetsequencewhichthenstillneedstobedetected.Thisalsolimitstheuseinintactcells.Atthispoint it is worth saying that chemical reactions triggered by the hybridisation ofoligonucleotides to target nucleic acid sequences can take place inside cells as there aremultiple potentialmethods for getting oligonucleotides into the desired cell.5‐7 Note, thatnoteachchemicalreactionisbiocompatible.

FullSangersequencingisanucleicaciddetectionmethodovercomingsomeoftheshort‐comingsofPCR.Itisatechniquewhichallowseverynucleotidetobeindentifiedonebyone.


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However, full Sanger sequencing is also an enzymatic method. Note, that although fullSangersequencingisanenzymaticmethoditsefficiencyisincreasinganddroppingincost.8 Because of the previously mentioned drawbacks of enzymatic methods, scientist arecontinuouslydevelopingnewnon‐enzymaticmethodsforselectiveandsensitivenucleicaciddetection. A promising candidate is the use of Molecular Beacons (MB) in which uponbindingofaoligonucleotideprobestothetargetnucleicacidsequence,thefluorescenceofaninternallyquenchedfluorophoreisrestored.9Although,MBarebasedonoligonucleotideprobesandcanthusprofitfromthetechniquetointroduceitintocells,5‐7itonlyyieldsonesignal per target which can give problems for detection if only a small number of targetmoleculesarepresent,orifthereisahighbackgroundsignal.ForMBtobeusefulinnucleicacid detection, amethod for the amplification of the signal has to be applied. Templatedchemicalreactionshowever,couldinprinciplereachmultipleturnoverpertargetmoleculeand thus amplifying the signal in situ without the need of an additional amplificationmethods. Basedonthebriefpreviousdiscussion,templatedchemicalreactionsbelongtothemostpromisingcandidatesforsensingofnucleicacidsequences.Intemplatedreactionsthesignalis amplified without the need of an additional step. The use of a chemical reaction forobtainingasignaloffersalsothepossibilitytodesignareactionthatisorthogonaltootherprocesses occurring in the sample obtaining a potentially clear on‐off signal ratio.10‐11 Theaimofthispaperistodiscussrecentadvancedinthedevelopmentofcatalyticreactionsthataretriggeredbythepresenceofatargetnucleicacidsequence.First,PCRisbrieflydiscussedfollowedbyadiscussionofwhyfluorescentreactionstriggeredbythehybridisationofshortDNAstrands–oligonucleotides–offeradvantagesandsolveproblemsobservedwithothermethods.


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2. BasicPrinciplesofNucleicAcidTemplateReactions In the previous section it was briefly discussedwhy templated chemical reactions arepromisingcandidatesincomparisonwithPCRandMB.Itwashowevernotsaidwhynucleicacid templated reactions are highly selective. In this section the basic principles andimportantconsiderationsofnucleicacidtemplatedreactionswillbediscussed.

2.1. BasicPrinciple

Template reactions are performed frequently in synthetic chemistry and nucleic acidchemistrytoperformchemicalreactionsthatarenotefficientintheabsenceofaso‐calledtemplate. A template is a particle (molecule or atom) which binds non‐covalently toreactants. Upon binding the reactive groups are aligned which results in an increasedeffectivemolarity(Figure1).Thisincreaseintheeffectivemolarityresultsinaccelerationoftemplate reactions and allows selective reactions to be performed at low reactantconcentrations comparedwith conventional synthesis, the reaction is catalyzed. Innucleicacid templated reactions the template is representedby the targetnucleicacid sequence,DNAorRNA.Inprincipletheprobe(s)couldberepresentedbyanymoleculethatcanbindtoanucleicacidtarget.However,inthisstudytheprobesareshortsyntheticoligonucleotidesstrandswhichbindwithahighpredictableaffinityandhighselectiverecognitiontonucleicacidsequences.10‐11

Figure1:schematicrepresentationofaNucleicAcidTemplatedReaction.theshortsyntheticoligonucleotidesrecognizesauniquenucleicacidsequenceandbindstoitviaWatson‐Crickbaseparing–hybridisation.Uponhybridisationthefunctionalgroupswillreact.

2.1.1. SelectivityofNucleicAcidTemplatereactions With the discovery of DNA molecules in 1953 by James Watson and Francis Crick itturnedout thatDNA isbuild from fourdifferentbuildingblocksand is responsible for thestorageofgeneticinformation.1Inmanycellularorganismsandviruses,RNAisalsousedasgeneticmaterial.Additionally,RNAperformsmanycriticalcellularfunctions.BothDNAandRNAare linearpolymers, callednucleic acidmolecules,witha fixedbackbone fromwhichprotrudevariablesubstituents.Thebackboneisbuiltofrepeatingsugar‐phosphateunits.InDNA the sugars are molecules of deoxyribose from which the name DNA is derived(Deoxyribonucleic acid), whereas in RNA (Ribonucleic acid) the sugar is referred to asriboses.InbothDNAandRNAthesugarsarelabelledwithoneoffourpossiblebases.InDNAeach of the deoxyribose is linkedwith one of the four possible bases; Adenine, Cytosine,GuanineandThymine(Figure2).InRNAThymineisreplacedbyUracil.Thus,eachmonomer


