Two‐Component 10–23 DNA Enzymes

A new strategy for engineering of catalytic two‐component constructions based on 10–23 DNAzyme was proposed. The using of a combination of shortened DNAzyme with 2′‐O‐methyl oligomers as effectors significantly increased the catalytic activity of this DNAzyme.     

Structural rearrangements of the 10-23 DNAzyme to beta 3 integrin subunit mRNA induced by cations and their relations to the catalytic activity

The intracellular ability of the "10-23" DNAzyme to efficiently inhibit expression of targeted proteins has been evidenced by in vitro and in vivo studies. However, standard conditions for kinetic measurements of the DNAzyme catalytic activity in vitro include 25 mM Mg2+, a concentration that is very unlikely to be achieved intracellularly. To study this discrepancy, we analyzed the folding transitions of the 10-23 DNAzyme induced by Mg2+. For this purpose, spectroscopic analyzes such as fluorescence resonance energy transfer, fluorescence anisotropy, circular dichroism, and surface plasmon resonance measurements were performed. The global geometry of the DNAzyme in the absence of added Mg2+ seems to be essentially extended, has no catalytic activity, and shows a very low binding affinity to its RNA substrate. The folding of the DNAzyme induced by binding of Mg2+ may occur in several distinct stages. The first stage, observed at 0.5 mM Mg2+, corresponds to the formation of a compact structure with limited binding properties and without catalytic activity. Then, at 5 mM Mg2+, flanking arms are projected at right position and angles to bind RNA. In such a state, DNAzyme shows substantial binding to its substrate and significant catalytic activity. Finally, the transition occurring at 15 mM Mg2+ leads to the formation of the catalytic domain, and DNAzyme shows high binding affinity toward substrate and efficient catalytic activity. Under conditions simulating intracellular conditions, the DNAzyme was only partially folded, did not bind to its substrate, and showed only residual catalytic activity, suggesting that it may be inactive in the transfected cells and behave like antisense oligodeoxynucleotide.     

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.     

Insights into how nucleotide supplements enhance the peroxidase-mimicking DNAzyme activity of the G-quadruplex/hemin system

Since the initial discovery of the catalytic capability of short DNA fragments, this peculiar enzyme-like property (termed DNAzyme) has continued to garner much interest in the scientific community because of the virtually unlimited applications in developing new molecular devices. Alongside the exponential rise in the number of DNAzyme applications in the last past years, the search for convenient ways to improve its overall efficiency has only started to emerge. Credence has been lent to this strategy by the recent demonstration that the quadruplex-based DNAzyme proficiency can be enhanced by ATP supplements. Herein, we have made a further leap along this path, trying first of all to decipher the actual DNAzyme catalytic cycle (to gain insights into the steps ATP may influence), and subsequently investigating in detail the influence of all the parameters that govern the catalytic efficiency. We have extended this study to other nucleotides and quadruplexes, thus demonstrating the versatility and broad applicability of such an approach. The defined exquisitely efficient DNAzyme protocols were exploited to highlight the enticing advantages of this method via a 96-well plate experiment that enables the detection of nanomolar DNA concentrations in real-time with the naked-eye (see movie as Supplementary Data).     

DNAzyme-mediated catalysis with only guanosine and cytidine nucleotides

Single-stranded DNA molecules have the capacity to adopt catalytically active structures known as DNAzymes, although the fundamental limits of this ability have not been determined. Starting with a parent DNAzyme composed of all four types of standard nucleotides, we conducted a search of the surrounding sequence space to identify functional derivatives with catalytic cores composed of only three, and subsequently only two types of nucleotides. We provide the first report of a DNAzyme that contains only guanosine and cytidine deoxyribonucleotides in its catalytic domain, which consists of just 13 nucleotides. This DNAzyme catalyzes the Mn²⁺-dependent cleavage of an RNA phosphodiester bond ~5300-fold faster than the corresponding uncatalyzed reaction, but ~10 000-fold slower than the parent. The demonstration of a catalytic DNA molecule made from a binary nucleotide alphabet broadens our understanding of the fundamental limits of nucleic-acid-mediated catalysis.     

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.     

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.     

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.     

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.     

Sensitive dual DNAzymes-based sensors designed by grafting self-blocked G-quadruplex DNAzymes to the substrates of metal ion-triggered DNA/RNA-cleaving DNAzymes

A universal label-free metal ion sensor design strategy was developed on the basis of a metal ion-specific DNA/RNA-cleaving DNAzyme and a G-quadruplex DNAzyme. In this strategy, the substrate strand of the DNA/RNA-cleaving DNAzyme was designed as an intramolecular stem-loop structure, and a G-rich sequence was caged in the double-stranded stem and could not form catalytically active G-quadruplex DNAzyme. The metal ion-triggered cleavage of the substrate strand could result in the release of the G-rich sequence and subsequent formation of a catalytic G-quadruplex DNAzyme. The self-blocking mechanism of the G-quadruplex DNAzyme provided the sensing system with a low background signal. The signal amplifications of both the DNA/RNA-cleaving DNAzyme and the G-quadruplex DNAzyme provided the sensing system with a high level of sensitivity. This sensor design strategy can be used for metal ions with reported specific DNA/RNA-cleaving DNAzymes and extended for metal ions with unique properties. As examples, dual DNAzymes-based Cu(2+), Pb(2+) and Hg(2+) sensors were designed. These "turn-on" colorimetric sensors can simply detect Cu(2+), Pb(2+) and Hg(2+) with high levels of sensitivity and selectivity, with detection limits of 4nM, 14nM and 4nM, respectively.     

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.     

