Ligase-mediated synthesis of CuII-responsive allosteric DNAzyme with bifacial 5-carboxyuracil nucleobases

A CuII-responsive allosteric DNAzyme has been developed by introducing bifacial 5-carboxyuracil (caU) nucleobases, which form both hydrogen-bonded caU–A and metal-mediated caU–CuII–caU base pairs. The base sequence was logically designed based on a known RNA-cleaving DNAzyme so that the caU-modified DNAzyme (caU-DNAzyme) can form a catalytically inactive structure containing three caU–A base pairs and an active form with three caU–CuII–caU pairs. The caU-DNAzyme was synthesized by joining short caU-containing fragments with a standard DNA ligase. The activity of caU-DNAzyme was suppressed without CuII, but enhanced 21-fold with the addition of CuII. Furthermore, the DNAzyme activity was turned on and off during the reaction by the addition and removal of CuII ions. Both ligase-mediated synthesis and CuII-dependent allosteric regulation were achieved by the bifacial base pairing properties of caU. This study provides a new strategy for designing stimuli-responsive DNA molecular systems.


Introduction
The highly sophisticated molecular recognition abilities of nucleic acids based on complementary hydrogen-bonded base pairing have led to a dramatic growth in the research eld recognized as DNA nanotechnology. 1Numerous DNA nanodevices, sensors, and molecular machines have been created by controlling DNA hybridization and structures in response to stimuli such as DNA/RNA binding, pH changes, and light irradiation. 2Metal ions also serve as external stimuli to regulate the DNA structure and function, particularly by exploiting metalmediated unnatural base pairing. 3Metal-mediated articial base pairs are formed between two opposing ligand-type nucleobase analogs by complexation with a bridging metal ion.Metal-mediated base pairing generally stabilizes DNA duplexes, thus controlling DNA hybridization in a metaldependent manner.
5][6][7] Bifacial bases such as 5-hydroxyuracil (U OH ) 4,5 and 5-carboxyuracil (caU) 6 are designed to form metal-mediated self-base pairs (e.g., U OH -Gd III -U OH ) in the presence of certain metal ions and to form Watson-Crick-like base pairs with a natural nucleobase in DNA duplexes (e.g., U OH -A).Based on the switching between U OH -A and U OH -Gd III -U OH base pairs, Gd III -triggered DNA strand displacement reactions were demonstrated and Gd III -mediated control of DNA tweezer structures and DNAzyme functions was successfully achieved. 5n this study, a metal-responsive DNAzyme was newly developed by utilizing bifacial 5-carboxyuracil (caU) bases as metal binding sites (Fig. 1).DNAzymes are DNA molecules with catalytic activity that have been widely applied for the

EDGE ARTICLE
development of DNA-based biosensors and molecular machines. 8Of particular interest is the rational design of allosteric DNAzymes whose activity can be controlled in response to specic stimuli.Such stimuli-responsive DNAzymes are versatile components for building up deformable DNA nanoarchitectures as well as DNA reaction networks.Metalresponsive DNAzymes have been developed previously by incorporating metal-mediated base pairs such as Cu II -mediated hydroxypyridone base pairs (H-Cu II -H). 9 The bifacial caU bases used in this study not only form hydrogen-bonded caU-A base pairs, but also Cu II -mediated caU-Cu II -caU base pairs with high metal selectivity. 6The caU-Cu II -caU base pairing signicantly stabilizes the DNA duplexes (DT m = +30.7 °C for a duplex with three caU-Cu II -caU pairs), whereas the caU-A base pairs are destabilized by the addition of Cu II ions (DT m = −7.3°C for a duplex with three caU-A pairs by using 6 equiv. of Cu II ions).Therefore, carefully designed DNAzymes containing caU bases were expected to exhibit good responsiveness to Cu II ions.To prepare DNAzyme strands containing multiple caU nucleotides, enzymatic synthesis using a DNA ligase was also investigated.A standard T4 DNA ligase was utilized because it was reported to tolerate modied base pairs and backbones.9b,d,10 Thus, we expected that caU-containing short fragments could be enzymatically ligated to give long DNA strands modied with caU bases.

