An allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities Dazhi Jiang*, Jiacui Xu*, Yongjie Sheng, Yanhong Sun and Jin Zhang Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, China Introduction DNAzymes are efficient biological catalysts that strengthen the catalytic power of nucleic acids [1,2]. To date, a series of DNAzymes with RNA-cleaving (or DNA-cleaving) activity have been obtained by in vitro selection. Some investigations have focused on the improvement of specific characteristics and functions of these DNAzymes through rational design, including the following: using oligo-DNAs [3,4] or different wavelengths of light [5–8] as effectors to control the catalytic activity of the DNAymes; engineering DNA- zyme-based sensors for Mg 2+ [9,10], Cu 2+ [11], Hg 2+ [12,13], Pb 2+ [14], and UO 2 2+ [15]; and constructing molecular logic gates and nanomotors [16–20]. How- ever, engineering an allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities is very challenging. To our knowledge, such a DNAzyme has not been reported. In this article, we report on a new catalytic activity in a DNAzyme scaffold generated by rational recon- struction, and the regulation of catalytic activity by a conformational transition. We prepared a DNA-cleav- ing DNAzyme, using a deoxyribonucleotide residue grafting strategy, as a model system for designing a bifunctional DNAzyme that undergoes the self-cleav- age reaction, but also possesses the ability to catalyze the cleavage of an RNA substrate (RS). An oligo- RNA molecule played a double role as both the substrate for the RNA-cleaving activity of the recon- structed DNAzyme and as a ‘negative’ effector for controlling the self-cleavage activity of the DNAzyme. Keywords activity; allosteric; DNAzyme; grafting; regulation Correspondence J. Zhang, Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, 130021 China Fax: +86 431 88980440 Tel: +86 431 88980440 E-mail: zhangjin@jlu.edu.cn *These authors contributed equally to this work (Received 4 November 2009, revised 21 March 2010, accepted 1 April 2010) doi:10.1111/j.1742-4658.2010.07669.x A series of RNA-cleaving or DNA-cleaving DNAzymes have been obtained by in vitro selection. However, engineering an allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities is very challenging. We used an in vitro-selected pistol-like (PL) DNAzyme as a DNA scaffold for designing a DNAzyme with dual catalytic activities. We prepared the 46-nucleotide DNAzyme with DNA-cleaving activity (PL DNAzyme), and then grafted the deoxyribonucleotide residues from an 8–17 variant DNAzyme into the region of stem–loop I and the catalytic core of the PL DNAzyme scaffold. This deoxyribonucleotide residue graft- ing resulted in a DNAzyme with dual RNA-cleaving and DNA-cleaving activities (DRc DNAzyme). Drc DNAzyme has properties different from those of the original PL DNAzyme, including DNA cleavage sites and the required metal ion concentration. Interestingly, the RNA substrate and RNase A can act as effectors to mediate the DNA cleavage. Our results show that RNA-cleaving and DNA-cleaving activities simultaneously coex- ist in DRc DNAzyme, and the DNA cleavage activity can be reversibly regulated by a conformational transition. Abbreviations DRc DNAzyme, DNA-cleaving and RNA-cleaving DNAzyme; PL, pistol-like; RS, RNA substrate. FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS 2543 RNase was prepared as a ‘positive’ effector to reacti- vate the DNAzyme via degradation of the ‘negative’ RNA. Results and Discussion Design of the DNA-cleaving