Metal ion as both a cofactor and a probe of metal-binding sites in a uranyl-specific DNAzyme: a uranyl photocleavage study

DNAzymes are known to bind metal ions specifically to carry out catalytic functions. Despite many studies since DNAzymes were discovered nearly two decades ago, the metal-binding sites in DNAzymes are not fully understood. Herein, we adopt uranyl photocleavage to probe specific uranyl-binding sites in the 39E DNAzyme with catalytically relevant concentrations of uranyl. The results indicate that uranyl binds between T23 and C25 in the bulge loop, G11 and T12 in the stem loop of the enzyme strand, as well as between T2.4 and G3 close to the cleavage site in the substrate strand. Control experiments using two 39E DNAzyme mutants revealed a different cleavage pattern of the mutated region. Another DNAzyme, the 8–17 DNAzyme, which has a similar secondary structure but shows no activity in the presence of uranyl, indicated a different uranyl-dependent photocleavage as well. In addition, a close correlation between the concentration-dependent photocleavage and enzymatic activities is also demonstrated. Together, these experiments suggest that uranyl photocleavage has been successfully used to probe catalytically relevant uranyl-binding sites in the 39E DNAzyme. As uranyl is the cofactor of the 39E DNAzyme as well as the probe, specific uranyl binding has now been identified without disruption of the structure.     

Studies on the Effect of Thymine-Mercury-Thymine Stem as a Structural or Functional Motif in DNAzymes

T-Hg-T base pair formation has been demonstrated to be compatible with duplex DNA context, with considerable thermal stability contribution. Here, the T-Hg-T stem in two small DNAzymes 8–17 and 10–23 was studied for its structural and functional roles. The recognition arm 5′ to the cleavage site of 10–23 DNAzyme complex and the stem in the catalytic loop of 8–17 DNAzyme could be replaced by consecutive T-Hg-T stem of different length. The linear relationship between the activity of the complex 10–23DZ-6T+D19–6T and the concentration of Hg²⁺ demonstrated that the T-Hg-T stem contributes thermal stability of the recognition arm binding. The effect of T-Hg-T stem in the catalytic core of 8–17 DNAzyme and the position-dependent effect in 10–23 DNAzyme demonstrated that T-Hg-T base pair is not compatible with canonical base pairs in playing the functions of nucleic acids.     

Targeting insulin-like growth factor I with 10-23 DNAzymes: 2'-O-methyl modifications in the catalytic core enhance mRNA cleavage

Insulin-like growth factor I (IGF-I) and its cognate receptor (IGF-1R) contribute to normal cell function and to tumorigenesis. The role of IGF-I signaling in tumor growth has been demonstrated in vivo using nucleic acid-based strategies. Here, we designed the first 10-23 DNAzymes directed against IGF-I mRNA. Unlike antisense approaches and RNA interference that require protein catalysis, DNAzymes catalyze protein-free RNA cleavage. We identified target sequences and measured catalytic properties of differently designed DNAzymes on short synthetic RNA targets and on in vitro transcribed IGF-I mRNA. The most efficient cleavers were then transfected into cells, and their inhibitory effect was analyzed using reporter gene assays. We found that increasing the size of DNAzyme flanking sequences and modifications of the termini with 2'-O-methyl residues improved cleavage rates of target RNAs. Modification of the catalytic loop with six 2'-O-methyl ribonucleotides at nonessential positions increased or decreased catalytic efficiency depending on the mRNA target site. In cells, DNAzymes with 2'-O-methyl-modified catalytic cores and flanking sequences were able to inhibit reporter gene activity because of specific recognition and cleavage of IGF-I mRNA sequences. Mutant DNAzymes with inactive catalytic cores were unable to block reporter gene expression, demonstrating that the RNA cleaving ability of 10-23 DNAzymes contributed to inhibitory mechanisms. Our results show that nuclease-resistant 2'-O-methyl-modified DNAzymes with high catalytic efficiencies are useful for inhibiting IGF-I gene function in cells.     

