两种物质同时检测的意义

&G-QuadruplexesIon-Responsive Hemin–G-Quadruplexes for Switchable DNAzyme and Enzyme FunctionsMiguel Angel Aleman-Garcia,Ron Orbach,and Itamar Willner*[a]IntroductionThe discovery of the horseradish peroxidase (HRP)mimicking activities of hemin–G-quadruplex [1]enabled numerous applica-tions of the DNAzyme as amplifying label for sensing [2]and as a catalyst for various chemical transformations,such as the H 2O 2-mediated oxidation of aniline to polyaniline,[3]or thiols to disulfide,[4]or the aerobic oxidation of NADH.[5]Different hemin–G-quadruplex nanostructures have been examined over the years,including the parallel and antiparallel formation of hemin–G-quadruplexes,[6]the self-assembly of G-quadruplex subunits into a catalytically active hemin–G-quadruplex DNA-zyme wires,[7]and the formation of supramolecular complexes consisting of different numbers of G-quadruplex layers,for ex-ample,hemin–telomeric G-quadruplexes.[8]Although all of the hemin–G-quadruplexes revealed HRP-like catalytic functions,the degree of catalytic activity was controlled by the specific hemin–G-quadruplex configurations.Stimuli-responsive func-tions of DNAzymes,and particularly of hemin–G-quadruplexes,have been studied in recent years,and different applications of these systems were suggested.For example,the pH-pro-grammed or ion/ligand-induced assembly or disassembly of DNAzyme subunits were implemented to switch the catalytic functions of the Mg 2+-dependent DNAzyme.[9]Also,the switch-able formation and dissolution of catalytic hydrogels by cross-linking (or separation)of acrylamide chains modified with G-quadruplex subunits was reported.[10]In the latter system,the K +-induced formation of the hemin–G-quadruplex-cross-linked hydrogel led to a catalytic hydrogel matrix that was switched off by a crown ether-mediated elimination of K +ions and the separation of the G-quadruplex-stabilized hydrogel matrix.Here we report the K +–crown ether-or cryptand-induced switchable activation of the hemin–G-quadruplex DNAzyme structure.We demonstrate that this DNAzyme switch allows the ON/OFF functional control of two coupled DNAzymes or DNAzyme/enzyme nanostructures,as well as the DNAzyme transduction of a switchable DNA tweezers machine.Results and DiscussionFigure 1A outlines the design of the bi-DNAzyme switching system consisting of the hemin–G-quadruplex and Mg 2+-de-pendent DNAzymes.The switching system is based on K +-ion induced formation of the G-quadruplex structure (switch ON)and separation of the G-quadruplex by elimination of the K +ions through the formation of the energetically favored K +–crown ether complex (switch OFF).The subunits corre-sponding to the G-quadruplex and Mg 2+-dependent DNAzyme are encoded in the nucleic acid strands 1and 2.Domains I and I’in 1and 2represent subunits of the G-quadruplex with par-tial complementarity,and domains II and III represent the two parts of the Mg 2+-dependent DNAzyme.Also,the fluorophore-quencher-modified ribonucleobase-containing oligonucleotide 3that acts as substrate for the Mg 2+-dependent DNAzyme is included in the system.In the absence of K +ions,the G-quad-ruplex cannot be formed,and strands 1,2,and 3are separat-ed,thus the formation of neither the hemin–G-quadruplex nor the Mg 2+-dependent DNAzyme is induced,and the two DNA-[a]Dr.M.A.Aleman-Garcia,R.Orbach,Prof.I.WillnerInstitute of Chemistry,The Hebrew University of Jerusalem Jerusalem 91904(Israel)E-mail:willnea@vms.huji.ac.ilSupporting information for this article is available on the WWW under /10.1002/chem.201304702.5619Full PaperDOI:10.1002/chem.201304702zymes are switched OFF.Addition of K +ions