and RNA-cleaving DNAzyme (DRc DNAzyme) We elected to use an in vitro-selected pistol-like (PL) DNAzyme as a DNA scaffold for designing a DNA- zyme with dual catalytic activities. The PL DNAzyme (Fig. 1A) can efficiently catalyze Cu 2+ -dependent self- cleavage, and is composed of a catalytic core spanning nucleotides 27–46 and two base-paired structural ele- ments (stems I and II) flanked by regions of ssDNA [21]. The 5¢-arm of the enzyme binds the cleavable sequence via Watson–Crick base pairs and the 3¢-arm through formation of a DNA triplex. The catalytic residues were derived from the 8–17 variant DNAzyme (Fig. 1B). The original 8–17 DNA- zyme was isolated by in vitro evolution; this enzyme can efficiently cleave RNA to provide 2¢,3¢-cyclic phos- phate and the 5¢-hydroxyl termini of RNA fragments [22]. In its catalytic core, the dinucleotides A6G7 of a terminal AGC loop and C13G14 of a bulge loop are essential, and serve as the key deoxyribonucleotide residues involved in the cleavage of the RNA phospho- diester bond [23–25]. Deoxyribonucleotides A12 and A15 of a bulge loop are not conserved. A12 can be changed to T12, and A15 can be changed to G15. When 8–17 DNAzyme binds its substrate, a gÆT wobble pair can be formed, and is considered to be significant and crucial for the catalytic activity. When the C deoxyribonucleotide is inserted in the 5¢-T28G29G30-3¢ sequence of PL DNAzyme, the sequence is changed to 5¢-TCGG-3¢, which is the same as the bulge sequence of 8–17 variant DNAzyme. The stem and terminal loop (5¢-AGC-3¢) of 8–17 variant DNAzyme replaces stem–loop I of the PL DNAzyme scaffold, and the T deoxyribonucleotide of 8–17 vari- ant DNAzyme is inserted between deoxyribonucleo- tides 13 and 14 of the scaffold (Fig. 1C). Characterization of DRc DNAzyme Like the parent PL DNAzyme, DRc DNAzyme was shown to catalyze self-cleavage in the presence of aCu 2+ (Fig. 2A). Other metal ions, including Mg 2+ ,Ca 2+ ,Mn 2+ ,Co 2+ ,Ni 2+ ,Cd 2+ ,Zn 2+ , and Ba 2+ , failed to facilitate the cleavage activity. The reconstructed DNAzyme was shown to use divalent copper ions with high specificity, despite replacement of the right domain of the DNAzyme scaffold. Incuba- tion of DRc DNAzyme yielded two distinct DNA cleavage products (P a and P b ). To map the cleavage site of the self-cleaving DNAzyme, we used denaturing PAGE and ran the gel until the reaction products were clearly separated (Fig. 2B). The P a and P b cleavage fragments were produced upon DRc DNAzyme scission at C14 and C24, respectively. The control, PL DNAzyme, displayed four cleavage fragments (P a ,P b1 ,P b2 , and P b3 ) which were cleaved at A14, T28, G29, and G30, respectively. To study the rate of DRc cleavage directly, the DNAzyme was incubated at pH 7.0 and 23 °C. The DRc DNAzyme exhibited a narrow functional range for the concentration of Cu 2+ , with optimum activity A C B 5′ 5′ Fig. 1. Sequence and predicted secondary structures of original DNAzymes and the reconstructed DNAzyme. (A) Sequence and secondary structure of a 46-nucleotide self-cleaving PL DNAzyme. A triple helix interaction (dots) occurs between the four base pairs of stem II and four consecutive pyrimidine residues near the 5¢-DNA. The major site of DNA cleavage is indicated by the black arrowhead. (B) Sequence and secondary structure of 8–17 variant DNAzyme. The capital letters represent deoxyribonucleotides, and the small letters represent ribonucleotides. (C) Sequence and secondary structure of the reconstructed DRc DNAzyme. The DRc DNAzyme can form the DNA or RNA cleavage