Coarse-Grained Brownian Dynamics Simulations of the 10-23 DNAzyme

Deoxyribozymes (DNAzymes) are single-stranded DNA that catalyze nucleic acid biochemistry. Although a number of DNAzymes have been discovered by in vitro selection, the relationship between their tertiary structure and function remains unknown. We focus here on the well-studied 10-23 DNAzyme, which cleaves mRNA with a catalytic efficiency approaching that of RNase A. Using coarse-grained Brownian dynamics simulations, we find that the DNAzyme bends its substrate away from the cleavage point, exposing the reactive site and buckling the DNAzyme catalytic core. This hypothesized transition state provides microscopic insights into experimental observations concerning the size of the DNAzyme/substrate complex, the impact of the recognition arm length, and the sensitivity of the enzymatic activity to point mutations of the catalytic core. Upon cleaving the pertinent backbone bond in the substrate, we find that the catalytic core of the DNAzyme unwinds and the overall complex rapidly extends, in agreement with experiments on the related 8-17 DNAzyme. The results presented here provide a starting point for interpreting experimental data on DNAzyme kinetics, as well as developing more detailed simulation models. The results also demonstrate the limitations of using a simple physical model to understand the role of point mutations.     

RNA cleaving '10-23' DNAzymes with enhanced stability and activity

‘10-23’ DNAzymes can be used to cleave any target RNA in a sequence-specific manner. For applications in vivo, they have to be stabilised against nucleolytic attack by the introduction of modified nucleotides without obstructing cleavage activity. In this study, we optimise the design of a DNAzyme targeting the 5′-non-translated region of the human rhinovirus 14, a common cold virus, with regard to its kinetic properties and its stability against nucleases. We compare a large number of DNAzymes against the same target site that are stabilised by the use of a 3′-3′-inverted thymidine, phosphorothioate linkages, 2′-O-methyl RNA and locked nucleic acids, respectively. Both cleavage activity and nuclease stability were significantly enhanced by optimisation of arm length and content of modified nucleotides. Furthermore, we introduced modified nucleotides into the catalytic core to enhance stability against endonucleolytic degradation without abolishing catalytic activity. Our findings enabled us to establish a design for DNAzymes containing nucleotide modifications both in the binding arms and in the catalytic core, yielding a species with up to 10-fold enhanced activity and significantly elevated stability against nucleolytic cleavage. When transferring the design to a DNAzyme against a different target, only a slight modification was necessary to retain activity.     

Characterization of non-8-17 sequences uncovers structurally diverse RNA-cleaving deoxyribozymes

RNA-cleaving deoxyribozymes (DNAzymes) can be isolated from random-sequence DNA pools via the process of in vitro selection. However, small and simple catalytic motifs, such as the 8-17 DNAzyme, are commonly observed in sequence space, presenting a challenge in discovering large and complex DNAzymes. In an effort to investigate underrepresented molecular species derived from in vitro selection, in this study we sought to characterize non-8-17 sequences obtained from a previous in vitro selection experiment wherein the 8-17 deoxyribozyme was the dominant motif. We examined 9 sequence families from 21 motifs by characterizing their structural and functional features. We discovered 9 novel deoxyribozyme classes with large catalytic domains (>40 nucleotides) utilizing three-way or four-way junction structural frameworks. Kinetic studies revealed that these deoxyribozymes exhibit moderate to excellent catalytic rates (k(obs) from 0.003 to 1 min(-1)), compared to other known RNA-cleaving DNAzymes. Although chemical probing experiments, site-directed mutational analyses, and metal cofactor dependency tests suggest unique catalytic cores for each deoxyribozyme, common dinucleotide junction selectivity was observed between DNAzymes with similar secondary structural features. Together, our findings indicate that larger, structurally more complex, and diverse catalytic motifs are able to survive the process of in vitro selection despite a sequence space dominated by smaller and structurally simpler catalysts.     

Deletion analysis in the catalytic region of the 10-23 DNA enzyme

In this study, the functional relevance of the core nucleotides of the RNA cleaving 10-23 DNA enzyme (DNAzyme) was investigated. Systematic deletion studies revealed that DNAzymes lacking thymine at position 8 (T8) retain catalytic activity comparable to that of the wild-type enzyme. Deletion of the adjacent cytosine at position 7 (C7) also resulted in a highly active enzyme and even the double deletion mutant C7/T8 displayed cleavage activity, although the catalytic rate under multiple turnover conditions was found to be reduced by one order of magnitude. The identification of non-essential nucleotides in the catalytic core might help to stabilize the DNAzyme against nucleolytic degradation and to overcome problems in elucidating its three-dimensional structure.     

