Metal ion-directed dynamic splicing of DNA through global conformational change by intramolecular complexation

Chemically engineered DNAs—in which global conformation can be modulated in response to specific stimuli—could be allosteric functional DNAs themselves or work as a modulator of the functional nucleic acids such as DNAzymes and aptamers. Here, we show that two terpyridines built in the DNA backbone form a stable intramolecular 1:2 complex, [M(terpy)2]2+, with divalent transition metal ions. Upon complexation, the DNA conjugates adopt a Ω-shape structure, in which two distal sequences located outside the terpyridines connect with each other to form a continuous segment with a specific structure or sequence. Such a DNA structure is globally controlled by local metal complexation events that can be rationally designed based on general coordination chemistry. This method is regarded as metal ion-directed dynamic sequence edition or DNA splicing. DNAzymes with peroxidase-like activity can thus be regulated by several transition metal ions through sequence edition techniques based on the Ω-motif. Higher-order structured DNA molecules can be manipulated to carry out specific enzymatic functions. Here the authors demonstrate the metal ion-directed global conformational control of DNA structure, using intramolecular coordination chemistry to manipulate the DNAzyme activity.

R ecently, several research groups have reported on the fabrication of specific DNA structures using metal iondirected techniques  . For example, two thymines and cytosines coordinate to Hg 2 þ and Ag þ , respectively, to form a M-DNA (metal ion DNA complex) duplex through the formation of metal-centred nucleobase dimers 7,8,[12][13][14][15] . A silver ion can be accommodated in the nucleobase triplet CGC of a DNA triplex by substitution of the N3 proton of cytosine on the third strand, which stabilizes the triplex structure even under neutral pH 10,11 . The knowledge of the specific coordination of nucleobases to these soft metal ions enabled us to prepare stable duplexes or triplexes by designing the sequences. On the other hand, several DNA analogues carrying metal chelators instead of natural nucleobases have been reported. Chelators, such as catechol, phenylenediamine, aminophenol and hydroxypyridone, selectively coordinate to certain transition metal ions to form stable M-DNA duplexes or triplexes [4][5][6] . The DNA conjugates with chelator molecules on their backbone have also been reported to form unique structures 9,16-21 . In the past decade, a number of functional nucleic acids, such as ribozymes, DNAzymes and aptamers, have been isolated using techniques of molecular evolution 22,23 . These nucleic acids have been applied to analytical systems for various biomolecules as functional modules for analyte recognition and signal amplification [24][25][26][27] . Prescribed higher-ordered structures are critically important for these nucleic acids to express their intrinsic functions. Therefore, techniques that control formation of their structures should be useful to modulate their functions. If the chemical groups, which respond to certain stimuli by changing their structure, are incorporated into the backbone of the functional DNAs or their complementary sequences, we could regulate their functions by controlling conformational changes in the DNAs. This strategy is general in the sense that it targets the higher-ordered structure of functional nucleic acids rather than each of their particular functions themselves. The techniques for drastic changes in DNA conformation could also be applied to regulate gene expression by certain stimuli such as decoy, antisense and RNAi strategies.
Herein, we synthesized 2,2 0 :6 0 ,2 00 -terpyridine (terpy)-based amidite reagents. Terpy units can be incorporated into any positions in the DNA backbone with these amidite reagents using automated DNA synthesis. Terpy forms a stable complex with Fe 2 þ and some other divalent transition metal ions 28,29 . We expected that the stiffness and conformation of the DNA backbone could be controlled through complexation with certain metal ions. Then, it would enable us to regulate the activity of functional nucleic acids. We have already reported the effect of metal ions on the stability of DNA structures consisting of the conjugates with one terpy (synthesized using 1 mentioned below). The terpy incorporated in the stem moiety of the duplex stabilized the structure by complexation, which confers entropic benefit on the DNA. In contrast, the structure of bimolecular triplexes carrying a terpy unit in its loop moiety significantly destabilizes on complexation with divalent transition metal ions 30 . This can be explained by the change in the length of the terpy unit in the loop of the triplex by metal coordination; the metal-bound terpy connecting two pyrimidine strands is too short to cap the base triplet in the structure of the DNA triplex.
In the present study, two terpy units are incorporated into the backbone of a DNA strand to form terpy 2 -DNA conjugates, in which two terpys are located at distant positions from each other with an intervening sequence. It is expected that transition metal ions, such as Fe 2 þ , forms a 1:2 complex with the two terpys in a terpy 2 -DNA conjugate. To form such an intramolecular complex, the backbone of terpy 2 -DNA conjugates make adopt an O-shaped conformation, where the two distal sequences outside both terpys in the sequence are directly connected with each other to form a continuous stretch of the specific structure or sequence. Such global conformational control of DNA would be regarded as metal ion-directed sequence edition or reversible splicing of DNA. We perform allosteric regulation of catalytic activity of DNAzyme through metal ion-directed sequence edition of DNA.