stimulates theself-assembly of domains I and I’of 1and 2into a stable G-quadruplex structure,which brings together sequences II and III,corresponding to Mg 2+–DNAzyme (switch ON).The associa-tion of substrate 3with the respective domains of 1and 2yields the supramolecular DNA structure consisting of the G-quadruplex and the Mg 2+-dependent DNAzyme sequence.Theassociation of hemin with the G-quadruplex or the Mg 2+to the self-assembled loop structure yields the two DNAzyme units.The hemin–G-quadruplex catalyzes the H 2O 2oxidation of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),ABTS 2À,to the colored product ABTS C À,whereas the Mg 2+-dependent DNA-zyme cleaves substrate 3,and the fluorophore-labeled frag-mented product provides a readout signal for its activity.Itshould be noted that the bifunctional DNAzyme structure is formed by synergistic cooperative interactions.The K +-stimu-lated assembly of the hemin–G-quadruplex cooperatively sta-bilizes the binding of 3to the Mg 2+-dependent DNAzyme structure.Treatment of the bifunctional DNAzyme structure with crown ether (18-crown-6)extracts the K +ions from the G-quadruplex,resulting in the separation of the G-quadruplex,and the concomitant unstable duplex of subunits 1,2,and substrate 3.That is,the bifunctional DNAzyme structure is sep-arated into the units 1,2,and 3,and both DNAzymes are switched OFF.At point a (Figure 1B,curve I),K +is added to the system,whereas at point b the crown ether is added.Thecyclic ON/OFF switchable activa-tion of the Mg 2+-dependent DNAzyme is clearly demonstrat-ed.Figure 1B (curve II)shows the selectivity of the bifunctional DNAzyme system for K +ions.Accordingly,the addition of Na +does not induce the formation of the G-quadruplex structure,thus supporting the fact that K +ions are required for the forma-tion of the structure.Further-more,curve (III)shows the effect of K +and crown ether on a system consisting of strands 4and 5,and substrate 3.Strands 4and 5include the Mg 2+-depen-dent DNAzyme sequences pres-ent in 1and 2but are conjugat-ed to tandem strands that cannot form the G-quadruplex.Evidently,addition of K +–crown ether does not activate the Mg 2+-dependent DNAzyme,thus sup-porting the fact that the K +-stimulated self-assembly of the hemin–G-quadruplex indeed sta-bilizes the formation of theactive Mg 2+-dependent DNA-zyme.Figure 1C shows the switchable hemin–G-quadruplexDNAzyme activities towards the oxidation of ABTS 2Àby H 2O 2upon subjecting the system consisting of 1,2and 3,in thepresence of hemin,to K +ions or crown ether,respectively.In the presence of K +ions,the effective hemin–G-quadruplex-cat-alyzed oxidation of ABTS 2Àby H 2O 2proceeds.Binding of theK +ions to the crown ether receptor dissociates the G-quadru-plex,leading to inefficient oxidation of ABTS 2À,switch OFF (the slow oxidation of ABTS 2Àis due to free-hemin-catalyzed oxida-tion by H 2O 2).In the next step we examined the possibility to switch theproteolytic activity of an enzyme ON and OFF.Many aptamersagainst proteins form G-quadruplex structures (e.g.,against thrombin or VEGF).It was found that binding of anti-thrombin aptamer to thrombin inhibits the proteolytic activity of the enzyme.