Position-specific modification with imidazolyl group on10–23 DNAzyme realized catalytic activity enhancement

Nucleoside analogues with imidazolyl and histidinyl groups were synthesized for site-specific modification on the catalytic core of 10–23 DNAzyme. The distinct position-dependent effect of imidazolyl group was observed. Positive effect at A9 position was always observed. The pH- and Mg²⁺-dependence of the imidazolyl-modified DNAzymes suggested that imidazolyl group in 10–23 DNAzyme probably plays a dual role, its hydrogen bonding ability and spacial occupation play the favorable influence on the catalytic conformation of the modified DNAzymes. This research demonstrated that the catalytic performance of DNAzymes could be enhanced by incorporation of additional functional groups. Chemical modification is a feasible approach toward more efficient DNAzymes for therapeutic and biotechnological applications.     

2′-Functional group of adenosine in 10–23 DNAzyme promotes catalytic activity

10–23 DNAzyme is an artificially selected catalytic DNA molecule. Its great potential as genetic therapeutics promoted chemical modifications for more efficient DNAzymes. Here, 10–23 DNAzyme was modified on its six deoxyadenosine residues (A5, A9, A11, A12, A15 in the catalytic domain and A0 of the recognition arm next to the cleavage site) with compound 1, an adenosine analogue with 2′-O-[N-(aminoethyl)carbamoyl]methyl group. A positive effect of compound 1 at A15 was observed (HJDS-05, k_obs = 0.0111 min⁻¹). Compared to the effect of 2′-H and 2′-OMe at A15, this result provided an approach for more efficient DNAzyme by combining 2′-substituted amino group of adenosine with A15 as the lead structure.     

10-23型DNA酶作为鉴定mRNA靶点有效性的新工具

10-23DNA酶是能主动切割mRNA的一类反义寡核苷酸.利用10-23DNA酶的直接切割作用验证mRNA结构靶点的有效性.对筛选的绿色荧光蛋白(GFP)基因mRNA的4个靶点平行设计了4条反义寡核苷酸和4条10-23DNA酶,对照组反义寡核苷酸将最佳靶点——靶点2的反义寡核苷酸突变2个碱基,对照组10-23DNA酶将靶点2的10-23DNA酶结合臂中央突变2个碱基.体外4条10-23DNA酶切割mRNA的结果和相应的4条反义寡核苷酸依赖的RNaseH降解结果完全相似,细胞内4条10-23DNA酶对绿色荧光蛋白的表达抑制作用与相应的4条反义寡核苷酸相似,表明10-23DNA酶显示的最佳作用靶点同样是最佳作用效果的反义寡核苷酸结合靶.10-23DNA酶可以作为评价mRNA结构靶点有效性的新工具.     

Introduction of guanidinium-modified deoxyuridine into the substrate binding regions of DNAzyme 10–23 to enhance target affinity: Implications for DNAzyme design

Deoxyribozymes (DNAzymes) are important catalysts for potential therapeutic RNA destruction and no DNAzyme has received as much notoriety in terms of therapeutic use as the Mg²⁺-dependent RNA-cleaving DNAzyme 10–23 (Dz10–23). As such, we have investigated the synthetic modification of Dz10–23 with a guanidinium group, a functionality that reduces the anionic nature and can potentially enhance the membrane permeability of oligonucleotides. To accomplish this, we synthesized a heretofore unknown phosphoramidite, 5-(N,N′-biscyanoethoxycarbonyl)-guanidinoallyl-2′-deoxyuridine and then incorporated it into oligonucleotides via solid phase synthesis to study duplex stability and its effect on Dz10–23. This particular modification was chosen as it had been used in the selection of Mg²⁺-free self-cleaving DNAzymes; as such this will enable the eventual comparison of modified DNAzymes that do or do not depend on Mg²⁺ for catalysis. Consistent with antecedent studies that have incorporated guanidinium groups into DNA oligonucleotides, this guanidinium-modified deoxyuridine enhanced the thermal stability of resulting duplexes. Surprisingly however, Dz10–23, when synthesized with modified residues in the substrate binding regions, was found to be somewhat less active than its non-modified counterpart. This work suggests that this particular system exhibits uniform binding with respect to ground state and transition state and provides insight into the challenge of re-engineering a Mg²⁺-dependent DNAzyme with enhanced catalytic activity.     