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of DNA or RNA consists of a sugar‐phosphate unit with one of the four possible basesattachedtothesugar.Eachmonomeriscalledanucleotide,thereforeDNAandRNAarealsoreferredasnucleicacidmolecules.Thebasescanbearranged inanyorderalongtheDNAandRNAstrand.Thisvariablesequenceofbasesactsasanveryefficientwayof storinggenetic information,aseachdistinctsequencecanrepresentadifferentmessage.1

MostDNAstrandsareaccompaniedbyasecondstrand(Figure3).ThestructurethatWatsonandCrickproposedis composed of two intertwined DNA‐strands where thebasesare locatedon the insideand the sugar‐phosphatebackbone on the outside– this the famous double helixstructure(Figure3).Thehelicalstructureisaresultofthefactthateachsugarpointsinthesamedirection.Themostimportant observation deduced from this double helix isthe formation of specific base pairs which are heldtogether by hydrogen bonds (Figure 2). The base paringonlyoccursbetweenspecificbases:Adenine(A)pairswithThymine(T)andGuanine(G)bindswithCytosine(C).Itcanbe imagined that because of this paring, onlycomplementarystrandscanformadoublehelixstructureInotherwords,thesequencealongonestrandcompletelydeterminesthesequencealongtheotherstrand.It isthisspecificbasepairingwhichmakesnucleicacidtemplatereactionshighlyselective.Innucleicacid templated reactions the oligonucleotide probes can only bind with a strand that iscomplementary to eachown sequence.With this inmind, theoligonucleotideprobeswillbindwithahighaffinitytoitstargetsequences.Key‐processesincellularorganismdependontheself‐recognitionofnucleicacidssuchasreplication,transcriptionandtranslation.1

Figure3:Thedouble‐helicalstructureofDNAproposedbyWatsonandCrick.Thesugar‐phosphatebackboneofthetwochainsisshowninredandthebasesarehighlightedwiththeirrespectiveabbreviation.Thestrandsareantiparallel,runninginoppositedirectionswithrespecttotheaxisofthedoublehelixstructure.

2.1.2.ParametersinNucleicAcidDetectionTable1.Parametersthatareof importanceinnucleicacidtemplatedreactionsforthedetectionofnucleicacids11Target Reactionconditions EnvironmentDoublestrandedSinglestrandedGeometricstructure

TemperaturePHIonicStrength

SolutionSolidSupportCellularenvironment

Figure2:thespecificbasepairingbetween the four distinct bases:Adenine, Thymine, Guanine andCytosine.


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Forthedetectionofspecificnucleicacidsequences,thetargetnucleicacidsequenceisused as the template and triggers a chemical reaction. Because of the inherent nature ofnucleic acidmolecules, this approach is highly selective andproduct is only formed if thetargetsequenceispresent.Forthetemplatedapproachtobeofuseinnucleicaciddetectionafewfactorshavetobeconsidered.

The first important factor that has to be considered is whether the target analyte iseitheraDNAorRNAstrand.Additionally,itisimportantifthetargetstrandsaredoubleorsingle stranded. RNA‐strands have the potential to bindmore strongly to oligonucleotideprobes than DNA strands and therefore product inhibition can form a problem in thedetection of RNA.1 Secondly, DNA strands are mostly accompanied by a complementarystrand.IfthetargetstrandisalreadyinvolvedinWatson‐Crickbaseparingitismoredifficulttouseitinnucleicacidtemplatedreactions.Therecognitionstepoftheoligonucleotidesisinhibited.11Tosolvethisproblem,syntheticoligonucleotideprobeswithastrongeraffinitytothetargetstrandcanbeused,thatisthefreeenergyofbindingwiththeprobesislarger.Another procedure which can be used to overcome the previous mentioned problem ispartialdenaturationofthetargetstrandbeforeoneaddstheoligonucleotideprobes.1Ascanbeseeninfigure2,thespecificbasepairingoccursbecauseofhydrogenbonding.Increasingthetemperatureandhencetheenergyofthestrandswilldisruptthehydrogenbondingandthestrandswillcleavefromeachother.1,11Notehowever,thatadenaturationsteppriortodetection is time‐consuming. This articlewill focusmainly on single stranded nucleic acidsequences.Inthiscase,however,stilltheoccurrenceofstemsandloopsinasinglestrand,inwhich strong base pairing occurs, has to be considered – the geometric structure of thetargetanalytehasaninfluenceonthedetection.1,12‐13

A second remark concerns the physical condition in which the templated catalyticreactionsarecarriedout.Themostimportantphysicalconditionthathastobeconsideredisthe temperature. At higher temperatures, strands with single basemismatching can alsohybridizewiththeprobesandfunctionastemplateforthechemicalreaction.Ontheotherhand, at lower temperatures, the probes can hybridize with the target strand butsubsequentlydonotcleaveand the templated reactionbecomes inhibited.Asa result,anoptimumtemperaturehastobefind.1,11Secondly,theacidandionicstrengthofthereactionmixtureisofimportanceasthesecaninfluencethehybridisationoftheprobestothetargetstrand and the subsequent triggered chemical reaction.1,11 Also the environment inwhichthetemplatedchemicalreactionisperformedmustbekeptinmind.Forexample,acellularenvironmentiscompletelydifferentthansensingthatiscarriedoutonasolidsupportwhichcanbewashedseveraltimesbeforetheidentificationoftheDNAorRNA.1,11