[11]Accordingly,we designed an aptameric G-quadru-plex-stimulated switching of thrombin using K +/crown ether asON/OFF switching triggers (Figure 2A).In the presence of K +ions,formation of the thrombin aptamer G-quadruplex takes place,resulting in binding the aptamer to thrombin and the in-hibition of its proteolytic functions.In turn,the crown ether-in-duced elimination of K +from the aptamer weakens G-quadru-plex,and the aptamer dissociated from the protein.This results in activation of the enzyme toward hydrolysis of its substrate(SensoLyte 520thrombin activity assay kit).The peptide sub-strate is modified with a fluorophore/quencher pair,and fluo-rescence is quenched in the peptide structure.Cleavage ofthe Figure 1.A)Schematic switchable ON/OFF activation and deactivation of the Mg 2+-dependent DNAzyme.The DNAzyme is formed by the K +-ion-stimulated bridging of the DNAzyme subunits through the formation of a G-quadruplex.The Mg 2+-dependent DNAzyme is separated to its subunits by the crown ether-induced eliminationof the K +ions from the G-quadruplex.The switchable supramolecular system is followed by the Mg 2+-dependentDNAzyme cleavage of a fluorophore/quencher-functionalized substrate,and by the catalytic activity of the result-ing hemin–G-quadruplex DNAzyme.B)Time-dependent fluorescence changes:(I)the ON/OFF activation of the Mg 2+-dependent DNAzyme,(a)addition of K +ions,(b)crown ether-stimulated elimination of K +ions;(II)thetreatment of the system consisting of 1/2/3with Na +ions that substitute the K +ions;(III)treatment of subunits4and 5in the presence of 3with K +ions,where 4and 5cannot assemble into a G-quadruplex bridge.C)Follow-ing the K +-induced formation of the G-quadruplex bridged structure of subunits 1and 2by the incorporation ofhemin into the G-quadruplex bridge,resulting in the formation of the hemin–G-quadruplex DNAzyme that cata-lyzes the H 2O 2-mediated oxidation of ABTS 2Àto ABTS C À:(a)addition of K +ions and formation of the 1/2/3catalyti-cally active structure (switch ON);(b)the crown ether-induced separation of the catalytically active nanostructure.5620peptide triggers the fluorescence of the fluorophore,and thisprovides a readout signal for the thrombin-catalyzed hydrolytic reaction.Figure 2B depicts the K +/crown ether switchable ON/OFF activation of thrombin in the presence of aptamer 6.In the absence of K +,hydrolysis of the substrate proceeds,as evi-dent by the time-dependent increase in fluorescence.At point a K +is added to the system and this results in the forma-tion of the aptameric G-quadruplex that binds to thrombin,a process that inhibits the enzyme.This results in the switching OFF of the hydrolysis of the peptide.At point b the crown ether is added to the system,the elimination of K +from the quadruplex separates the aptameric G-quadruplex,resulting in the switching ON of the biocatalytic hydrolysis of the peptide.By elimination of K +from the quadruplex by addition of crownether and readdition of K +ions,the system is switched be-tween ON and OFF states.Control experiments indicate that in the presence of a foreign non-aptameric G-quadruplex con-taining sequence 7,the addition of K +or crown ether does not have any effect on thrombin activity,and the hydrolysis ofthe peptide proceeded continuously (Figure S1in the Support-ing Information).These results show that the switching of thethrombin activity is due to the specific interactions of K +/crown ether with the aptamer.The formation of the G-quadruplex,upon addition of K +to the peptide/thrombinsystem,and the separation of the G-quadruplex in the pres-ence of the crown ether were further confirmed by the addi-tion of hemin (Figure 2C).The addition of K +induces the for-mation of an active DNAzyme structure that catalyzes the oxi-dation of ABTS 2Àto the colored product ABTS C À,whereas treat-ment of the resulting complex with crown ether eliminates the K +ions from the G-quadruplex,resulting in its dissociation and the release of the G-quadruplex associated hemin.The low cata-lytic activity of the system toward the oxidation of ABTS 2Àby