folded motifs under different reaction conditions. An RNA-cleaving and DNA-cleaving DNAzyme D. Jiang et al. 2544 FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS being reached at 100 lm. The rate of DNA cleavage was highly dependent on the concentration of Cu 2+ used in the reaction mixture. When the concentration of Cu 2+ was higher or lower than 100 lm, the cleav- age activity rapidly decreased (Fig. 3A). The original PL DNAzyme showed a bell-shaped dependence on Cu 2+ concentration from 10 mm to 1 nm, with cleav- age essentially going to completion at 10 lm. DRc DNAzyme and PL DNAzyme were incubated at 23 °C in the presence of 100 lm Cu 2+ and buffers of varying pH (5.0–8.5). The DRc DNAzyme cleavage product increased to a maximum yield of 47% at pH 7.5 (Fig. 3B). A similar pattern was observed for PL DNAzyme. To evaluate the effect of temperature on DNA cleavage, we incubated DRc DNAzyme under standard cleavage conditions while varying the temper- ature. As compared with PL DNAzyme, DRc DNA- zyme function seemed to have decreased sensitivity to the reaction temperature. DRc DNAzyme exhibited a broad functional range for temperature, with optimum activity being reached at 23 °C (Fig. 3C). After characterizing the DNA-cleaving activity of DRc DNAzyme, we continued to study its RNA- cleaving activity (Fig. 3D–F). Improved cleavage activity has been observed upon replacement of Mg 2+ with Mn 2+ . To obtain RNA-cleaving rates over a broad range of metal concentrations, cleavage reac- tions in the presence of Mn 2+ (100–200 mm) were performed at pH 7.5. The cleavage activity exhibited a sharp metal concentration dependence, with maximal activity at 1 mm (Fig. 3D). DRc DNAzyme was mod- erately perturbed in its RNA-cleaving activity relative to the 8–17 variant. Although the Cu 2+ and ascorbate were important for the DNA-cleaving activity of DRc DNAzyme, they did not support the RNA-cleaving activity of DRc DNAzyme under our reaction conditions (100 lm Cu 2+ ,10lm ascorbate, 10 mm Mn 2+ , and 50 mm Tris ⁄ HCl, pH 7.5). To investigate the effect of pH on RNA cleavage, the pH dependence of DRc DNAzyme was analyzed between pH 4.92 and pH 9.18 in the presence of 1 mm Mn 2+ (Fig. 3E), and was very similar to that of the 8–17 variant DNAzyme. It was not feasible to obtain a quantitatively meaningful rate versus pH, because, at high pH, Mn 2+ precipitation occurred. The RNA- cleaving activity of DRc DNAzyme was assayed at A B C D Fig. 2. The DNA-cleaving activity and cleavage sites of DRc DNAzyme. (A) 5¢- 32 P-labeled DRc DNAzyme was incubated in buffer A at 23 °C with 10 l M various divalent metal ions, which generated two labeled products (P a and P b ) in the presence of Cu 2+ . Control reactions were incubated in the absence of metal ions. Reaction products were separated by 20% denaturing PAGE and imaged by autoradiography. (B) Trace amounts of 5¢- 32 P-labeled PL DNAzyme or DRc DNAzyme were incubated in buffer A, containing 10 lM CuCl 2 , 0.3 M NaCl, 10 lM L -ascorbate (except for PL–), and 30 mM Hepes at 23 °C. PL+ and PL) represent the presence and absence, respectively, of L-ascorbate in the reaction. Lanes M I and M II were loaded with 5¢- 32 P-labeled synthetic DNAs of different lengths as indicated, each with a sequence that corresponded to the respective 5¢-terminus of the substrate DNA. The letters indicate the 3¢-termini of these radiolabeled marker DNAs. (C, D) Schemes for the substrate cleavage sites of PL DNAzyme and DRc DNAzyme, respectively. The arrowheads and asterisks denote the positions of