Finally, the length of the synthetic oligonucleotides is of influence on the templatedchemical reaction. The synthetic oligonucleotides strand must be long enough such thatsufficientlystrongbindingoccurs.Astrongerbindingbetweentheprobesandtargetallowsfora longer timescale inwhich theprobescanbind to thesametargetandsubsequentlyallow the chemical reaction to occur.14 Additionally, the probe must have an number ofoligonucleotides that isat least longenoughsuchthat itonlybinds toaspecific target,asshortoligonucleotidesequencescanoccurmorefrequently inDNAorRNAstrands.Probesconsistingofatleast18nucleotidesarenecessarytoacquirespecificrecognition.However,theprobemustnotbetoolongasalongerprobesbindsmorestronglytothetargetandarelesssequencespecific, that is the longer theprobethemorebasemismatchescanoccur.6Also,longerprobesaremorecostlyinpreparation.15


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2.2. TheTriggeredChemicalReactions

Although high‐rate accelerations may be obtained for the chemical reaction incomparisonwith the reaction in theabsenceof the template, turnoverof the reactants isoften impeded because the reaction may give products with a higher affinity for thetemplate. Product inhibition is the most significant problem in nucleic acid templatereactions.Itwasmentionedintheprevioussectionthattheoligonucleotideprobemustnotbe to long but long enough that it can only bind to specific regions of a target strand.Nevertheless, a high turnover number is needed for a sufficient signal amplification.Hereopportunitieslietodesignasysteminwhichproductinhibitioniscircumvented.10‐11Becausetheultimategoal is todesignageneral system for thedetectionofnucleicacids inwhicheachrandomsequencecouldinprinciplebedetected,reactiondesignratherthantemplatedesign has to be explored. Different reaction types have been explored in recent yearsincluding ligation, cleavage, hydrolysis and transfer reactions as well as organometalmediated reactions. In the remainder of this paper these different reaction types will beexplored with the focus on those reactions in which a fluorescent group is formed oractivated.

3. FluorogenicTemplateChemicalReactions Intheprevioussectionthebasicprinciplesofnucleicacidtemplatedreactionshavebeendiscussed,and itwasarguedthatnucleicacidtemplatedreactionsarehighlyselectiveandsensitive.Severalchemicalreactionshavebeenexploredinnucleicacidtemplatedreactionswiththepurposeofultrasensitivenucleicaciddetection.Fluorogenicreactionshavealreadybeen proven to be a excellent tool in biodetection schemes. Fluorogenic systems are forexample extensively used for the labelling of proteins and visualisation of biologicallyprocesses.19‐22Asaresult,fluorogenicreactionshavealsobeeninvestigatedinthesearchofultrasensitivenucleicaciddetection.Besidesfluorogenicreactionsareparticularlyappealingbecause these can significantly shorten and simplify the detection protocol by obviatingseveralpurificationstepsincludingtheextractionofDNAorRNAstrandsoutofthecells.23‐26Itwasalsomentionedinprevioussectionsthatitishighlydesirabletohaveareactionwherethe template can reach a turnover number to provide a sufficient fluorescent signalwithsingle nucleotidemismatch resolution. To date, fluorescent reaction canbe classified intotwo types of reactive probes. First, non‐fluorescent probes recognize a target and uponhybridisationareactionistriggeredwhichresultsinatheactivationofafluorescentproduct.The second type of reactive probe relies on the cleavage of a quencher moiety uponhybridisationoftheprobesandsubsequentenhancementofthefluorescence.

3.1. ActivationofaFluorescein

Thefirstclassoffluorescenttemplatedreactionstobeexploredarethosereactiontypesin which a fluorescein is activated upon hybridisation of the probes. Although there aremany fluorescent functional groups in chemistry which can be activated by a chemicalreaction,notallchemicalreactionsaresuitedfortemplatedreactions.Inviewofnucleicaciddetection two types of chemical reactions are extensively used. The first reaction type to


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consider is the Staudinger reaction which offers some significant advantages over otherreaction types. The second reaction which will briefly be considered aremetal mediatedreactions.

3.1.1. StaudingerReaction

An important chemical reaction used for the detection of nucleic acids by fluorescentmeansistheStaudingerreaction.InaStaudingerreactionanazidereactswithaphosphineto produce an aza‐ylide intermediate. spontaneous hydrolisation yields an amine and theoxidized formof thephosphorusgroup.16Bertozzihad shown in2003 that theStaudingerreactioncouldbeusedtoactivateafluorophoreasaresultofthephosphorusoxidation.17In2004Taylorandco‐workerspresentedaconceptinwhichtheStaudingerreactionwasusedto switch on a fluorescent signal. Here, an ortho‐acylated fluorescein‐probe was able toreactwithanazidethatwasconnectedtoasecondprobe.18Whenevertheprobesbindthetemplateonadjacentpositions,theacylmoietyistransferredtotheazideyieldinganamideandafluorophore(Figure4).Inotherwordstheazideisreducedbythetriphenylphosphatemoietygeneratingafluorescentsignal.A188‐foldincreaseintherateofthereactioninthepresence of theDNA template is observed. additionally, the reaction showed amismatchdiscriminationwitha30‐fold increase in initial ratefora fullymatchedversusmismatchedtemplate.However,thereactionhasnotbeendescribedwithsubstoichiometricamountsofthetemplate.