Results and discussion
The Cu II -responsive allosteric DNAzyme was logically designed by modifying the base sequence of the known RNA-cleaving NaA43 DNAzyme 11 (Fig. 2a) in a manner similar to the Gd IIIresponsive DNAzyme with U OH bases. 5The NaA43 DNAzyme was chosen because it does not require metal cofactors that can be trapped by common chelators used to selectively remove Cu II ions for reversible regulation of DNAzyme activity (vide infra).Since duplex stabilization is more pronounced when three or more consecutive caU-Cu II -caU pairs are used, 6 we decided to incorporate three caU-Cu II -caU pairs into the parent NaA43 DNAzyme.Three pairs of caU bases were introduced into the stem region and the surrounding bases (shown in orange) were redesigned to form a different secondary structure in the absence of Cu II ions.The caU-modied DNAzyme (caU-DNAzyme) was expected to undergo a structural change upon addition of Cu II ions, from a catalytically inactive structure with three caU-A base pairs to an active form with three caU-Cu II -caU base pairs (Fig. 2b).The plausible secondary structure was simulated using the NUPACK soware 12 (Fig. S1 †).The caU bases were replaced with natural T bases to calculate the structure in the absence of Cu II , and the potential caU-Cu II -caU pairs were changed to G-C base pairs to simulate the structure in the presence of Cu II .The results indicated that both the inactive and the active structures can be stably formed via the formation of caU-A and caU-Cu II -caU base pairs, respectively.
A caU-modied DNAzyme strand (58-nt) was synthesized by ligating short DNA fragments (Fig. 3a).The caU-containing strands can be chemically synthesized based on the conventional phosphoramidite chemistry, 6 but the chemical synthesis requires an additional protecting group on the carboxylate of the caU bases.Although the coupling yield is sufficiently high, incomplete deprotection oen makes purication of long oligonucleotides containing multiple caU bases very difficult.Therefore, we expected that the ligase-mediated synthesis would be a suitable strategy to synthesize caU-modied DNAzymes.Since caU nucleobases can form caU-A base pairs with adenine bases on the complementary DNA, caU-modied oligonucleotides were expected to assemble on the splint strand and to be ligated by a standard T4 DNA ligase.A DNA tetramer 5 0 -caUcaUcaUG-3 0 (1) containing three caU nucleotides was used to introduce three consecutive caU bases into the resulting DNA strands.A natural nucleotide G was added at the 3 0 -terminal so that an additional G-C base pairing would facilitate the hybridization of tetramer 1 to the splint DNA 5.The caU-containing fragment 1 was prepared using an automated DNA synthesizer according to the reported procedure. 6Prior to the ligation reaction, fragments 1, 2, and 3 were treated with T4 polynucleotide kinase (T4 PNK) to introduce a phosphate group at the 5 0 end (step i).Aer all the DNA fragments were hybridized to splint 5 (step ii), a ligation reaction was performed using T4 DNA ligase (step iii).Reaction products were analyzed by  denaturing polyacrylamide gel electrophoresis (PAGE) (Fig. 3b).Aer incubation at 16 °C for 18 h, fragment 4 was efficiently consumed and a low mobility band appeared.The mobility of the new band was nearly identical to that of a chemically synthesized T-DNAzyme strand (58-nt) in which the caU nucleotides of the caU-DNAzyme were replaced with natural thymidines (T).Comparing the band intensities, the reaction yield reached 70% or more, conrming that the ligase reaction was proceeding well.The formation of the desired caU-DNAzyme strand was conrmed by MALDI mass spectrometry aer isolation ([M-2H] 2− : calcd 9150.31,found 9150.67,Fig. S2 †).It was demonstrated that T4 PNK and T4 DNA ligase successfully phosphorylated and ligated the short strand 1 (5 0 -caUcaUcaUG-3 0 ) despite the presence of a modied caU base at the 5 0 end.Since the desired product can be easily isolated by denaturing PAGE, the ligase-mediated synthesis was shown to be a powerful alternative method to incorporate multiple caU bases into functional DNA sequences.
Using the resulting caU-modied DNAzyme strand, we examined whether the catalytic activity of caU-DNAzyme can be regulated in response to the addition of Cu II ions.DNAzymecatalyzed RNA-cleaving reactions were performed using 10 equiv. of substrates labeled with a uorescent dye (FAM).Substrate cleavage was quantitatively evaluated by denaturing PAGE analysis (Fig. S3 †).Fig. 4a compares the RNA cleavage reaction catalyzed by caU-DNAzyme in the presence of varying concentrations of Cu II ions.In the absence of Cu II ions, the RNA-cleaving activity of the caU-DNAzyme was greatly suppressed.The DNAzyme activity was found to be enhanced by the addition of Cu II ions; the highest activity was observed when 9 equiv. of Cu II ions were added.Time-course analysis further conrmed the Cu II -dependent activation of caU-DNAzyme (Fig. 4b and S4 †).Under the same conditions, the catalytic activity of the unmodied NaA43 DNAzyme was reduced by adding Cu II ions (Fig. S5 †).A control T-DNAzyme containing natural T bases in place of caU showed no RNA-cleaving activity both in the absence and presence of Cu II ions.These results clearly demonstrate that the addition of Cu II ions enhances the catalytic activity of the caU-modied DNAzyme.In the presence of Cu II ions, the caU-DNAzyme cleaved approximately 70% of the substrate (i.e., 7 equiv.) in 20 h, conrming that the modi-ed DNAzyme maintains multiple turnover ability.