Results DNA preparation and system design. In this study, we aimed to regulate the function of DNAzyme with peroxidase-like activity 31,32 by conformational control of the terpy 2 -DNA conjugate. To incorporate terpy units into the backbone of the DNA, terpybased amidite reagents were prepared. We synthesized two amidite reagents 1 and 2 ( Fig. 1a) according to the schemes shown in Supplementary Information. Although both of them have a 2,2 0 :6 0 ,2 00 -terpyridine structure with two ethylene linker chains connecting to phosphoramidite and dimethoxytritylprotected hydroxyl groups, the substitution position of the linkers are different. 1 and 2 tether the two linker chains from 6,6 00 -and 5,5 00 -positions of terpy, respectively.
The sequences of the DNAs used in this study are shown in Fig. 1b. The four terpy 2 -DNA conjugates, tp 2 hp1, tp 2 hp2, tp 2 tmp1 and tp 2 tmp2, were prepared using 1 and 2 according to the standard phosphoramidite method shown in Supplementary Information. The last letter in the names of the terpy 2 -DNA conjugates, '1' or '2', stands for the amidite reagents 1 and 2 built in their sequences, respectively. We designed two systems for regulation of oxidation reactions by DNAzyme. In one system, we used the modified DNAzyme, tp 2 hp1 and tp 2 hp2, which can adopt a hairpin form by intramolecular metal complexation. The sequence of DNAzyme was split into two parts 33 , and each half was attached to both ends of a part of the complimentary sequence of the H-ras gene 34 through terpys to form tp 2 hp1 and tp 2 hp2. The catalytic activity of tp 2 hp1 and tp 2 hp2 were expected to be restored when forming a hairpin conformation induced by intramolecular 1:2 metal complexation (Fig. 1c). Reconstruction of an integrated active DNAzyme on the allosteric template, tp 2 tmp1 and tp 2 tmp2, was expected in another system. The same DNAzyme mentioned above was also split into two parts, and then the short binding arms were attached to the end of these parts to form a pair of the half DNAzymes 33 , zymR and zymL. The outside two sequences in tp 2 tmp1 and tp 2 tmp2 were designed to be complementary to the binding arms of zymR and zymL. Therefore, tp 2 tmp1 and tp 2 tmp2 are expected to work as an effective template when both outside sequences come in proximity with each other by forming an O-shaped conformation through 1:2-type intramolecular metal complexation (Fig. 1d). The loop sequences between the two terpys of tp 2 tmp1 and tp 2 tmp2 are designed to be complementary to miR-34 (microRNA) 35 .
Preliminary studies of complexation. Complexation of the terpy 2 -DNA conjugates, tp 2 tmp1 and tp 2 tmp2, with metal ions were investigated by ultraviolet-visible (vis) titrations. Four divalent transition metal ions, Fe 2 þ , Ni 2 þ , Cu 2 þ and Zn 2 þ , were used in this study (Fig. 2). The aliquots of transition metal ions were added into the conjugate solutions, and their ultraviolet-vis spectrum was recorded after each addition. The spectral changes and the plots of absorbance around the peak with Fe 2 þ titration are shown in Fig. 2a,b. The complexation behaviour of the two conjugates was completely different. tp 2 tmp1 showed stoichiometric complexation with Fe 2 þ . The absorbance increased monotonously and saturated at the point of (Fig. 2a), indicating that each of the two terpys on a tp 2 tmp1 bound a Fe 2 þ to form two [Fe(terpy)] 2 þ on ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7640 a tp 2 tmp1. In contrast, the stoichiometry of tp 2 tmp2 complexation was clearly shown to be 1.0 ( ¼ [Fe 2 þ ]/[tp 2 tmp2]) (Fig. 2b), indicating that the terpy units formed [Fe(terpy) 2 ] 2 þ on tp 2 tmp2. Further change was not observed in the spectra even with more additional Fe 2 þ . The characteristic band was observed at 530 nm. This is attributed to the metal-to-ligand chargetransfer band of the 1:2 complex, [Fe(terpy) 2 ] 2 þ (refs 28,29). Interestingly, this meant that the complex [Fe(terpy) 2 ] 2 þ did not form on tp 2 tmp1 but exclusively formed on tp 2 tmp2. All of the results observed in the ultraviolet-vis titrations were reproduced in the presence of the complementary DNA, ctmp, which is the tandem sequence complementary to the two distal sequences outside of the two terpys.