Figure4:ActivationofaFluorophorebyatemplatedStaudingerreaction.

The results of Taylor and co‐workers show that the Staudinger reaction is particularlywelladaptedtotemplatedsynthesisandcouldbeusedtoactivateafluorescein.Therefore,researcheshavebeenutilizingtheStaudingerreactionextensivelyintheviewofnucleicaciddetection. Pianowski and Winssinger have used the Staudinger reaction to unmask a 7‐azidocoumarinfluorophore.27Alsohereadetectionschemewithsinglenucleotideresolutionandsignalamplificationbyvirtueofthetemplateturnoverisobserved.Itwasfoundtoshowa10‐folddifference in fluorescence intensity for theperfectmatched template relative tosingle nucleotidemismatched template using catalytic amounts of template. The reactionshowedaconversionof50%within15minutesandconcentrationsdownto1nMcouldbedetected.


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From the obtained results it could beconcluded that the Staudinger reaction hasparticularly good kinetics which is desired forfast detection. Additionally, the Staudingerreactioncanbeusedforsignalamplificationbyvirtueoftemplateturnover.AnotherimportantremarkisthattheStaudingerreactiondoesnotrequireadditionalreagentsandisorthogonaltobiological processes meaning that it has thepotentialtobeusedinintactcells.

AlthoughthetemplatedStaudingerreactionhas certain advantages over conventionaltechniqueslikePCR,itstillcontainssomepointsof consideration. To date, known examples that include the Staudinger reaction for theactivationofafluoresceinarelimitedtoazidebasedfluoresceins.Theseexamplesinclude7‐azidocoumarin and azidesubstituted rhodamines. However, certain drawbacks limit thescopeofthesestructures.First,thephosphinemoietyisparticularlypronetooxidationandsecondlytheesteriseasilyhydrolyzed.Itisverylikelythatthesedegradationreactionscausetheoftenobserved lowcatalytic turnoverof templatedStaudinger reactions.Besides, thisinstabilityhinderstheuseoftheStaudingerreactioninintactcells.28

3.1.2. MetalMediatedActivationofaFluorescein Metal complexes can provide useful functionalities for the activation of a fluoresceinuponbindingoftheprobestothetemplateortargetstrand.Besides,mostorganometalliccomplexes are characterised by unique chemical reactivities and also utilize metal‐ligandinteractions that are inert tophysiological conditions.As a result, organometal complexesmay provide stable, bioorthogonal reactantsthat are capable of performing reactions inliving cells. Franzini et al. has explored theuse of nucleic acid templated reactionbetween an organomercury‐oligonucleotideprobe and a masked fluorophore linked tothe second probe (Figure 5).29 Theorganomercury‐ probe is composed ofPhenylmercury group linked to anoligonucleotide strand. The maskedfluorophore is the Rhodamine Bphenylthiosemicarbazide. When the twoprobesbindonadjacentpositiononthetemplate, thepresenceof themercury functionalgroupmediatestheoccurrenceofacyclizationreactionofthethiosemicarbazidefunctionalgroup generating anoxadiazole.Upon formationof theoxadiazole thehighly fluorescentrhodamine spirolactam ring is unmasked. The reaction mediated by the presence of amercuryfunctionalgroupiscompletelybiostableandthereforefuturestudiesforincelluseisneeded.Itwasfoundthatafter110minofreactiontime,thefluorescenceinthepresenceof the complementary template was 11.3 times the fluorescence of background signal.

Figure 5: Chemistry of DNA‐Templated Activationof rhodium Fluorescence by Phenylmercury‐DNAConjugates

Figure 5: Staudinger reaction between anazidocoumarin (39) and a phosphin‐modified PNAprobe(40).27


