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1.
In vitro selection of RNA-cleaving DNAzymes is a powerful method for isolating metal-specific DNA. A few successful examples are known, but it is still difficult to target some thiophilic metals such as Cd2+ due to limited functional groups in DNA. While using modified bases expands the chemical functionality of DNA, a single phosphorothioate modification might boost its affinity for thiophilic metals without complicating the selection process or using bases that are not commercially available. In this work, the first such in vitro selection for Cd2+ is reported. After using a blocking DNA and negative selections to rationally direct the library outcome, a highly specific DNAzyme with only 12 nucleotides in the catalytic loop is isolated. This DNAzyme has a cleavage rate of 0.12 min−1 with 10 μM Cd2+ at pH 6.0. The Rp form of the substrate is cleaved ∼100-fold faster than the Sp form. The DNAzyme is most active with Cd2+ and its selectivity against Zn2+ is over 100 000-fold. Its application in detecting Cd2+ is also demonstrated. The idea of introducing single modifications in the fixed region expands the scope of DNA/metal interactions with minimal perturbation of DNA structure and property. 相似文献
2.
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. 相似文献3.
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). 相似文献
4.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2017,1861(8):1913-1920
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. 相似文献
5.
Schubert S Gül DC Grunert HP Zeichhardt H Erdmann VA Kurreck J 《Nucleic acids research》2003,31(20):5982-5992
‘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. 相似文献
6.
Many DNAzymes have been isolated from synthetic DNA pools to cleave natural RNA (D-RNA) substrates and some have been utilized for the design of aptazyme biosensors for bioanalytical applications. Even though these biosensors perform well in simple sample matrices, they do not function effectively in complex biological samples due to ubiquitous RNases that can efficiently cleave D-RNA substrates. To overcome this issue, we set out to develop DNAzymes that cleave L-RNA, the enantiomer of D-RNA, which is known to be completely resistant to RNases. Through in vitro selection we isolated three L-RNA-cleaving DNAzymes from a random-sequence DNA pool. The most active DNAzyme exhibits a catalytic rate constant ~3 min-1 and has a structure that contains a kissing loop, a structural motif that has never been observed with D-RNA-cleaving DNAzymes. Furthermore we have used this DNAzyme and a well-known ATP-binding DNA aptamer to construct an aptazyme sensor and demonstrated that this biosensor can achieve ATP detection in biological samples that contain RNases. The current work lays the foundation for exploring RNA-cleaving DNAzymes for engineering biosensors that are compatible with complex biological samples. 相似文献
7.
Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities 总被引:2,自引:3,他引:2
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. 相似文献
8.
pH6DZ1 is a synthetic deoxyribozyme that is able to couple catalysis with fluorescence signal generation. This deoxyribozyme has the ability to cleave itself at a lone ribonucleotide that is present between a pair of deoxyribothymidines, one modified with a fluorophore (fluorescein) and the other with a quencher (DABCYL). Herein, we report on the sequence truncation and secondary structure characterization of pH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicate that pH6DZ1 has a four-way, junction-like secondary structure comprised of four short duplexes, three hairpin loops, and three interhelical unpaired elements. Ten nucleotides, all located in two separate single-stranded regions, were identified as functionally indispensable nucleotides (complete loss of the catalytic function was obtained upon mutation). Nine nucleotides, most of which are also distributed in three single-stranded DNA elements, were identified as functionally vital nucleotides (at least a 1000-fold activity reduction was obtained upon mutation). Our study has shown that pH6DZ1 has a secondary structure that is more complex than those reported for other RNA-cleaving deoxyribozymes. The identification of functionally important nucleotides lays the foundation for future mechanistic studies on this DNAzyme. The elucidation of the secondary structure of pH6DZ1 should facilitate the future exploration of this unique DNAzyme for the development of DNAzyme-based biosensors. 相似文献
9.
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. 相似文献
10.