The only difference between tp 2 tmp1 and tp 2 tmp2 is the tethering positions of the terpys connected to DNA backbones. We calculated the structures of both complexes using the density functional theory (DFT) method (B3LYP/6-31G*). The optimized structures of [Fe(terpy) 2 ] 2 þ are shown in Fig. 3. The structure of Fig. 3a is the complex consisting of a Fe 2 þ and two 6,6 00 -tethered terpys and Fig. 3b is of a Fe 2 þ and two 5,5 00 -tethered terpys, each of which is the complex expected to form on tp 2 tmp1 (or tp 2 tmp1/ctmp) and tp 2 tmp2 (or tp 2 tmp2/ctmp), respectively. In the structure of [Fe(terpy) 2 ] 2 þ with 6,6 00 -tethered terpys, the linker chains on a terpy almost touches the central pyridine ring of another terpy in the complex. This steric hindrance should make the complex unstable or impossible to form. In contrast, this hindrance was not observed in the complex consisting of 5,5 00 -tethered terpys. This would explain the difference in complexation behaviour between tp 2 tmp1 and tp 2 tmp2 observed in ultraviolet-vis titration.
To identify the complex, an equimolar mixture of terpy 2 -DNA and Fe 2 þ was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The short terpy 2 -DNA conjugate (18-mer DNA conjugate carrying two terpy units) was prepared for this study using the terpy amidite 2.
Using the conjugate with a lower molecular weight, we can obtain accurate and precise m/z values of the complexes, even for multimeric DNAs. The result are shown in Supplementary Fig. 4   Metallo-regulation of thermal stability of the duplexes. The melting behaviour was studied for the duplexes consisting of the terpy 2 -DNA conjugates and the complementary DNA, ctmp.
The ultraviolet melting curves of the duplexes, tp 2 tmp1/ctmp and tp 2 tmp2/ctmp, recorded in the presence of the divalent transition metal ions are shown in Fig. 4. The melting curves of tp 2 tmp1/ ctmp were scarcely affected by any transition metal ions (Fig. 4a).
In contrast, the duplex structure of tp 2 tmp2/ctmp was apparently stabilized by the addition of an equimolar amount of transition metal ions (Fig. 4b). In addition, the metal ions appeared to change the shape of the melting curves of tp 2 tmp2/ctmp to be more cooperative, indicating that the two sequences outside of the terpys cooperatively dissociated in a narrow temperature range in the presence of the metal ions. The highly cooperative melting behaviour also suggests that [Fe(terpy) 2 ] 2 þ observed in ultraviolet-vis titration for tp 2 tmp2 was an intramolecular complex formed on tp 2 tmp2Fe 2 þ . That is, tp 2 tmp2 formed an O-shaped conformation through intramolecular 1:2 complexation and, concomitantly, the distal two segments located outside of two terpys came close in proximity with each other to form a continuous stretch of the complementary sequence for ctmp (Fig. 4c). The duplexes would be stabilized by entropic benefit through metal ion-directed global conformational change of DNA. This could be regarded as dynamic sequence edition. Meanwhile, the complex formation does not affect the global conformation of tp 2 tmp1, because each of the terpys binds Fe 2 þ independently, which is the reason the metal ions scarcely stabilize the duplex tp 2 tmp1/ctmp.
The melting temperature of czym/ctmp was 38°C under the same conditions. Although the O-shaped conformation of tp 2 tmp2Fe 2 þ gave the same sequence with czym, the duplex stability of tp 2 tmp2Fe 2 þ /ctmp did not come up to czym/ctmp. The structure of tp 2 tmp2Fe 2 þ could be improved for its structural continuity. Comparable results were commonly observed for chemical ligation products with non-natural structures at the connecting points 37 . It could be improved by optimization of the linker chain structure of terpy units in terpy 2 -DNA.