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Unfortunately, Franzini did not show the results for single nucleotide mismatching andsubstoichiometricamountsoftemplate.However,thefactthatmetalmediatedreaction isbioorthogonalandbiostableispromising. Othermetalmediatedorcatalysedtemplatedreactionshavebeenreported.30However,these systems do not produce a fluorescent product. Consequently, the detection assayreliesonothertechniques,whichoftenrequiresmultiplystepsoradvancedtechniques. Recently, a completely different approach for generating strong fluorescence uponhybridisation of the probe molecules to the target strand, and the subsequent chemicalreaction, has been proposed byHerrmann and co‐workers.31 Herein, a nucleic acid (DNA)templated generationof a potent fluorophoreby thePd‐catalyzeddisplacementof iodinefrom a Boron dipyrromethane (BODIPY) core, a Heck‐reaction, is reported. BODIPYchromophores exhibit high fluorescencequantumyields, and in addition, their absorptionand fluorescence spectra can be easily changed by chemical modifications of the pyrrolerings.Additionally, BODIPYderivatives havebeen shown to exhibit heavy‐atomquenchingeffects. Upon removal of the Iodine by utilizing the Heck‐reaction the fluorescence isrestored.Theyobserveda20‐foldincreaseinfluorescenceintensityforfullymatchedversussinglebase‐mismatched templateswhere90%of the fluorescencemaximumwas reachedafter10min.Completesaturationwasachievedwithin20minwherethedetectionlimitwasfoundtobe10pM. The obtained detection limit by Herrmann and co‐workers is exceptionally low andpromisingforfuturedetectionschemesofnucleicacids.However,becauseofthefactthataPd‐metal is needed for the reaction to occur is difficult to be used in intact cells.Nevertheless, themethodoffersthepossibility for fastdetectionofnucleicacidmoleculesthatarepresentinlowamounts.

3.2. Fluorescentenhancement The second class of probes used for fluorescent detection of nucleic acids is theenhancementofthefluorescencethroughtransferorcleavageofaquenchermoiety.Inthisviewtwogeneralapproachesexist.Thefirstapproachtoconsiderisareactiontypeinwhicha reportergroup is transferred fromoneprobemolecule to theanotherprobemolecule,the so‐called transfer reactions. The second type used for the enhancement of afluorescencesignalwidelyemployedistheremovalorcleavageofquenchermoietyfromaoligonucleotideprobe.

3.2.1. TransferReactions

Transfer reactions should be considered in view of templated reactions and is apromisingreactiontypesincethenumberofpairedoligonucleotidesremainsunchangedandproductinhibitionisunlikely,thatisboththereactantandproductswillshowsimilaraffinityforthetemplatewiththeresultthattheexchangemayproceedquickly.Transferreactionsallowthemodificationoftheprobesalongwiththetransferofareportergroup.Grossmannetal.usedanucleicacidcontrolledreaction to transfera fluorescencequencher (Q) fromoneprobetotheother.32‐33Upontransferofthedabsylquenchertheemissionoffluorescein(F)isturnedonwhilethefluorescenceof(T)isswitchedoff(Figure6).Thetransferreaction


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proceededviaanativechemicallikefashion.ThehybridisationoftheprobestothetemplatetriggersatransthioesterficationfollowedbyanirreversibleS→N‐acylmigration.Asaresultthe reporter group, the quencher, is transferred. In the design of the template reactionGrossmann has indeed fashioned reactants and probeswith similar affinity. The templatereaction showed 402 turnovers at 104‐fold excess of the probe after 24 h but moreimportantly a useful 7‐fold fluorescence enhancement after 5h. A signal was obtained atrelatively low probe concentrations (100 nM) where most other nucleic acid templatereactions require higher probe concentrations (10μM). Additionally, the transfer reactionshowed low background signal (3.4% non‐templated transfer). These result could beobtained in the presence of 0.1 nM of template (target DNA). The method reported byGrossmann is highly selective but takes arelatively long 5 h. to obtain a usefulfluorescencesignal.

Note that although Grossmann used aquencher as the reported group, otherreported groups could be envisionedresultingindifferentreadoutstrategies.Thisis well illustrated by Grossmann in 2007whereanRNA‐catalyzed transferofabiotinallowed for the pre‐amplification for anenzyme‐basedreadout(Figure7).34Here,thebiotin reported group is transferred fromoneprobetoaprobebearingaterminalHistag (His6). The nucleic acid templatedtransferreactionofbiotinshoweda102‐foldamplification,whichiscomparablewithPCR.After the transfer one probe bears both abiotin label and a His tag. As His‐tags arecapable of forming strong interactions withnickel, it ispossibleto immobilizetheprobeon a surface of Nickel‐coated well plates.Afterwashing,thewallswereincubatedwiththe enzyme horse‐radish peroxidase‐streptavidin conjugate (HRP‐SA). TheHRP‐SAbinds only with the biotin reported group,and catalyses the oxidation of colourlesstetramethylbenzidine to the colouredquinoid.Consequently,theoxidationreactionis only observed when the biotin istransferred via the template reaction to theprobe bearing the His tag. Bymeans of thisdouble‐amplification strategy a sensitivitywasreacheddownto500attomol.AlthoughthisapproachishighlysensitiveandclaimstobecomparablewithPCRitstillcontainssomedrawbacks. The method makes use of anenzyme‐based readout resulting in the need

Figure 7: Double Signal Amplification using RNA‐catalyzedbiotin transfer reactionaspreamplificationandamodifiedELISAasthefinalamplification.

Figure 6: Transfer of a fluorescence quencher (Q)fromafluorescein(F)toafluoroscein(T).


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ofspecificenvironmentsfortheenzymetobeefficientandreachahighturnovernumber.Besides,theprocedurerequiresseveralwashingstepsmakingittime‐consumingandcostly.Despite these drawbacks, nucleic acid template transfer reactions again show to be apotentialmethodforultrasensitivenucleicaciddetection.