Light-regulated catalysis by an RNA-cleaving deoxyribozyme

We describe light-induced switches for the catalytic activity of the small, RNA-cleaving 8-17 deoxyribozyme (DNAzyme), based on photochemically induced cis-trans isomerization of azobenzene (Az) moieties covalently tethered at various locations within the DNAzyme. Prior studies have shown that trans-azobenzene is able to stack comfortably within a DNA double helix, stabilizing it, while cis-azobenzene has a helix-destabilizing effect. We designed two classes of Az-modified 8-17DNAzyme constructs, in each of which two azobenzene molecules substituted for nucleotides, either in the substrate-binding arm (SBA); or, within the catalytic core. Measurement of single-turnover kinetics for RNA cleavage revealed that in the SBA constructs Ell and E13, five- to sixfold higher catalytic rates were obtained when the reaction mixture was irradiated with visible light (favouring trans-Az) as compared to ultraviolet light (which promotes cis-Az), consistent with trans-Az in these constructs stabilizing the enzyme-substrate complex. Surprisingly, the reverse result was obtained with the catalytic core construct E17, where ultraviolet irradiation resulted in a five- to sixfold faster catalytic activity relative to visible light irradiation. The development of such light-responsive nucleic acid enzymes may open new possibilities of using light as the activating or repressing agent in the control of gene expression within living cells and organisms.     

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.     

Relations between the loop transposition of DNA G-quadruplex and the catalytic function of DNAzyme

The structures of DNA G-quadruplexes are essential for their functions in vivo and in vitro. Our present study revealed that sequential order of the three G-quadruplex loops, that is, loop transposition, could be a critical factor to determinate the G-quadruplex conformation and consequently improved the catalytic function of G-quadruplex based DNAzyme. In the presence of 100 mM K⁺, loop transposition induced one of the G-quadruplex isomers which shared identical loops but differed in the sequential order of loops into a hybrid topology while the others into predominately parallel topologies. ¹D NMR spectroscopy and mutation analysis suggested that the hydrogen bonding from loops residues with nucleotides in flanking sequences may be responsible for the stabilization of the different conformations. A well-known DNAzyme consisting of G-quadruplex and hemin (Ferriprotoporphyrin IX chloride) was chosen to test the catalytic function. We found that the loop transposition could enhance the reaction rate obviously by increasing the hemin binding affinity to G-quadruplex. These findings disclose the relations between the loop transposition, G-quadruplex conformation and catalytic function of DNAzyme.     

The use of 10-23 DNAzyme to selectively destroy the allele of mRNA with a unique purine-pyrimidine dinucleotide

10-23 DNAzyme is an oligodeoxyribonucleotide-based ribonuclease. It consists of a 15-nt catalytic domain flanked by two target-specific complementary arms. It has been shown to cleave target mRNA effectively at purine (R)-pyrimidine (Y) dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of a given allele at a unique RY dinucleotide while leaving the mRNA encoded from other alleles of the same gene intact. In this study, a p53-R249S (AGG-->AGT) mutant was tested. 10-23 DNAzyme was used to cut mutant mRNA at GT dinucleotide of codon 249. Both in vitro and in vivo studies showed that this DNAzyme could specifically cut the mutant p53 allele, leaving the wild-type unaffected. This proof-of-concept experiment provided a new way to knock down expression of a given allele with special single-base transversion.     

Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities

Two modified 2′-deoxynucleoside 5′-triphosphates have been used for the in vitro selection of a modified deoxyribozyme (DNAzyme) capable of the sequence-specific cleavage of a 12 nt RNA target in the absence of divalent metal ions. The modified nucleotides, a C5-imidazolyl-modified dUTP and 3-(aminopropynyl)-7-deaza-dATP were used in place of TTP and dATP during the selection and incorporate two extra protein-like functionalities, namely, imidazolyl (histidine analogue) and primary amino (lysine analogue) into the DNAzyme. The functional groups are analogous to the catalytic Lys and His residues employed during the metal-independent cleavage of RNA by the protein enzyme RNaseA. The DNAzyme requires no divalent metal ions or other cofactors for catalysis, remains active at physiological pH and ionic strength and can recognize and cleave a 12 nt RNA substrate with sequence specificity. This is the first example of a functionalized, metal-independent DNAzyme that recognizes and cleaves an all-RNA target in a sequence-specific manner. The selected DNAzyme is two orders of magnitude more efficient in its cleavage of RNA than an unmodified DNAzyme in the absence of metal ions and represents a rate enhancement of 10⁵ compared with the uncatalysed hydrolysis of RNA.     

Efficient silencing of gene expression by an ASON-bulge-DNAzyme complex

Background

DNAzymes are DNA molecules that can directly cleave cognate mRNA, and have been developed to silence gene expression for research and clinical purposes. The advantage of DNAzymes over ribozymes is that they are inexpensive to produce and exhibit good stability. The “10-23 DNA enzyme” is composed of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of approximately eight nucleotides in each direction, which provides the complementary sequence required for specific binding to RNA substrates. However, these eight nucleotides might not afford sufficient binding energy to hold the RNA substrate along with the DNAzyme, which would interfere with the efficiency of the DNAzyme or cause side effects, such as the cleavage of non-cognate mRNAs.

Methodology

In this study, we inserted a nonpairing bulge at the 5′ end of the “10–23 DNA enzyme” to enhance its efficiency and specificity. Different sizes of bulges were inserted at different positions in the 5′ end of the DNAzyme. The non-matching bulge will avoid strong binding between the DNAzyme and target mRNA, which may interfere with the efficiency of the DNAzyme.

Conclusions

Our novel DNAzyme constructs could efficiently silence the expression of target genes, proving a powerful tool for gene silencing. The results showed that the six oligo bulge was the most effective when the six oligo bulge was 12–15 bp away from the core catalytic domain.     

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.     

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.