Brief communication: stability and catalytic activity of novel circular DNAzymes

DNAzymes represent a new generation of catalytic nucleic acids for specific RNA targeting in order to inhibit protein translation from the specifically cleaved mRNA. The 10-23 DNAzyme was found to hydrolyze RNA in a sequence-specific manner both in vitro and in vivo. Although single-stranded DNAzymes may represent the most effective nucleic acid drug to date, they are nevertheless sensitive to nuclease degradation and require modifications for in vivo application. However, previously used stabilization of DNAzymes by site-specific phosphorothioate (PT) modifications reduces the catalytic activity, and the PTO displays toxic side effects when applied in vivo. Thus, improving the stability of DNAzymes without reducing their catalytic activity is essential if the potential of these compounds should be realized in vivo. RESULTS: The Circozyme was tested targeting the mRNA of the most common genetic rearrangement in pediatric acute lymphoblastic leukemia TEL/AML1 (ETV6/RUNX1). The Circozyme exhibits a stability comparable to PTO-modified DNAzymes without reduction of catalytic activity and specificity and may represent a promising tool for DNAzyme in vivo applications. CONCLUSION: The inclusion of the catalytic site and the specific mRNA binding sequence of the DNAzyme into a circular loop-stem-loop structure (Circozyme) of approximately 70 bases presented here represents a new effective possibility of DNAzyme stabilization.     

Brief Communication: Stability and Catalytic Activity of Novel Circular DNAzymes

DNAzymes represent a new generation of catalytic nucleic acids for specific RNA targeting in order to inhibit protein translation from the specifically cleaved mRNA. The 10–23 DNAzyme was found to hydrolyze RNA in a sequence-specific manner both in vitro and in vivo. Although single-stranded DNAzymes may represent the most effective nucleic acid drug to date, they are nevertheless sensitive to nuclease degradation and require modifications for in vivo application. However, previously used stabilization of DNAzymes by site-specific phosphorothioate (PT) modifications reduces the catalytic activity, and the PTO displays toxic side effects when applied in vivo. Thus, improving the stability of DNAzymes without reducing their catalytic activity is essential if the potential of these compounds should be realized in vivo. Results: The Circozyme was tested targeting the mRNA of the most common genetic rearrangement in pediatric acute lymphoblastic leukemia TEL/AML1 (ETV6/RUNX1). The Circozyme exhibits a stability comparable to PTO-modified DNAzymes without reduction of catalytic activity and specificity and may represent a promising tool for DNAzyme in vivo applications. Conclusion: The inclusion of the catalytic site and the specific mRNA binding sequence of the DNAzyme into a circular loop-stem-loop structure (Circozyme) of approximately 70 bases presented here represents a new effective possibility of DNAzyme stabilization.     

A general strategy for effector-mediated control of RNA-cleaving ribozymes and DNA enzymes

A novel and general approach is described for generating versions of RNA-cleaving ribozymes (RNA enzymes) and DNAzymes (DNA enzymes), whose catalytic activity can be controlled by the binding of activator molecules. Variants of the RNA-cleaving 10-23 DNAzyme and 8-17 DNAzyme were created, whose catalysis was activated by up to approximately 35-fold by the binding of the effector adenosine. The design of such variants was possible even though the tertiary folding of the two DNAzymes is not known. Variants of the hammerhead ribozyme were constructed, to respond to the effectors ATP and flavin mononucleotide. Whereas in conventional allosteric ribozymes, effector-binding modulates the chemical step of catalysis, here, effectors exercise their effect upon the substrate-binding step, by stabilizing the enzyme-substrate complex. Because such an approach for controlling the activity of DNAzymes/ribozymes requires no prior knowledge of the enzyme's secondary or tertiary folding, this regulatory strategy should be generally applicable to any RNA-cleaving ribozyme or DNAzyme, natural or in vitro selected, provided substrate-recognition is achieved by Watson-Crick base-pairing.     