A novel replicating circular DNAzyme 总被引:6,自引:1,他引:5
10–23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. However, the dependence on exogenous delivery limits its applications. The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector. By cloning the 10–23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz7 and C-Dz482, targeting the β-lactamase mRNA. These circular DNAzymes showed in vitro catalytic efficiencies (kcat/KM) of 7.82 × 106 and 1.36 × 107 M–1·min–1, respectively. Their dependence on divalent metal ions is similar to that found with linear 10–23 DNAzyme. Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting β-lactamase and bacterial growth. This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications. 相似文献
11.
A self-cleaving DNA enzyme modified with amines, guanidines and imidazoles operates independently of divalent metal cations (M2+)
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Marcel Hollenstein Christopher J. Hipolito Curtis H. Lam David M. Perrin 《Nucleic acids research》2009,37(5):1638-1649
The selection of modified DNAzymes represents an important endeavor in expanding the chemical and catalytic properties of catalytic nucleic acids. Few examples of such exist and to date, there is no example where three different modified bases have been simultaneously incorporated for catalytic activity. Herein, dCTP, dATP and dUTP bearing, respectively, a cationic amine, an imidazole and a cationic guanidine, were enzymatically polymerized on a DNA template for the selection of a highly functionalized DNAzyme, called DNAzyme 9-86, that catalyzed (M2+)-independent self-cleavage under physiological conditions at a single ribo(cytosine)phosphodiester linkage with a rate constant of (0.134 ± 0.026) min−1. A pH rate profile analysis revealed pKa's of 7.4 and 8.1, consistent with both general acid and base catalysis. The presence of guanidinium cations permits cleavage at significantly higher temperatures than previously observed for DNAzymes with only amines and imidazoles. Qualitatively, DNAzyme 9-86 presents an unprecedented ensemble of synthetic functionalities while quantitatively it expresses one of the highest reported values for any self-cleaving nucleic acid when investigated under M2+-free conditions at 37°C. 相似文献
12.
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. 相似文献
13.
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. 相似文献
14.
Yang Liu Gaofeng Liu Zhiwen Li Wei Rong Jianping Yu Ying Zhou 《Nucleosides, nucleotides & nucleic acids》2013,32(10):645-655
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. 相似文献
15.
Structural and functional study of a simple,rapid, and label‐free DNAzyme‐based DNA biosensor for optimization activity
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Peroxidase‐mimicking DNAzyme has a potential to self‐assemble into a G‐quadruplex and shows peroxidase activity. In comparison to proteins, peroxidase‐mimicking DNAzyme is less expensive and more stable. Herein, it is used in fabricating non‐labeling biosensors. This paper investigates the structural and functional properties of a DNA biosensor based on split DNAzyme with a detection limit in nM range (9.48 nM). Two halves of DNAzyme were linked by a complementary sequence of DNA target. Hybridization of the DNA target pulled two DNAzyme halves apart and peroxidase activity decreased. This study can be divided into 3 stages. First, the characteristics of DNAzyme were studied by Circular Dichroism technique and UV–Vis spectroscopy to find out DNAzyme's optimum activity. It is worth to note that some divalent cations were used to form G‐quadruplex, in addition to common monovalent cations. Furthermore, the hemin incubation was also optimized. Secondly, the structural and functional properties of two types of split DNAzyme were compared with DNAzyme. Thirdly, the hybridization of DNA target was monitored. The results revealed that peroxidase activities of split types decreased by half without any specific conformational changes. Interestingly, the catalytic activities of split DNAzymes could be promoted by adding Mg2+. Besides, it was demonstrated that the structure, peroxidation reaction, and DNA target hybridization of 2:2 and 3:1 split modes were almost alike. It was also illustrated that magnesium promoted the possibility of hybridization. 相似文献
16.