cleavage sites. D. Jiang et al. An RNA-cleaving and DNA-cleaving DNAzyme FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS 2545 different temperatures. The DNAzyme (Fig. 3F) showed a linear temperature dependence between 25.8 and 53.7 °C. When evaluating the optimal design for a DNAzyme with respect to DNA-cleaving and RNA-cleaving activities, we found that a number of nucleotides within the catalytic core and substrate-binding arm of PL DNAzymes were not highly conserved and could be substituted by the structural domain derived from the 8–17 variant DNAzyme. We wanted to combine our optimization of the arm design and modification of the catalytic domain to yield an enhanced DNAzyme. Unfortunately, other designed DNAzymes (DA1–DA3) suggested that DNA-cleaving and RNA-cleaving activi- ties could not coexist within a DNA motif, and the double activities competed with each other. Because DRc DNAzyme has comparatively high activities, we focused substantial effort on its characterization. The DRc DNAzyme with new catalytic activity can arise from an existing DNAzyme scaffold, indicating that a single DNA sequence can catalyze the two respective reactions and assume either of two DNAzyme folds. RNAzymes previously investigated have shown similar properties [26,27]. The characterization data collected (Fig. 3) provide a number of indications and constraints for future modeling studies on the active structure and evolution of the DNAzymes. The regulating effects of RS and RNase on the DNA cleavage of DRc DNAzyme After characterizing the DNA-cleaving and RNA- cleaving activities of DRc DNAzyme, we found that an RS could act as an effector to control the DNA cleavage of DRc DNAzyme (Fig. 4A). Regulation proceeds via an effector-generated rearrangement of the active site, where the DNA-cleaving active site of DRc DNAzyme is hindered through the binding of RS. The self-cleaving DRc DNAzyme was incubated in reaction buffer B, containing 100 lm CuCl 2 , 0.3 m NaCl, 10 lml-ascorbate, and 30 mm Hepes (pH 7.0) at 23 °C. The self-cleavage of DRc DNAzyme was A BC FED Fig. 3. Characterization of the DNA-cleaving and RNA-cleaving reactions catalyzed by DRc DNAzyme. (A–C) Analyses of DNA cleavage at dif- ferent Cu 2+ concentrations, pH values, and temperatures, respectively. All reactions were conducted using trace amounts of 5¢- 32 P-labeled DRc DNAzyme. In (A), the CuCl 2 concentration was varied from 1 to 10 mM. The reactions were conducted at pH 7.0 (30 mM Hepes) and 23 °C with 0.3 M NaCl and 10 lML-ascorbate. In (B), the reactions were conducted under different pH conditions with 0.3 M NaCl, 10 lM L -ascorbate, and 10 lM CuCl 2 , and were incubated at 23 °C. In (C), the effect of reaction temperature on DRc DNAzyme function was assessed with cleavage assays conducted as described in (A), except that 10 l M CuCl 2 was present and the temperature was varied from 12 to 40.7 °C. (D–F) Analyses of RNA cleavage at with different Mn 2+ concentration, reaction pH values, and temperatures, respectively. All reactions were conducted using 20 n M DNAzyme and 2 nM 5¢- 32 P-labeled RS. In (D), the MnCl 2 concentration was varied from 0.1 to 200 m M. The reactions were conducted at 37 °C and pH 7.5 (50 mM Tris ⁄ HCl). In (E), reactions were conducted under different pH condi- tions with 10 m M Mn 2+ , and were incubated at 37 °C. In (F), the reaction temperature was varied from 25.8 to 53.7 °C. The reactions were conducted at pH 7.5 (50 m M Tris ⁄ HCl) with 10 mM Mn 2+ . An RNA-cleaving and DNA-cleaving DNAzyme D. Jiang et al. 2546 FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS measured in the presence of various concentration of RS regulator, and the percentage cleavage versus RS concentration yielded a sigmoidal curve (Fig. 4B). The presence of a 12-nucleotide RNA regulator that is complementary to the RS recognition domain dramati- cally decreased the rate of DNA self-cleavage. We systematically varied the length of the RS to determine the effects on regulation of the DNA self-cleaving activity. Enlarging the RS to 18 bp essentially abol- ished the catalytic activity, as expected. This might be attributed to steric interference in the DNA-cleaving catalytic domain of DRc DNAzyme. Reducing the RS to 6 bp increased the rate of DNA cleavage, which approached the self-cleaving rate of DRc DNAzyme in the absence of RS. RSs with greater lengths were more efficient at decreasing the DNA self-cleavage of DRc DNAzyme. Here, the RS actually acted as a ‘negative’ effector to regulate the DNA-cleaving activity of DRc DNA- zyme. We were interested in constructing a reversible control for catalytic activity. RNase is a type of nucle- ase that catalyzes the degradation of RNA into smaller components. Bishop and Klavins reported that RNase H had been used to reverse binding in a deoxy- ribozyme nanomotor [20]. In our study, RNase A and RNase H were selected as ‘positive’ effectors to elimi- nate the RS regulation. As shown in Fig. 4C, RNase A was more efficient than RNase H in triggering the DNA-cleaving activity of DRc DNAzyme. Under the conditions of the DNA-cleaving regulated system, most RSs exist in a single-stranded state, and a few RSs can form a DNAÆRNA duplex with the RNA substrate recognition domain of DRc DNAzyme. RNase A cleaved ssRNA and RNase H specifically A CB Fig. 4. The regulating effects of RS and RNase on DNA cleavage. (A) Scheme for reversible modulation of DNA self-cleavage. (B) The RS acted as a ‘negative’ effector to decrease the DNA cleavage. The data fit the Boltzmann equation and the curve is sigmoidal. (C) RNase A or RNase H acted as ‘positive’ effectors to renew the DNA cleavage. D. Jiang et al. An RNA-cleaving and DNA-cleaving DNAzyme FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS 2547 hydrolyzed RNA in a DNAÆRNA duplex. We specu- lated that this difference between RNase A and RNase H might lead to different efficiencies in ‘positive’ regulation. When compared with RNase H, RNase A was better suited as a ‘positive’ effector. We used a simple and invasive method to reversibly modulate the DNA cleavage rate of DRc DNAzyme with RNA substrate and RNase A. Such regulatory biocatalyst systems offer several advantages, including the ease of RNA substrate and DNAzyme synthesis without any chemical modification, and convenient use of RNase A as a common bioreagent, an attractive property for DNAzymes that have various applications. In conclusion, we have described a key residue graft- ing strategy for generating RNA-cleaving activity in a self-cleaving DNAzyme. The DNAzyme with DNA- cleaving and RNA-cleaving activities was constructed by incorporating the catalytic domain of 8–17 variant DNAzyme into the right domain of the secondary structure of PL DNAzyme, demonstrating that dual activities can coexist in a small DNA scaffold. Using the RNA substrate and RNase A, we constructed a simple conformational switch to control the DNA- cleaving activity of DRc DNAzyme. The generation of a new active site within a DNAzyme scaffold and reg- ulation of the catalytic activity provide further insights into the engineering