At the point of equimolar ratio ([M 2 þ ]/[tp 2 tmp2] ¼ 1), all transition metal ions used in the melting study stabilized the duplex tp 2 tmp2/ctmp to a similar extent. However, the dependences of the stabilization effects on the feeding ratio were different for each of the metal ions. For Fe 2 þ and Ni 2 þ , the duplex remained stable at [M 2 þ ]/[tp 2 tmp2] ¼ 2 and 3. In contrast, the duplex was destabilized at higher feeding ratios for Cu 2 þ and Zn 2 þ (Fig. 4d). The results essentially coincide with those obtained in the ultraviolet-vis titration. The duplex stability of tp 2 tmp2/ctmp was maintained even in the presence of the excess amount of Fe 2 þ and Ni 2 þ , because the O-shaped conformation of tp 2 tmp2 would be preserved due to the binding properties of the metal ions with terpy, K 1 oK 2 . Meanwhile, for Cu 2 þ and Zn 2 þ , the global conformation of tp 2 tmp2 would be changed from Oto a linear-form accompanying the transition of the complex types formed on tp 2 tmp2 from [M(terpy) 2 ] 2 þ (on tp 2 tmp2M 2 þ ) to 2[M(terpy)] 2 þ (on tp 2 tmp22M 2 þ ) with increasing amounts of the ions due to their magnitudes of the two successive binding constants with terpy, K 1 4K 2 (ref. 36). Especially, the binding of Cu 2 þ with endocyclic nitrogens of nucleobases is strong 38,39 . This would explain the significant inhibition of the duplex formation by Cu 2 þ at higher [Cu 2 þ ]/ [tp 2 tmp2] ratios.
Allosteric regulation of DNAzyme activity. We constructed the two systems to regulate the activity of DNAzyme by specific coordination of metal ions. The active form of the DNAzyme is a G-quadruplex (G4) structure that requires a hemin as a cofactor 31,32 . The catalytic activity was monitored by oxidation of ABTS (2,2 0 -Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), which shows green in colour after oxidation 40 . Figure 5a shows the time course of the reaction profiles for the conjugates, tp 2 hp1 or tp 2 hp2, in the presence of an equivalent amount of Fe 2 þ . The activity of tp 2 hp2 was apparently restored by Fe 2 þ . Such activation was not observed for tp 2 hp1. As expected from the preliminary studies mentioned above, only tp 2 hp2 seemed to adopt a hairpin structure by intramolecular [Fe(terpy) 2 ] 2 þ formation and restored the active form of DNAzyme (Fig. 1c). The conjugate tp 2 hp2 could be regarded as a semi-artificial metallo-DNAzyme. However, the activity level of tp 2 hp2Fe 2 þ was very little compared with the system of tp 2 tmp2. The G4-structure formed on tp 2 hp2Fe 2 þ might not be ideal as a catalyst. This could be improved by fine-tuning of the structures around terpys.
Another allosteric system was also tested to control the activity of DNAzyme. Here tp 2 tmp2 was used as the tunable template to activate the split DNAzyme (zymR and zymL) as shown in     Fig. 5b. Equivalent concentrations of Fe 2 þ and Ni 2 þ restored the activity of the split DNAzyme in the presence of tp 2 tmp2. The effect of metal ions could be observed by naked eyes (Fig. 5c). The regained activity level was comparable to that of the positive control, zymR/zymL/czym (see Supplementary Fig. 6). Cu 2 þ and Zn 2 þ also showed moderate effect in restoration of split DNAzyme activity (see Supplementary Fig. 5). As we expected, global conformation of tp 2 tmp2 was fixed to a O-shape by intramolecular formation of [M(terpy) 2 ] 2 þ . Then, tp 2 tmp2M 2 þ worked as an effective template to reconstruct the integrated active form of DNAzyme.
The activity was almost completely suppressed by a large excess amount of EDTA and an equimolar amount of the singlestranded 23-mer DNA (miR) with an miR-34 sequence, which is complementary to the loop sequence (Fig. 5b). The results of these inhibition experiments confirmed our scheme for the regulation of DNAzyme activity. EDTA removed Fe 2 þ from the complex as a masking reagent. The single-stranded DNA with the miR-34 sequence straightened the loop region of tp 2 tmp2 by hybridization and made it impossible to form [Fe(terpy) 2 ] 2 þ .
The ternary DNA complex zymR/zymL/tp 2 tmp2 was subjected to circular dichroism (CD) spectroscopy. Figure 5d shows the differential CD spectra. To show only the contribution of the DNAzyme moiety, the CD spectrum of tp 2 tmp2/ctmp (duplex þ loop) was subtracted from those of zymR/zymL/tp 2 tmp2 (reconstituted DNAzyme þ duplex þ loop) obtained in the absence and in the presence of Fe 2 þ . The characteristic CD band for the mixed-type G4-structure (or the mixture of parallel and antiparallel folded species) was induced by the addition of Fe 2 þ (refs 41,42). This suggests that Fe 2 þ activated the DNAzyme by induction of a valid higher-ordered structure through sequence edition of the template, tp 2 tmp2.