It is most likely that the activity of caU-DNAzyme was switched based on the changes in the base-pairing partners of the caU bases.This was supported by a model experiment using a FAM-labeled strand containing three caU bases in the middle (1U) (Fig. 4c and S6 †).The DNA 1U was annealed with two complementary strands 2U and 2A containing three caU or A bases, respectively, and the hybridization products were analyzed by native PAGE (Fig. 4d).Under Cu II -free conditions, only the duplex 1U$2A with caU-A pairs was formed.In the presence of Cu II ions, the duplex 1U$2U with caU-Cu II -caU pairs was formed (up to about 60%).These results clearly show the Cu II -mediated change in the hybridization partners, which is the driving force behind the allosteric regulation of the caU-DNAzyme.
The catalytically active form contains three caU-Cu II -caU base pairs, but the maximum activity was observed in the presence of 9 equiv. of Cu II ions (Fig. 4a).This inconsistency can be explained by the stability of the caU-A base pairs in addition to the caU-Cu II -caU pairs. 6Melting experiments (Fig. S7 †) showed that the model 15-bp duplex 1U 0 $2U, containing three caU-caU pairs, exhibited the highest melting temperature (T m ) in the presence of 3 equiv.of Cu II ions, due to the quantitative formation of caU-Cu II -caU pairs.On the other hand, the stability of duplex 1U 0 $2A 0 with three caU-A pairs decreases with increasing amounts of Cu II ions, possibly due to the binding of Cu II ions to the caU bases. 6The difference in the T m values of duplexes 1U 0 $2U and 1U 0 $2A 0 was maximal when more than 3 equiv.of Cu II were added.In fact, the hybridization experiments (Fig. 4d) showed that an excess of Cu II ions are required to change the hybridization partners of the caU bases.Therefore, the requirement for an excess of Cu II ions to activate the caU-DNAzyme suggests that the DNAzyme functions through both Cu II -mediated destabilization of the caU-A pairs (inactive state) and Cu II -mediated caU-Cu II -caU base pair formation (active state) exactly as designed.
The apparent rst-order rate constants (k obs ) for the DNAzyme reactions were estimated from the initial rates (Table 1).The catalytic activity of caU-DNAzyme was found to increase by approximately 21-fold with the addition of 9 equiv. of Cu II ions.The maximum activity of the caU-DNAzyme (k obs = 4.2 × 10 −2 h −1 ) was lower than that of the unmodied NaA43 DNAzyme (k obs = 4.7 × 10 −1 h −1 ).This may be due not only to the incomplete transformation into the active state, but also to the structural distortion caused by the caU-Cu II -caU base pairs. 6As indicated by circular dichroism (CD) analysis in the previous study, 6 caU-Cu II -caU base pairing can unwind the stem duplex to some extent.Introducing the caU base at a position more distant from the catalytic core would reduce the negative effect on the DNAzyme activity.It is noteworthy that the Cu II -mediated activation of caU-DNAzyme (21-fold) was much more efficient than that of a Cu II -responsive H-modied DNAzyme (5.9-fold), which was developed by incorporating an H-Cu II -H base pair into the same NaA43 DNAzyme.The results clearly show that the bifacial caU nucleobases, which form both hydrogenbonded caU-A and metal-mediated caU-Cu II -caU base pairs, are useful for metal-responsive switching of DNA functions.
We further carried out the DNAzyme reactions in the presence of Hg II ions that can mediate caU-Hg II -caU base pairing 6 (Fig. S8 †).In contrast to Cu II ions, the addition of Hg II ions did not activate caU-DNAzyme at all.Note that the activity of the unmodied NaA43 DNAzyme was signicantly reduced by the addition of Hg II ions.This is probably due to the undesired binding of Hg II ions to the natural T bases.These results clearly demonstrate the advantage of using metal ions that do not interact strongly with natural bases (e.g., Cu II ), especially with long oligonucleotides such as DNAzymes.
The RNA-cleaving activity of caU-DNAzyme was reversibly regulated in response to Cu II ions during the reaction (Fig. 5).The reaction was initiated in the absence of Cu II ions, and aer 4 h, Cu II ions (9 equiv.)were added.The reaction rate was immediately increased to k obs = 3.9 × 10 −2 h −1 , which is comparable to the rate observed when the reaction was initiated with Cu II ions (Fig. 5a).In a similar manner, removal of Cu II ions was shown to inactivate caU-DNAzyme.Addition of the chelating agent EDTA or Cu II -binding peptide (GHK) 13 in equimolar amounts with Cu II ions (9 equiv.)immediately slowed the reaction (k obs = 2.5 × 10 −3 h −1 and 5.3 × 10 −3 h −1 , respectively) (Fig. 5b).The addition of sodium ascorbate, which can reduce Cu II to Cu I , also decreased the activity of caU-DNAzyme to k obs = 3.6 × 10 −3 h −1 (Fig. S9 †).These results demonstrate that the activity of caU-DNAzyme can be rapidly switched by the addition, removal, and reduction of Cu II ions under isothermal conditions.Alternate addition of Cu II ions (9 equiv.)and EDTA (9 equiv.)cycled the on-off switching of caU-DNAzyme (Fig. 5c).The k obs values in each step demonstrate a clear switching in the caU-DNAzyme activity in response to Cu II (Fig. 5d).