Herpes Simplex Virus Processivity Factor UL42 Imparts Increased DNA-Binding Specificity to the Viral DNA Polymerase and Decreased Dissociation from Primer-Template without Reducing the Elongation Rate 总被引:5,自引:2,他引:3
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Herpes simplex virus DNA polymerase consists of a catalytic subunit, Pol, and a processivity subunit, UL42, that, unlike other established processivity factors, binds DNA directly. We used gel retardation and filter-binding assays to investigate how UL42 affects the polymerase-DNA interaction. The Pol/UL42 heterodimer bound more tightly to DNA in a primer-template configuration than to single-stranded DNA (ssDNA), while Pol alone bound more tightly to ssDNA than to DNA in a primer-template configuration. The affinity of Pol/UL42 for ssDNA was reduced severalfold relative to that of Pol, while the affinity of Pol/UL42 for primer-template DNA was increased ~15-fold relative to that of Pol. The affinity of Pol/UL42 for circular double-stranded DNA (dsDNA) was reduced drastically relative to that of UL42, but the affinity of Pol/UL42 for short primer-templates was increased modestly relative to that of UL42. Pol/UL42 associated with primer-template DNA ~2-fold faster than did Pol and dissociated ~10-fold more slowly, resulting in a half-life of 2 h and a subnanomolar Kd. Despite such stable binding, rapid-quench analysis revealed that the rates of elongation of Pol/UL42 and Pol were essentially the same, ~30 nucleotides/s. Taken together, these studies indicate that (i) Pol/UL42 is more likely than its subunits to associate with DNA in a primer-template configuration rather than nonspecifically to either ssDNA or dsDNA, and (ii) UL42 reduces the rate of dissociation from primer-template DNA but not the rate of elongation. Two models of polymerase-DNA interactions during replication that may explain these findings are presented. 相似文献
17.
Martin Kenward 《Biophysical journal》2009,97(10):2785-2793
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. 相似文献
18.
We previously demonstrated—through the isolation of RNA-cleaving deoxyribozymes by in vitro selection that are catalytically active in highly acidic solutions—that DNA, despite its chemical simplicity, could perform catalysis under challenging chemical conditions [Liu,Z., Mei,S.H., Brennan,J.D. and Li,Y. (2003) J. Am. Chem. Soc. 125, 7539–7545]. One remarkable DNA molecule therefrom is pH4DZ1, a self-cleaving deoxyribozyme that exhibits a kobs of ~1 min−1 at pH 3.8. In this study, we carried out a series of experiments to examine the sequence and catalytic properties of this acidic deoxyribozyme. Extensive nucleotide truncation experiments indicated that pH4DZ1 was a considerably large deoxyribozyme, requiring ~80 out of the original 123 nt for the optimal catalytic activity. A reselection experiment identified ten absolutely conserved nucleotides that are distributed in three catalytically crucial sequence elements. In addition, a trans deoxyribozyme was successfully designed. Comparison of the observed rate constant of pH4DZ1 with experimentally determined rate constant for the uncatalyzed reaction revealed that pH4DZ1 achieved a rate enhancement of ~106-fold. This study provides valuable information about this low-pH-functional deoxyribozyme and paves way for further structural and mechanistic characterization of this unique catalytic DNA. 相似文献
19.
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. 相似文献
20.
A novel one-step electrochemical method for DNA detection is described. The procedure utilizes a reaction catalyzed by a peroxidase-mimicking DNAzyme to produce a product, which forms an insoluble precipitation layer on the surface of an electrode. A rationally designed forward primer, conjugated with a peroxidase DNAzyme complementary sequence at its 5′-end, is used for PCR amplification of target DNA. As a result, the DNAzyme sequence is produced by amplification only when the target DNA is present in the sample. The PCR product is then subjected to the precipitation reaction on the electrode surface using an electrolyte assay buffer containing 4-chloronaphthol, hydrogen peroxide, ferrocenemethanol, hemin, and 5′-lambdaexonuclease. Finally, analysis is carried out using Faradaic impedance spectroscopy. The impedance value was found to greatly increase when target DNA is present owing to the formation of a precipitation layer on the electrode surface caused by the catalytic action of the DNAzyme. In contrast, no impedance increase is observed when a control sample not containing target DNA is utilized. By employing this strategy, target DNA from Chlamydia trachomatis was reliably detected within a 10 min period following precipitation without the need for complicated secondary procedures. This effort has led to the development of a highly convenient electrochemical one-step method for DNA detection that utilizes a peroxidase-mimicking DNAzyme, which is specifically designed to undergo amplification during PCR of target DNA. 相似文献