of DNAzymes. Experimental procedures Materials The DNA sequences (PL DNAzyme, 5¢-GAATTCTAATAC GACTCAGAATGAGTCTGGGCCTCTTTTTAAGAAC-3¢; 8–17 variant DNAzyme, 5¢-AATACTCCGAGCCGGTCG GGCCTC-3¢; DRc DNAzyme, 5¢-GAATTCTAATACTCC GAGCCGGTCGGGCCTCTTTTTAAGAAC-3¢) were pre- pared by automated synthesis, and purified by 16% denaturing PAGE (Sangon, Shanghai, China). The RS (5¢-gaggcagguauu-3¢) was also prepared by automated syn- thesis and purified by HPLC (TaKaRa, Dalian, China). [ 32 P]ATP[cP] was purchased from Furui. T4 polynucleotide kinase was purchased from TaKaRa. RNase A and RNase H were purchased from MBI. All chemical reagents were purchased from BBI. Activity assays for the DNAzyme To assess the cleavage activity of the DNAzyme, radiola- beled RNA or DNA were first generated by enzymatically tagging the 5¢-termini of synthetic RSs or self-cleaving DNAzymes. The reaction mixture contained 10 mm MgCl 2 , 5mm dithiothreitol, 2 lm RS or self-cleaving DNAzyme, 0.4 lm [ 32 P]ATP[cP] ( 20 lCi; 1 Ci = 37 GBq), and 0.5 lL of T4 PNK (10 UÆlL )1 ), and the mixture was incu- bated at 37 °C for 1 h. Prior to the self-cleavage activity assays, the DNAzyme was first denatured by heating to 90 °C for 2 min, and then incubated at 0 °C for 5 min. Trace amounts of 5¢- 32 P-labeled DNAzyme were incubated in reaction buffer A, containing 10 lm CuCl 2 , 0.3 m NaCl, 10 lml-ascorbate and 30 mm Hepes (pH 7.0) at 23 °C. To assess the RNA-cleaving activ- ity of the DNAzyme, cleavage reactions were performed by combining 20 nm DNAzyme and 2 nm 5¢- 32 P-labeled RS in the presence of 10 mm Mn 2+ in 50 mm Tris ⁄ HCl (pH 7.5). Mixtures were incubated at 37 °C for 30 min. The reaction was terminated after a designated period of time by the addition of stop solution containing 60 mm EDTA, 8 m urea, 0.02% (w ⁄ v) xylene cyanol, and 0.02% (w ⁄ v) bromophenyl blue solution. Cleavage products were separated by 20% denaturing PAGE and visualized by autoradiography. Regulation of the DNA cleavage of the DNAzyme For ‘negative’ regulation assays, reactions were initiated by the addition of a mixture of 1 nm 5¢- 32 P-labeled DNAzyme, reaction buffer B (100 lm CuCl 2 , 0.3 m NaCl, 10 lm l-ascorbate, 30 mm Hepes, pH 7.0) and varying concentra- tions (0–100 nm) of RS (as a ‘negative’ effector) at 23 °C. For ‘positive’ regulation assays, reactions were con- ducted at 23 °C for a designated period of time, using 1 nm 5¢- 32 P-labeled DNAzyme, 100 nm RS, reaction buffer B, and 10 ng of RNase A (or 5 U of RNase H). Cleavage products were separated by denaturing PAGE and imaged by autoradiography. Acknowledgement This work was supported by grants from the National Natural Science Foundation of China (General Pro- gram No. 30770479). References 1 Emilsson GM & Breaker RR (2002) Deoxyribozymes: new activities and new applications. Cell Mol Life Sci 59, 596–607. 2Ho ¨ bartner C & Silverman SK (2007) Recent advances in DNA catalysis. Biopolymers 87, 279–292. 3 Wang DY & Sen D (2001) A novel mode of regulation of an RNA-cleaving DNAzyme by effectors that bind to both enzyme and substrate. J Mol Biol 310, 723–734. 4 Wang DY, Lai BHY & Sen D (2002) A general strategy for effector-mediated control of RNA-cleaving ribo- zymes and DNA enzymes. J Mol Biol 318, 33–43. 5 Liu Y & Sen D (2004) Light-regulated catalysis by an RNA-cleaving deoxyribozyme. J Mol Biol 341, 887–892. An RNA-cleaving and DNA-cleaving DNAzyme D. Jiang et al. 2548 FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS 6 Ting R, Lermer L & Perrin DM (2004) Triggering DNAzymes with light: a photoactive C 8 thioether-linked adenosine. J Am Chem Soc 126, 12720–12721. 