Conclusions
In summary, a Cu II -responsive allosteric DNAzyme was developed by introducing 5-carboxyuracil (caU) nucleobases into a known DNAzyme sequence.The caU-modied DNAzyme (caU-DNAzyme) was enzymatically synthesized by joining short caUcontaining fragments with a standard T4 DNA ligase.The ligasemediated synthesis was possible because the caU base was structurally similar to the natural T base and could form a Watson-Crick-like base pair with the A base on the splint DNA.The base sequence of caU-DNAzyme is logically designed to form both the catalytically inactive structure by caU-A base pairing and the active form by metal-mediated caU-Cu II -caU base pairing.The activity of caU-DNAzyme was enhanced 21fold by the addition of Cu II ions and could be turned on and off during the reaction by the addition and removal (or reduction) of Cu II ions.These results demonstrate that the caU-modied DNAzyme was allosterically regulated through metal-mediated base-pair switching between caU-A and caU-Cu II -caU.The use of Cu II is essential to induce base-pair switching of the caU base.The caU base can form other types of metal-mediated base pairs such as caU-Hg II -T, caU-Ag I -C, and caU-Cu II -G. 6Therefore, caU-modied DNAs are expected to be further applied in constructing more complex DNA systems responsive to multiple metal ions.
This study conrms that metal-responsive DNA systems can be logically designed based on metal-mediated base-pair switching of bifacial caU nucleobases.The strategic design of caU-modied DNAzymes is expected to be applied to other types of DNAzymes 14 and other functional DNAs as well.Ligase-   mediated synthesis provides a simple way to incorporate caU bases into longer DNA sequences and is advantageous for sequence screening.Thus, it is suggested that the incorporation of bifacial caU bases is a powerful strategy for creating a variety of Cu II -responsive DNA molecular systems.The range of metal ions used could be expanded by developing other types of bifacial nucleobases with a different metal coordinating functionality at the 5-position of pyrimidine bases.In fact, Gd IIIresponsive DNA systems have been developed using cognate U OH nucleobases. 5The bifacial 5-modied pyrimidine bases are expected to be introduced into DNA via the ligase-mediated synthesis, similar to the case with caU.Therefore, metalmediated base-pair switching of bifacial nucleobases and the ligase-mediated synthesis have the potential to be versatile tools for building DNA-based stimuli-responsive systems such as biosensors, molecular machines, and computing devices.Further applications of bifacial caU nucleobases to metaltriggered operation of DNA nanoarchitectures and DNA logic circuits are currently under investigation.