3.2.2. QuenchedAutoligation

Innucleicacidcontrolledligationreactionstheoligonucleotideprobesarelabelledwithmutually reactive groups and connect the two oligonucleotides upon hybridisation to thetemplate.Inprinciplethetemplatecanactascatalystfortheligationproduct,howeverthetemplatedligationreactionssufferfromstronginhibitionoftheproducts.Thisisduetothefacttheproductsofligationreactionsoftenshowastrongeraffinityforthetemplate–theproductformedconsistsofcoupledoligonucleotideprobesandhencehasahigherbindingenergy.Toreachasufficientconversionofthereactantsstoichiometricamountsoftemplateareneeded.Thechallengeistodecreasetheaffinityoftheligationproductwithoutaffectingtheaffinityoftheprobemolecules.35

Figure8:ChemistryofQUALprobes.Afterhybridizationtotemplate,thephosphorothioatedisplacesthequencher,leadingtoself‐ligationofthetwoprobestrandsandunquenchingofthefluorophore.

Althoughproductinhibitionformsamajorprobleminligationreactionsforthedetection

of nucleic acids, there is method for the detection of nucleic acids based on ligationreactionscalledQuenchedAutoligation(QUAL).ThesignalofQUALprobesisaresultfromachemicalreactionbetweentwoprobesbindingonadjacentsidesofthetemplateortarget.Oneof theprobes is labelledwithan internal fluorophoreandaquencherattachedbyanelectrophilic sulfonic acid linkage,whereas the other probe is labelledwith a nucleophilicphosphorothioate (Figure 8).36‐37Whenever the probes bind on adjacent positions of thetemplate,thephosphorothioateisinapositiontoreplacethequencherontheotherprobeviaanucleophilicdisplacementreaction.Inthisreactiontheprobeswillbecomeligatedwiththeaccompaniedgenerationofa fluorescent signal. However, thisprocess resultsonly inone ligation product per target molecule because of product inhibition. Thereforestoichiometric amounts of the template is needed. To solve this problem Abe et al.introducedamodificationtothejustdescribedQUALmethod.38ByattachingtheQuenchertoashort‘universallinker’thatformsabulgeuponligation,thedissociationoftheligationproductfromthetargetincreasedsignificantlyand,inaddition,thereactionrateshoweda4‐fold increase.Usingtheuniversal linkertheyobserved100signalspertargetstrand.TheQUALapproachallowedalso for thedistinctionofclosely relatedsequencesbyapplyingatwo‐colour approach. Herein, two probes complementary to distinct targets are labelledwith either a red or green fluorescein and the accompanied quencher, respectively.Consequently,agreenor redsignal isobserved.BecauseQUALcanbeused todistinguishtwodistinctstrandsinsitu,itiswidelyusedforthedistinctionofcloselyrelatedbacteria.


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In 2009 Kool and co‐workers proposed a probe design for templated fluorescenceactivationthatcombinesthestrongfluorescenceenhancementandgeneralityofquencherreleaseprobessuchasQUAL,togetherwiththekineticadvantagesandbioorthogonalityoftheStaudingerreaction.39Theapproach,calledQ‐STAR,isbasedonthereleaseofadabsylquencher that is initiallyattached to theprobeviaaα‐azidoether (Figure9).Reductionofthe azide by triphenylphosphine triggers the cleavage of the α‐azidoether linker and theaccompanied release of the dabsyl quencher. Upon release of the quencher a strongenhancementofthefluorescenceisobserved.Thereactionshowed90%conversionafter32min and a 61‐fold increase of the fluorescence was observed after 115 min. With thismethodKoolandco‐workerscoulddetecttargetsequencesdownto2nM.

Figure9:DetectionofnucleicacidsbytemplatedfluorescenceactivationofQ‐STARprobes.Left)AschematicrepresentationofQ‐STAR.Theprobebearingthefluorophoreandquencher(Q‐STARprobe)bindtogetherwiththeprobebearing thetriphenylphosphine (TPP‐Probe) toacommontargetstrand (the template).Proximity‐inducedreductionoftheQ‐STARazidefunctionalityresultsincleavageoftheR‐azidoetherlinkerandreleaseofthequencher,yieldingafluorescenceturn‐onsignal.Subsequentprobeexchangeonthetemplateallowsformultipleturnovers;Right)Molecularstructuresof reactants, including (top) thequencher‐conjugatedreleaselinkerand(Right)theproductsafterreduction,cleavage,anddissociationfromthetemplate.39

3.3. NonetheredReagentsinNucleicAcidTemplateReactions The basic feature of nucleic acid template reactions is the increase of the effectivemolarity of the reactants. An alternative approach is based on the release of catalyticmoieties,eitherbychemicalmodificationorbynoncovalentbindinguponhybridisationoftheprobes.In2006Kramerandco‐workerspresentedamethodbasedonatargetinduced

Figure10:Left)DetectionofDNAbyallostericsignaltransductionandcatalyticamplificationwithformationofthefluorescentdye;Right)Reactionsolutioncontainingonlytheprobe(left)andtheprobeplusthetargetstrand(Right).40