7 Keiper S & Vyle JS (2006) Reversible photocontrol of deoxyribozyme-catalyzed RNA cleavage under multiple- turnover conditions. Angew Chem Int Ed Engl 45, 3306–3309. 8 Lusic H, Young DD, Lively MO & Deiters A (2007) Photochemical DNA activation. Org Lett 9, 1903–1906. 9 Chiuman W & Li Y (2007) Efficient signaling platforms built from a small catalytic DNA and doubly labeled fluorogenic substrates. Nucleic Acids Res 35, 401–405. 10 Chiuman W & Li Y (2007) Simple fluorescent sensors engineered with catalytic DNA ‘MgZ’ based on a non-classic allosteric design. PLoS ONE 11, e1124. 11 Liu J & Lu Y (2007) A DNAzyme catalytic beacon sensor for paramagnetic Cu 2+ ions in aqueous solution with high sensitivity and selectivity. J Am Chem Soc 129, 9838–9839. 12 Liu J & Lu Y (2007) Rational design of ‘turn-on’ allo- steric DNAzyme catalytic beacons for aqueous mercury ions with ultra high sensitivity and selectivity. Angew Chem Int Ed Engl 46, 7587–7590. 13 Hollenstein M, Hipolito C, Lam C, Dietrich D & Perrin DM (2008) A highly selective DNAzyme sensor for mercuric ions. Angew Chem Int Ed Engl 47, 4346–4350. 14 Li J & Lu Y (2000) A highly sensitive and selective catalytic DNA biosensor for lead ions. J Am Chem Soc 122, 10466–10467. 15 Liu J, Brown AK, Meng X, Cropek DM, Istok JD, Watson DB & Lu Y (2007) A catalytic beacon sensor for uranium with parts-pertrillion sensitivity and millionfold selectivity. Proc Natl Acad Sci USA 104, 2056–2061. 16 Chen X, Wang Y, Liu Q, Zhang Z, Fan C & He L (2006) Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl 45, 1759–1762. 17 Stojanovic MN, Semova S, Kolpashchikov D, Macdonald J, Morgan C & Stefanovic D (2005) Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc 127, 6914–6915. 18 Lederman H, Macdonald J, Stefanovic D & Stojanovic MN (2006) Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry 45, 1194–1199. 19 Macdonald J, Li Y, Sutovic M, Lederman H, Pendri K, Lu W, Andrews BL, Stefanovic D & Stojanovic MN (2006) Medium scale integration of molecular logic gates in an automaton. Nano Lett 6, 2598–2603. 20 Bishop JD & Klavins E (2007) An improved autonomous DNA nanomotor. Nano Lett 7, 2574– 2577. 21 Carmi N, Balkhi SR & Breaker RR (1998) Cleaving DNA with DNA. Proc Natl Acad Sci USA 95, 2233– 2237. 22 Santoro SW & Joyce GF (1997) A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci USA 94, 4262–4266. 23 Cruz RPG, Withers JB & Li Y (2004) Dinucleotide junction cleavage versatility of 8–17 deoxyribozyme. Chem Biol 11, 57–67. 24 Schlosser K, Gu J, Sule L & Li Y (2008) Sequence– function relationships provide new insight into the cleavage site selectivity of the 8–17 RNA- cleaving deoxyribozyme. Nucleic Acids Res 36, 1472–1481. 25 Peracchi A, Bonaccio M & Clerici M (2005) A muta- tional analysis of the 8–17 deoxyribozyme core. J Mol Biol 352, 783–794. 26 Curtis EA & Bartel DP (2005) New catalytic structures from an existing ribozyme. Nat Struct Mol Biol 12, 994–1000. 27 Schultes EA & Bartel DP (2000) One sequence, two ribozymes: implications for the emergence of new ribozyme folds. Science 289, 448–452. D. Jiang et al. An RNA-cleaving and DNA-cleaving DNAzyme FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS 2549 . An allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities Dazhi Jiang*, Jiacui Xu*, Yongjie Sheng, Yanhong Sun and Jin Zhang Key. Pb 2+ [14], and UO 2 2+ [15]; and constructing molecular logic gates and nanomotors [16–20]. How- ever, engineering an allosteric DNAzyme with dual RNA-cleaving and