Discussion
Our aim in this study was to induce a drastic conformational change of global DNA (secondary) structure and its application for the allosteric regulation of the function of nucleic acids by certain stimuli. Although several stimuli-responsive DNA conjugates that change their local conformation have been reported [43][44][45] , the systems of allosteric control of the global DNA structures are quite limited 9,17,46,47 . Even more, as far as we know, there is no precedent of conformational modulation of DNA aiming at sequence edition. The formation of a O-shape structure by intramolecular interaction between two specific points on a sequence could be regarded as stimuli-responsive artificial splicing. That is, the distal two segments are directly connected with each other by clipping out the intervening loop sequence.
In this study, the specific coordination of terpys to transition metal ions was chosen as a connecting reaction to show the availability of a O-motif for regulation of DNA function. Actually, while the phosphates in DNA backbone and nucleobases can coordinate to the transition metal ions 39 , the ions, especially Fe 2 þ and Ni 2 þ , selectively bind two terpys to form a O-shaped structure under experimental conditions. The common preference in coordination chemistry of terpy was retained even for complexation on DNA. Therefore, the O-shape structure formation could be designed based on general knowledge in coordination chemistry. The kind and concentration of the metal ions, the structure of the terpy and the design of the conjugate architecture could be effective factors to control the system. Especially, it was interesting that local structural differences (just as E1.4 Å) in terpy units, such as substitution positions, were critically important to define the global structure. We succeeded in the regulation of DNAzyme activity using the O-motif. Judging from the results of ultraviolet-vis titration, ultraviolet melting, CD spectroscopy, modelling analysis by DFT and ABTS oxidation and inhibition experiments, the activity of DNAzyme was regulated basically according to the proposed scheme shown in Fig. 1. However, the difference in melting temperatures of the duplex tp 2 tmp2/ctmp observed in the presence and absence of Fe 2 þ might not be enough to solely explain the sharp response in restoration of DNAzyme activity. At least, the CD spectra showed that Fe 2 þ induced the active G4structure of DNAzyme. Fe 2 þ could work as an essential factor not only to assemble the split DNAzymes but also to induce the active G4-structure through complexation.
To construct the system for artificial (non-enzymatic or chemical) splicing of DNA, we can choose appropriate clipping reactions from many bioorthogonal chemistries including click reactions 48,49 and photochemical reactions 37,[44][45][46]50 . The techniques could be applied for signal amplification in bioanalyses through DNA circuits based on autonomous succession of strand exchange 26,51 . The regulation of cell function through the control of gene expression would also be very compatible and a promising application.

Methods
Synthesis. Preparation 30 of the amidite reagents, 1 and 2, and DNAs are described in the Supplementary Information.
Ultraviolet melting. Thermal denaturation experiments were performed 9,54,55 in HEPES buffer solution (10 mM, pH 7.0) on a UV-1650 (Shimadzu) ultraviolet-vis spectrophotometer equipped with a Peltier thermal controller. Concentrations of each DNA strand and MCl 2 (M ¼ Fe 2 þ , Ni 2 þ , Cu 2 þ or Zn 2 þ ) were 1.0 mM. Prior to the beginning of each melt, the samples were degassed at 85°C for 5 min and then annealed by slowly cooling to 0°C. In the denaturation experiments, the solutions were heated at a rate of 0.5 deg min À 1 after equilibration for 20 min at 0°C.
The temperature that gave the maximum first derivative of the melting curve was used as the melting temperature, a measure of the thermal stability of the duplexes.
Circular dichroism measurement. CD 56,57 spectra were measured in HEPES buffer solution (20 mM, pH 7.0) containing 1.0 M NaCl and 200 mM KCl on a J-725 Spectropolarimeter (Jasco) equipped with a Peltier thermal controller, at 15°C. Concentrations of all DNA strands and FeCl 2 were 3.0 mM. Prior to the beginning of each melt, the samples were degassed at 85°C for 5 min and then annealed by slowly cooling to 15°C.
To only show the contribution of the DNAzyme moiety, the CD spectrum of tp 2 tmp2/ctmp (duplex þ loop) was subtracted from those of zymR/zymL/tp 2 tmp2 (reconstituted DNAzyme þ duplex þ loop) obtained in the absence and in the presence of Fe 2 þ . Molecular modelling. Both complex ([Fe(terpy) 2 ] 2 þ ) structures were calculated by the DFT method (B3LYP/6-31G*) using Spartan 10 (Wavefunction Inc., USA). The molecular models shown in Fig. 4c were constructed with MacroModel (ver. 9.5, Schrödinger Inc., USA). The initial structure was built as a standard double helix using the Maestro (Schrödinger Inc.) fragment library. The optimization was performed with an AMBER* force field with generalized Born/surface area treatment of solvation.