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allostericreleaseofCu(II)ions.40TheCupperionsservedasaco‐catalystfortheformationofa fluorescent product (Figure 10).Upon binding of the probe to the target sequence, theCu(II) ion is released from its initial situation where it was chelated by two terpyridinefragments attached to both ends of the oligonucleotide. After hybridisation the Cu(II) ionformsacomplexwiththephenanthroline,whichiscapableofcatalysingtheoxidationofthenon‐fluorescentmolecule2’,7’‐dichlorodihydrofluorescein(DCFH)tothefluorescentproduct2’,7’‐dichlorofluorescein (DCF). Formation of the product was observed by following theincrease in the relative fluorescence. It was observed that rate of oxidation increasesdramatically in the presence of template. The activity is about two thirds of that of atemplate‐free reactionmixture. The describedmethod is sensitive to single basematchesandtargetsequencesweredetectabledownto5nMwithaturnovernumberequalto20.Aslightdisadvantageofthisapproachisthattakes12hourstoobtainagoodsignal.4. Discussion4.1. NucleicAcidDetectioninLivingCells

In the previous sections different reaction types that are used in templated catalyticreactionstoproduceafluorescentsignalarediscussed.Itcanbeconcludedthatnucleicacidtemplated reactions promising candidates for the detection of nucleic acidmolecules fordiagnostic with detection limits down to 1 pM. Themost important reaction type that isexploited is the Staudinger reaction because of its bioorthogonality and reagent freecharacter.Thebioorthogonalityandreagentfreereactionmakesitparticularlyusefulforusein intact cells, in which procedure steps such as DNA or RNA extraction are obviated.Although this is true, limited researchhasbeenexploitedon theuseof templatecatalyticreactioninintactcellsdespitethenumberofmethodsthatareknownfortheintroductionofoligonucleotideprobesintocells.todateonlyarestrictedamountofresearchisperformedonamplifiedcatalyticreactionsinsideacell.

Fornucleictemplatereactionstobeawell‐adaptedtechniqueforusageincells,5‐7afewpointshavetobetaken intoaccount.First, theoligonucleotideprobeshaveatendencytoaggregate in physiological conditions which gives erratic results. Secondly, the intrinsicfluorescence of cellular organelles must not overlap which otherwise leads to highbackground fluorescence thatseverely compromises thesensitivity.11 Pianowski et al.proposedadetectionschemethatwas particularly well adapted forin cell use (Figure 11).41 Herein,the use of two cell‐permeableGaunidine‐based cell‐permeablepeptide nucleic acid (GNPA)probes, in which one probe islabelled with a aziderhodaminefluorescein and the other withtrialkylphosphine, enabled thedetection of cellular mRNA in

Figure11:SchematicrepresentationofthetemplatedunmaskingofazidorhodaminebyStaudingerreduction.


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intact cells. In the initial situation the rhodamine fluorophore is azide‐quenched. Uponhybridisation of the probes the rhodamine fluorophore is activated by a templatedStaudinger reduction. It was observed that the presence of a template let to a 30‐foldincreaseofthefluorescenceintensity.Therateoftemplatereactionyieldsa80%conversionin10minusing1equivalentoftemplate.Theuseoftherhodaminefluorophoreenabledtovisualize the detection of mRNA in intact cells (Figure 12) because the fluorescenceexcitation/emission falls in a different region of the electromagnetic spectrum than theexcitation/emission of the cellular organelles. Satisfactory signal to noise ratios wereobtainedwiththeuseof250nMofprobewithasignificantdecreaseinthereactionrateforprobes bearing a single base mismatch. Pianowski found that the signal increasedsignificantlybetween15and30minutesofincubationtimefortheprobesintothecellswithonlyamodestincreaseforlongerincubationtimes.Consequently,thedescribedprocedureallowedforarelativelyfastnucleicaciddetectionschemebyapplicationofaacidtemplatedStaudingerreaction.

Figure12:(top)White‐light,(middle)fluorescence,and(bottom)compositeimagesofcellsincubatedwiththeoligonucleotide labelledwithaaziderhodaminefluorescein for0,5,15,30,45,and60minandthentreatedwitholigonucleotidelabelledwiththetrialkylphosphine. Although,thePianowskidescribesoneofthefirstexperimentsfornucleicaciddetectionusingcatalyticsystemsinintactcellsonehastobecriticalandfurtherinvestigationsmustbeperformed. In the experiments, the template was a strand ofmRNA that was present inexcess inside the cells. Therefore, a further look has to be done on the sensitivity of thisapproachwhenusedinintactcellsandifindeedthenucleicacidtemplateapproachcanbeusedtodetectDNAorRNAfragmentsonlypresentinsmallamounts.Thelowsensitivityofthis technique is probably due to the fact the oligonucleotide probes aggregate in thecellularenvironment.