H 2O 2is due to the inefficient hemin-catalyzed oxidation of ABTS 2À,and/or to residues of the hemin–G-quadruplex that cata-lyze the oxidation of ABTS 2Àby H 2O 2.Finally,we implemented a switchable DNA tweezers ma-chine to reversibly activate and deactivate the hemin–G-quadru-plex DNAzyme.Different supra-molecular DNA nanostructures have been reported to mimic the functions of macroscopic machines,[12]such as structures mimicking the cyclic opening/closure of “tweezers”,[13]bidirec-tional “walkers”,[14]directionalmolecular rotors [15]or directional transitions of interlocked catenated rings.[16]Nucleic acid strands,[17]pH changes,[18]metal ion/ligands,[19]and light [20]have been used as fuel and antifuel components to drive these cyclic DNA machines.Here we im-plement the K +/2.2.2-cryptand system as cation and cation-binding ligand for the cyclic stimulated opening and closure ofDNA tweezers,and the concomitant formation of the hemin–G-quadruplex DNAzyme (switch ON)and its dissociation into a catalytically inactive structure (switch OFF;Figure 3).In this system 2.2.2-cryptand was used instead of 18-crown-6due itshigher affinity for K +ions (K a 105.3vs.102.05),[21]which is neces-sary since the concentration of K +is higher than in the previ-ous systems (40vs.20m m ).Note that cryptand cannot be used in the Mg 2+-dependent DNAzyme system because it also binds Mg 2+ions.The tweezers structure,T 1,consists of two nu-cleic acid “arms”,8and 9,bridged by two nucleic acids,10and 11,to form the closed tweezers structure.The bridgingunit 10is substituted at its ends with fluorophore/quencherunits.Domains I and II in the arms 8and 9include G-reach se-quences that are capable of forming the G-quadruplex-stabi-lized structure in the presence of K +ions.In the absence of K +,domains I and II are complementary to the respective bases as-sociated with the bridging element,and the tweezers exist inthe closed state,T 1.In the presence of K +ions,the stabilization of the G-quadruplexes opens the tweezers,to state T 2,withthe concomitant release of 11.Further addition of the cryptandligand eliminates K +ions from the G-quadruplex,resultinginFigure 2.A)Anti-thrombin aptameric G-quadruplex switchable activation and deactivation of the hydrolytic activi-ties of thrombin.The K +-ion-induced G-quadruplex aptamer binds to thrombin and inhibits the hydrolyticenzyme activity.The crown ether-stimulated elimination of K +ions dissociates the G-quadruplex,and therandom-coil aptamer detaches from thrombin,leading to the activation of the enzyme.The association of hemin to the K +-stabilized G-quadruplex bound to thrombin yields the active hemin–G-quadruplex DNAzyme that cata-lyzes the H 2O 2-mediated oxidation of ABTS 2Àto ABTS C À.B)Time-dependent fluorescence changes upon theswitchable activation/deactivation of thrombin in the presence of K +/crown ether:(a)addition of K +ions to thesystem,(b)addition of crown ether to the system.C)Time-dependent absorbance changes as a result of thehemin–aptamer G-quadruplex-catalyzed oxidation of ABTS 2Àby H 2O 2:(a)addition of K +ions to the system,(b)elimination of the K +ions from the G-quadruplex by the added crown ether.5621the K +–cryptand complex.This process dissociates the G-quad-ruplex,resulting in the rebinding of 11to the nucleic acid arms 8and 9,and the reclosure of the tweezers.Consequently,by cyclic treatment of tweezers T 1with K +,and subsequent treatment of the open K +-stabilized G-quadruplex tweezers T 2with cryptand,the DNA system reversibly switches between the open (T 2)and closed (T 1)states.The cyclic opening and closure of the tweezers was followed by the