Fig. 1
Fig. 1 Ligase-mediated synthesis of a Cu II -responsive DNAzyme containing bifacial 5-carboxyuracil (caU) bases, which form hydrogenbonded caU-A and metal-mediated caU-Cu II -caU base pairs.U in the figure represents a caU base.

Fig. 2
Fig. 2 Molecular design of a Cu II -responsive allosteric DNAzyme with caU nucleobases (caU-DNAzyme).(a) Sequence of the parent RNAcleaving DNAzyme (NaA43 DNAzyme).(b) Sequence of caU-DNAzyme.Both the active structure with caU-Cu II -caU base pairs and the inactive structure with caU-A base pairs are shown."rA" in the substrate indicates an adenosine ribonucleotide at the cleavage site.

Fig. 4
Fig. 4 (a) RNA-cleaving activity of caU-DNAzyme in the presence of varying concentrations of Cu II ions.The fractions of the cleaved substrate after a 6 h reaction are shown (see also Fig. S4 †).(b) RNAcleaving activity of caU-DNAzyme and the parent NaA43 DNAzyme in the absence and the presence of 9 equiv. of Cu II .T-DNAzyme, in which all caU bases are replaced with natural T bases, was used as a control.[DNAzyme] = 1.0 mM, [substrate] = 10 mM, [CuSO 4 ] = 0, 9.0 mM in 10 mM HEPES (pH 7.0), 100 mM NaCl, 25 °C.(c) Cu II -mediated change in the hybridization partners of the caU-containing strand (1U).(d) Native PAGE analysis of the hybridization product in the presence of varying amounts of Cu II ions.[DNA] = 2 mM each in 10 mM HEPES (pH 7.0) and 100 mM NaCl.The samples were annealed prior to the analysis.FAM detection.
DNAzyme modied with a pair of hydroxypyridone (H) nucleobases.9bb In the presence of 9 equiv. of Cu II ions.c In the presence of 1 equiv. of Cu II ions.

Fig. 5
Fig. 5 Cu II -dependent regulation of the RNA-cleaving activity of caU-DNAzyme.(a) Activation of caU-DNAzyme by the addition of Cu II ions (9 equiv.).(b) Deactivation of caU-DNAzyme by the removal of Cu II ions with a chelating agent EDTA or a Cu II -binding tripeptide (GHK) (9 equiv.).(c) Iterative switching of the DNAzyme activity.Cu II (9 equiv.)and EDTA (9 equiv.)were alternately added.[DNAzyme] = 1.0 mM, [substrate] = 10 mM, 25 °C.The activities of caU-DNAzyme in the absence (red dotted lines) and presence of Cu II ions (red solid lines) are also shown.(d) Apparent first-order rate constant (k obs ) for each step.N = 3. Error bars indicate standard errors.

Table 1
Apparent first-order rate constants (k obs ) for the DNAzymecatalyzed RNA cleavage reactions