4.2. SequenceSpecificFluorogenicDetectionofDoubleStrandedDNA DNA is most often present in the double helix structure. The obtained results forsequencespecific fluorogenicdetectionofnucleicacidssequences ismostlyperformedonsingle stranded DNA or RNA. Double stranded DNA (dsDNA) has received very limiting


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attention as a template for the of chemical reactions in order to amplify a signal. Theexamples that exist for the detection dsDNA include association of polyamides in thegrooves of the helical structure of DNAor oligonucleotide probes binding in a triple‐helixformation. However these methods show little or no fluorescence enhancement uponbinding of the probes. Recently, Kool and co‐workers described a template chemicalreaction for the detection of dsDNA.42 The method is based on a previously describedmethod(Q‐STAR) inwhichaquencher is releasedviaaStaudingerreaction (Figure13).Tosummarize, a fluorescein labelled probe contains a dabsyl quencher with at the sameterminus an α‐azidoether. A second probe carries a phosphine group. The templatechemistry allows the reactive groups to react because of increased effective molarity,resulting in an azide reduction and rapid linker hydrolysis. As a result the quencher isreleasedandenhancedfluorescenceisobserved.Theprobesweredesignedsuchthattheywillbindsidebysideinaparallelmannerbyapyrimidinemotiftriplehelixformation.Inthepyrimidinemotif triplehelix formationathyminerecognizesanA‐TbasepairandcytosinerecognizesaG‐Cbasepairformingso‐calledHoogsteenbaseparinginteractions.Toobtaincomplementary paring at physiological conditions the cytosine was replaced bypseudoisocytosines. Kool and co‐workers observed that the reaction is slower than itsanalogueonsinglestrandedsequences.TheyrelatedthisobservationtothefactthatwithdsDNAthereactivegroupsarenotoptimalalignedduetostericalhindranceofdsDNA.

Figure 13: Strategy for the detection of dsDNA by template‐mediated fluorescence activation of Q‐STARprobes.

The fact that no denaturation of the DNA strand is necessary to detect a specificsequence contributes to a highly simplified method, as special equipment to separatenucleicacidstrandsisnolongerneeded.Toconclude,thedescribedprobehasthepotentialtodetectsmallamountofDNA,althoughfurtherstudiesareneededasonlyidealdsDNAisexploitedbyKoolandco‐workers.Ifhowever,theproblemssterichindranceanddetectionlimit could be overcome denaturation steps to obtain single stranded DNA is no longerneeded.

5. FutureProspects It was mentioned in the introduction that the cancer disease can be detected usingnucleic acid template reactions because cancer is characterised by a specific nucleotidesequenceintheDNAstrandofcells.Insteadofatemplatechemicalreactionswhichtriggersthe formationofa fluorescentgroup, the targetstrandcould triggera reaction inwhichadrug is released to combat thedisease. If this couldbe realized, than thedrugneeded to


17

combat a disease is only released iftheDNAor RNA strand characteristicfor thedisease is present, that is thedrug is only released in cancer cells.Forcancerthiswouldmeanthatonlycancer cells will be killed whereashealthy cancer‐free cells areunaffected. This is in contrast topresent day cancer drugs where thedrug affects both the cancer ascancer‐freecells.Agoodexampleofatemplatereactionforthedetectionofa target strand and the accompanied particle‐induced cell death is that of Ogilby andGothelf.41 Herein, a strategy is presented that allows for the control of the synthesis ofsinglet oxygen (Figure 14). The photosensitiser pyropheophorbide‐α (P) was linked to aoligonucleotideprobe.Uponexcitationof thephotosensitisereP theconversionofoxygenfromitstripletstate(3O2)toitssingletstate(

1O2).Addingaoligonucleotideprobelabelledwith a quencher the conversion of 3O2 is

1O2 suppressed. To activate the photosensitiseragain,theoligonucleotideprobelabelledwiththequencherhastobecleaved.Thiscanbeachieved for example a by the presence of a complementary target strand. This methodproves that it is possible for nucleic acid catalyzed reactions to be used in drug deliverysystems. Anotherpointof interest is the fact that theabovedescribedmethods fornucleicaciddetectiononlygivesquantitativeresults.Itwouldbeinterestingifinthefuturethenotonlythepresenceofaparticularsequenceisknownbutalsotheamount.Secondly,themethodsthat exist to introduce oligonucleotides in cells is limited.Not each oligonucleotide probecanbeintroducedefficiently.Thereforefurtherstudiesareneeded.

6. Conclusion Astheabovediscussionpointsoutthereexistquiteanumberofdifferentreactiontypesforthedetectionandvisualisationofnucleiacidsequences,forbothinreactionmixturesasinintactcells.ThemostpromisingreactiontypefortheuseinintactcellsistheStaudingerreaction because of its bioorthogonality and reagent free character. Although, manyfluoresceinsareexploredtovisualizenucleicacidstrandsfurtherstudyisnecessaryintermsofsensitivity.Todate,thetemplatedreactiongivingthebestresults intermsofsensitivitystill relies on enzymatic methods. The non‐enzymatic methods can detect down thepicomolarregime.Additionally,onlystrandsthatarepresentinexcessinsidethecellcanbevisualized.Here, the technique for introducingoligonucleotideprobes in cellsneeds tobeimproved.Besides,itisfoundthatonlyRNAorsinglestrandedDNAcanbeeasilyvisualizedusing templated chemistry. To best of my knowledge only one article describes to thedetectionofdoublestrandedDNA,whereonlyidealstrandscanbedetected.

Figure 18: Schematic representation of the templated catalyticproductionofsingletoxygen.


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