fluorescence in-tensities of the fluorophore label associated with 10(Fig-ure 4A).The close proximity between the fluorophore/quench-er pair in structure T 1leads to effective quenching of the fluo-rophore,while the spatial separation of the fluorophore/quencher pair in state T 2leads to less efficient quenching.Indeed,Figure 4A shows that in the presence of K +ions,open-ing of the tweezers yields a high fluorescence,whereas addi-tion of the cryptand and the formation of the closed state T 1,yields effective quenching of the fluorophore.The switchable opening and closure of the tweezers through the K +/ligand-stimulated formation of the G-quadruplex and its dissociation were implemented to incorporate the HRP-mimicking DNA-zyme functions into the tweezers device.Association of hemin with the K +-stabilized G-quadruplex is known to generate the HRP-mimicking DNAzyme that catalyzes the H 2O 2-mediated ox-idation of Amplex Red to the resofurin fluorescence product.[22]Accordingly,the association of hemin to the G-quadruplexes connected with tweezers T 2is anticipated to form active DNA-zyme structures,whereas the closure of tweezers T 2is expect-ed to switch OFF the catalytic properties of the system.Fig-ure 4B depicts the cyclic and switchable activation and deacti-vation of the hemin–G-quadruplex DNAzyme upon the transi-tion of the tweezers system between the open and closed states.The time-dependent fluorescence changes at points a correspond to the DNAzyme-catalyzed oxidation of Amplex Red to resofurin,while in the closed tweezers,T 1,the G-quad-ruplex is “caged”in a catalytically inactive structure (points b).It should be noted that in the present system we used theDNAzyme-catalyzed oxidation of Amplex Red by H 2O 2to reso-furin as the chemical reaction that probes the activity of the hemin–G-quadruplex DNAzyme,rather than the DNAzyme-in-duced oxidation of ABTS 2Àby H 2O 2used in the previous system.This is because ABTS C Àreacts with cryptand and,there-fore,cannot be used to monitor the ON/OFF switch.ConclusionIn conclusion,the present study has demonstrated that the K +-induced formation of G-quadruplexes can be implemented to switch the catalytic activities of DNAzymes and enzymes and to trigger the opening/closure of a DNA machine (tweezers).The K +-induced formation of the G-quadruplex has enabled the association of hemin to the nanostructure while generat-ing the HRP-mimicking DNAzyme.The switching functions of the different systems were achieved by the K +-induced assem-bly of the G-quadruplex structures and their separation by the uptake of K +from the DNA structures using appropriate recep-tor ligands (crown ether or cryptand).Specifically,we have demonstrated that the K +-stimulated formation of the G-quad-ruplex structure consisting of the Mg 2+-dependent DNAzyme subunits led to the ON/OFF switchable DNAzyme.Similarly,theFigure 3.The K +ion/cryptand-triggered opening and closure of DNA tweez-ers.Opening of the tweezers by K +leads to the formation of K +-stabilized G-quadruplexes.The opening and closure of the tweezers is followed by the fluorescence changes of a fluorophore/quencher pair associated witha bridging unit linking the “arms”of the tweezers,and by the incorporation of hemin into the generated G-quadruplexes to yield the DNAzyme that cat-alyzes the H 2O 2-mediated oxidation of Amplex Red to the fluorescent reso-furinproduct.Figure 4.A)Fluorescence spectra corresponding to:(a)the closed tweezers,T 1;(b)the open tweezers,T 2.Inset:cyclic fluorescence changes of thesystem upon opening of the tweezers with K +ions to yield T 2and closure of the tweezers by the addition of the