Gaining target access for deoxyribozymes

Antisense oligonucleotides and ribozymes have been used widely to regulate gene expression by targeting mRNAs in a sequence-specific manner. Long RNAs, however, are highly structured molecules. Thus, up to 90% of putative cleavage sites have been shown to be inaccessible to classical RNA based ribozymes or DNAzymes. Here, we report the use of modified nucleotides to overcome barriers raised by internal structures of the target RNA. In our attempt to cleave a broad range of picornavirus RNAs, we generated a DNAzyme against a highly conserved sequence in the 5' untranslated region (5' UTR). While this DNAzyme was highly efficient against the 5' UTR of the human rhinovirus 14, it failed to cleave the identical target sequence within the RNA of the related coxsackievirus A21 (CAV-21). After introduction of 2'-O-methyl RNA or locked nucleic acid (LNA) monomers into the substrate recognition arms, the DNAzyme degraded the previously inaccessible virus RNA at a high catalytic rate even to completion, indicating that nucleotides with high target affinity were able to compete successfully with internal structures. We then adopted this strategy to two DNAzymes that we had found to be inactive in our earlier experiments. The modified DNAzymes proved to be highly effective against their respective target structures. Our approach may be useful for other ribozyme strategies struggling with accessibility problems, especially when being restricted to unique target sites.     

A lead-dependent DNAzyme with a two-step mechanism

A detailed biochemical and mechanistic study of in vitro selected variants of 8-17 DNAzymes is presented. Even though the 8-17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg(2+) (called 8-17), 0.5 mM Mg(2+)/50 mM histidine (called Mg5), or 100 microM Zn(2+) (called 17E), all variants are shown to be the most active with Pb(2+) (8-17: k(obs) approximately 0.5 min(-1); Mg5: k(obs) approximately 2 min(-1); 17E: k(obs) approximately 1 min(-1) with 200 microM Pb(2+) at pH 5.0). For the 17E variant of the 8-17 DNAzyme, the single-turnover rate constants followed the order of Pb(2+) > Zn(2+) > Mn(2+) approximately Co(2+) > Ni(2+) > Mg(2+) approximately Ca(2+) > Sr(2+) approximately Ba(2+). The catalytic rate is half-maximal at 13.5 microM Pb(2+), 0.97 mM Zn(2+), or 10.5 mM Mg(2+), suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb(2+) > Zn(2+) > Mg(2+). The Pb(2+)-dependent activity increases linearly with pH and the slope of the plot of log k(obs) versus pH is approximately 1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variations of the DNAzyme confirm the importance of the G*T wobble pair, the two loops and the intervening stem in maintaining the active conformation of the system. While Mg(2+) and Zn(2+) catalyze only a transesterification reaction with formation of a product containing a 2',3'-cyclic phosphate, Pb(2+) catalyzes a transesterification reaction followed by hydrolysis of the 2',3'-cyclic phosphate. Although this two-step mechanism has shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Therefore, the two-step mechanism is observed in metalloenzymes of all classes, and this 8-17 DNAzyme provides a simple, stable, and cost-effective model system for understanding the structure of Pb(2+)-binding sites and their roles in the two-step mechanism.     

An efficient RNA-cleaving DNA enzyme can specifically target the 5'-untranslated region of severe acute respiratory syndrome associated coronavirus (SARS-CoV)

BACKGROUND: The worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS-CoV. We report the use of DNAzyme (catalytic DNA) to target the 5'-untranslated region (5'UTR) of a highly conserved fragment in the SARS genome as an approach to suppression of SARS-CoV replication. A mono-DNA enzyme (Dz-104) possessing the 10-23 catalytic motif was synthesized and tested both in vitro and in cell culture. MATERIALS AND METHODS: SARS-CoV total RNA was isolated, extracted from the SARS-CoV-WHU strain and converted into cDNA. We designed a RNA-cleaving 10-23 DNAzyme targeting at the loop region of the 5'UTR of SARS-CoV. The designed DNAzyme, Dz-104, and its mutant version, Dz-104 (mut), as a control consist of 9 + 9 arm sequences with a 10-23 catalytic core. In vitro cleavage was performed using an in vitro transcribed 5'UTR RNA substrate. A vector containing a fused 5'UTR and enhanced green fluorescent protein (eGFP) was co-transfected with the DNAzyme into E6 cells and the cells expressing eGFP were visualized with fluorescence microscopy and analyzed by fluorescence-activated cell sorting (FACS). RESULTS AND CONCLUSIONS: Our results demonstrated that this DNAzyme could efficiently cleave the SARS-CoV RNA substrate in vitro and inhibit the expression of the SARS-CoV 5'UTR-eGFP fusion RNA in mammalian cells. This work presents a model system to rapidly screen effective DNAzymes targeting SARS and provides a basis for potential therapeutic use of DNA enzymes to combat the SARS infection.