cryptand to yield T 1.B)Time-depen-dent fluorescence changes as a result of the hemin–G-quadruplex-catalyzed oxidation of Amplex Red to the fluorescent resofurin:(a)addition of K +ions to yield tweezers,T 2;(b)addition of cryptand to regenerate tweezers,T 1.5622K+-induced formation of the G-quadruplex consisting of the anti-thrombin aptamer complex enabled the switching-OFF of the activity of thrombin.The ON/OFF switching of the throm-bin functions were then demonstrated by the cyclic formation and dissociation of the aptamer G-quadruplex structure.Finally, the K+-stimulated formation of the G-quadruplex structure was implemented to open a predesigned DNA tweezers structure, leading to the generation of active HRP-mimicking DNAzyme units.By the formation of the K+-stabilized G-quadruplex struc-tures,and their separation by means of the receptor ligands, the tweezers were cycled between open and closed states, leading to ON/OFF catalytic functions.The switching paradigm presented in this study is general and introduces a new means to assemble and dissociate programmed nanostructures of nu-cleic acids.Experimental SectionMaterials2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid sodium salt (HEPES),ABTS2À,sodium chloride,magnesium chloride,potassium acetate,and potassium nitrate were purchased from Sigma–Aldrich.DNA oligonucleotides were purchased from Integrated DNA Technologies Inc.,(Coralville,IA).The products were HPLC pure and characterized by mass spectrometry.Hydrogen peroxide solution was purchased from Fluka.Hemin was purchased from Porphyrin Products(Logan,UT).SensoLyte 520thrombin activity assay kit was purchased from Anaspec(Fermont,CA).Ultrapure water from NANOpure Diamond(Barnstead)source was used in all of the experiments.Amplex UltraRed reagent was purchased from Invitrogen,Lifetechnologies.The sequences of the oligonucleotides used in this study are:1:5’-TGGGTTAATGCCACCCATGTTATCCTA-3’2:5’-AGTACTCAGCGATGCATTATGGGTAGGGCGGG-3’3:5’-FAM-TAGGATATrAGGAGTACT-BHQ-2-3’4:5’-CTCACTCACCTACTCATCTTCACCCATGTTATCCTA-3’5:5’-AGTACTCAGCGATAAGATCACTCACTCCACTCC-3’6:5’-GGTGGTGTGGTTGG-3’7:5’-TAAGGGTAAGGGTAAGGGTAAGGG-3’8:5’-GTTGGAGCGACATTAGAGACTGGGTAAGGGTAAGGGTAAGGG-3’9:5’-AGGGTAAGGGTAAGGGTAAGGGTGGCCTGTCCTATCTATG-ATGG-3’10:5’-Cy5.5-TCTCTAATGTCGCTCCAACAACCATCATAGATAGGA-CAGG-IowaBlack RQ/-3’11:5’-CTCTTATTCTTACTCATAACACTCTTATTCTTACTC-3’InstrumentationFluorescence measurements were performed using a Cary Eclipse Fluorometer(Varian Inc.).The excitation of resofurin,FAM,and Cy5.5were performed at490,480,and680nm,respectively.Ab-sorbance measurements of ABTS CÀwere performed using a Shimad-zu UV-2401PC UV/Vis spectrophotometer.DNAzyme assayThe K+-switchable bifunctional DNAzyme nanostructure was stud-ied in a solution consisting of1,2,and3at a final concentration of3m m in a10m m HEPES buffer(20m m MgCl2,50m m NaCl, pH7.2).DNA samples were heated to908C for5min and slowly cooled to room temperature before measurement.The switchable HRP-mimicking activities of the hemin–G-quadru-plex system were analyzed using hemin and ABTS2Àwith a final concentrations of15n m and2m m,respectively.The reaction was initialized by addition of hydrogen peroxide(final concentration of 200m m).The ON/OFF system was generated by the addition of K+ (20m m as KOAc)or18-crown-6(25m m),respectively.DNAzyme/enzyme assayThe thrombin assay was conducted in a10m m HEPES buffer (100m m NaCl,5m m MgCl2,pH7.2).The final concentration of the aptamers6and7was1m m.After1.5h of incubation,thrombin (20n m)was added and allowed to equilibrate for30min in order to form the aptamer–substrate complex.For the fluorescence measurements thrombin substrate5.8 10À6m was used(Senso-Lyte 520thrombin activity assay kit).DNA tweezers assayThe K+-triggered DNA tweezers system was studied in a solution consisting of8,9,10,and11at a final concentration of1m m in a10m m HEPES buffer(5m m MgCl2,pH7.2).Hemin and Amplex UltraRed were added to a final concentration of15and100n m, respectively.The peroxidase-mimicking reaction was initialized by addition of hydrogen peroxide(final concentration of200m m).The ON/OFF system was generated by adding K+(40m m as KNO3)or 2.2.2cryptand(25m m),respectively. AcknowledgementsParts of this study were supported by the Volkswagen Founda-tion,Germany,and by the Israel Science Foundation. Keywords:crown ether·cryptand·G-quadruplex·thrombin·tweezers[1]a)P.Travascio,Y.F.Li,D.Sen,Chem.Biol.1998,5,505–517;b)P.K.Wit-ting,P.Travascio,D.Sen,A.G.Mauk,Inorg.Chem.2001,40,5017–5023;c)D.Sen,L.C.H.Poon,Crit.Rev.Biochem.Mol.Biol.2011,46,478–492.[2]a)Y.Xiao,V.Pavlov,T.Niazov,A.Dishon,M.Kotler,I.Willner,J.Am.Chem.Soc.2004,126,7430–7431;b)C.Teller,S.Shimron,I.Willner, Anal.Chem.2009,81,9114–9119;c)T.Li,L.L.Shi, E.K.Wang,S.J.Dong,Chem.Eur.J.2009,15,3347–3350;d)D.M.Kong,J.Xu,H.X.Shen,Anal.Chem.2010,82,6148–6153;e)S.Bi,L.Li,S.S.Zhang,Anal.Chem.2010,82,9447–9454;f)X.Liu,R.Freeman,E.Golub,I.Willner, ACS Nano2011,5,7648–7655;g)T.Li,E.Wang,S.Dong,Anal.Chem.2010,82,1515–1520;h)S.Shimron,F.Wang,R.Orbach,I.Willner,Anal.Chem.2012,84,1042–1048;i)F.Wang,R.Orbach,I.Willner,Chem.Eur.J.2012,18,16030–16036.[3]Z.G.Wang,P.Zhan,B.Ding,ACS Nano2013,7,1591–1598.[4]E.Golub,R.Freeman,I.Willner,Anal.Chem.2013,85,12126–12133.[5]E.Golub,R.Freeman,I.Willner,Angew.Chem.2011,123,11914–11918;Angew.Chem.Int.Ed.2011,50,11710–11714.[6]a)H.-W.Lee, D.J.-F.Chinnapen, D.Sen,Pure Appl.Chem.2004,76,1537–1545;b)P.R.Majhi,R.H.Shafer,Biopolymers2006,82,558–569;c)D.M.Kong,W.Yang,J.Wu,C.X.Li,H.X.Shen,Analyst2010,34,321–326.[7]a)L.Zhu,C.Li,Z.Zhu,D.Liu,Y.Zou,C.Wang,H.Fu,C.Yang,Anal.Chem.2012,84,8383–8390;b)S.Nakayama,J.X.Wang,H.O.Sintim, Chem.Eur.J.2011,17,5691–5698.5623[8]R.Freeman,E.Sharon,C.Teller,A.Henning,Y.Tzfati,I.Willner,ChemBio-Chem2010,11,2362–2367.[9]a)S.Shimron,J.Elbaz,A.Henning,I.Willner,mun.2010,46,3250–3252;b)J.Elbaz,S.shimron,I.Willner,mun.2010,46, 1209–1211.[10]C.H.Lu,X.J.Qi,R.Orbach,I.Mironi-Harpaz,D.Seliktar,I.Willner,NanoLett.2013,13,1298–1302.[11]a)L.C.Bock,L.C.Griffin,tham,E.H.Vermaas,J.J.Toole,Nature1992,355,564–566;b)R.F.Macaya,J.A.Waldron,B.A.Beutel,H.T.Gao,M.E.Joesten,M.H.Yang,R.Patel,A.H.Bertelsen,A.F.Cook,Bio-chemistry1995,34,4478–4492.[12]a)Y.Krishnan, F.C.Simmel,Angew.Chem.2011,123,3180–3215;Angew.Chem.Int.Ed.2011,50,3124–3156;b)C.Teller,I.Willner,Curr.Opin.Biotechnol.2010,21,376–391;c)J.Bath,A.J.Turberfield,Nat.Nanotechnol.2007,2,275–284;d)M.K.Beissenhirtz,I.Willner,Org.Biomol.Chem.2006,4,3392–3401.[13]B.Yurke,A.J.Turberfield,ls,F.C.Simmel,J.L.Neumann,Nature2000,406,605–608.[14]a)W.B.Sherman,N.C.Seeman,Nano.Lett.2004,4,1203–1207;b)Z.G.Wang,J.Elbaz,I.Willner,Nano Lett.2011,11,304–309.[15]C.H.Lu,A.Cecconello,J.Elbaz,A.Credi,I.Willner,Nano Lett.2013,13,2303–2308.[16]J.Elbaz,Z.G.Wang,F.Wang,I.Willner,Angew.Chem.2012,124,2399–2403;Angew.Chem.Int.Ed.2012,51,2349–2353.[17]J.S.Shin,N.A.Pierce,J.Am.Chem.Soc.2004,126,10834–10835.[18]J.Elbaz,Z.G.Wang,R.Orbach,I.Willner,Nano Lett.2009,9,4510–4514.[19]Z.G.Wang,J.Elbaz,F.Remacle,R.D.Levine,I.Willner,Proc.Natl.Acad.A2010,107,21996–22001.[20]X.Liang,H.Nishioka,N.Takenaka,H.Asanuma,ChemBioChem2008,9,702–705.[21]R.M.Izatt,J.S.Bradshaw,S.A.Nielsen,mb,J.J.Christensen,D.Sen,Chem.Rev.1985,85,271–339.[22]S.Nakayama,H.O.Sintim,Mol.BioSyst.2010,6,95–97.Received:December1,2013Revised:January20,2014Published online on March28,20145624。