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1.
Xiao Y  Chandra M  Silverman SK 《Biochemistry》2010,49(44):9630-9637
We recently reported the identification by in vitro selection of 10MD5, a deoxyribozyme that requires both Mn2+ and Zn2+ to hydrolyze a single-stranded DNA substrate with formation of 5′-phosphate and 3′-hydroxyl termini. DNA cleavage by 10MD5 proceeds with kobs=2.7 h(?1) and rate enhancement of 10(12) over the uncatalyzed P?O hydrolysis reaction. 10MD5 has a very sharp pH optimum near 7.5, with greatly reduced DNA cleavage rate and yield when the pH is changed by only 0.1 unit in either direction. Here we have optimized 10MD5 by reselection (in vitro evolution), leading to variants with broader pH tolerance, which is important for practical DNA cleavage applications. Because of the extensive Watson?Crick complementarity between deoxyribozyme and substrate, the parent 10MD5 is inherently sequence-specific; i.e., it is able to cleave one DNA substrate sequence in preference to other sequences. 10MD5 is also site-specific because only one phosphodiester bond within the DNA substrate is cleaved, although here we show that intentionally creating Watson?Crick mismatches near the cleavage site relaxes the site specificity. Newly evolved 10MD5 variants such as 9NL27 are also sequence-specific. However, the 9NL27 site specificity is relaxed for some substrate sequences even when full Watson?Crick complementarity is maintained, corresponding to a functional compromise between pH tolerance and site specificity. The site specificity of 9NL27 may be restored by expanding its “recognition site” from ATGT (as for 10MD5) to ATGTT or larger, i.e., by considering 9NL27 to have reduced substrate sequence tolerance relative to 10MD5. These findings provide fundamental insights into the interplay among key deoxyribozyme characteristics of tolerance and selectivity, with implications for ongoing development of practical DNA-catalyzed DNA hydrolysis.  相似文献   

2.
McManus SA  Li Y 《Biochemistry》2007,46(8):2198-2204
The catalytic and structural characteristics of two new self-phosphorylating deoxyribozymes (referred to as deoxyribozyme kinases), denoted "Dk3" and "Dk4", are compared to those of Dk2, a previously reported deoxyribozyme kinase. All three deoxyribozymes not only utilize GTP as the source of activated phosphate and Mn(II) as the divalent metal cofactor but also share a common secondary structure with significant sequence variations. Multiple Watson-Crick helices are identified within the secondary structure, and these helical interactions confine three extremely conserved sequence elements of 8, 5, and 14 nucleotides in length, presumably for the formation of the catalytic core for GTP binding and the self-phosphorylating reaction. The locations of the conserved regions suggest that these three deoxyribozymes arose independently from in vitro selection. The existence of three sequence variants of the same deoxyribozyme from the same in vitro selection experiment implies that these catalytic DNAs may represent the simplest structural solution for the DNA self-phosphorylation reaction when GTP is used as the substrate.  相似文献   

3.
Immobilized small deoxyribozyme to distinguish RNA secondary structures   总被引:3,自引:0,他引:3  
Okumoto Y  Ohmichi T  Sugimoto N 《Biochemistry》2002,41(8):2769-2773
The RNA folding variation due to one or more mutations leads to different RNA splicing, RNA processing, and translational controls as a result of differences in the primary and higher-ordered structures that interact with other cellular molecules. Thus, distinguishing RNA folding is one of the guides to detect the gene functions related to disease and drug responses. We found, previously, a small Ca(2+)-dependent deoxyribozyme with its site-specific RNA cleavage [Sugimoto, N., Okumoto, Y., and Ohmichi, T. (1999) J. Chem. Soc., Perkin Trans. 2, 1382-1388]. In this study, we report the potential of this deoxyribozyme as a useful tool to distinguish RNA foldings. It is found that the immobilized deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The systematic design for the target RNA hairpin loops shows that the immobilized deoxyribozyme is able to cleave them with a > or =17 nucleotide loop size at only one site under single-turnover conditions. Furthermore, an RNA cleavage reaction is detected using the immobilized deoxyribozyme on a surface plasmon resonance (SPR) sensor chip. These results show that the immobilized deoxyribozymes on a column and on an SPR sensor chip become a novel and useful tool to distinguish the RNA foldings.  相似文献   

4.
Target site selection for an RNA-cleaving catalytic DNA.   总被引:20,自引:0,他引:20  
A small catalytic DNA, known as the 10-23 DNA enzyme or deoxyribozyme, has been shown to efficiently hydrolyze RNA at purine-pyrimidine (R-Y) junctions in vitro. Although these potentially cleavable junctions are ubiquitous, they are often protected from deoxyribozyme activity by RNA secondary structure. We have developed a multiplex cleavage assay for screening the entire length of a target RNA molecule for deoxyribozyme cleavage sites that are efficient, both in terms of kinetics and accessibility. This strategy allowed us to simultaneously compare the RNA cleaving activity of 80 deoxyribozymes for a model target gene (HPV16 E6), and an additional 60 deoxyribozymes against the rat c-myc target. The human papilloma virus (HPV) target was used primarily to characterize the multiplex system and determine its validity. The c-myc target, coupled with a smooth muscle cell proliferation assay, allowed us to assess the relationship between in vitro cleavage efficiency and c-myc gene suppression in cell culture. The multiplex reaction approach streamlines the process of revealing effective deoxyribozymes in a functional assay and provides accessibility data that may also be applicable to site selection for other hybridization-based agents.  相似文献   

5.
Studies of catalytically active DNA sequences have expanded considerably since the first artificial deoxyribozyme was identified in 1994. Nevertheless, the field is still quite young, and advances in both fundamental understanding and practical applications of deoxyribozymes are still developing. Deoxyribozymes that either cleave or ligate two RNA substrates have been most widely investigated, and this review describes recent advances in the fundamental studies and applications of these DNA enzymes. Deoxyribozymes with catalytic activities other than RNA ligation and cleavage are also increasingly pursued, and this review covers several key examples.  相似文献   

6.
Shen Y  Brennan JD  Li Y 《Biochemistry》2005,44(36):12066-12076
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   

7.
A covalently branched nucleic acid can be synthesized by joining the 2′-hydroxyl of the branch-site ribonucleotide of a DNA or RNA strand to the activated 5′-phosphorus of a separate DNA or RNA strand. We have previously used deoxyribozymes to synthesize several types of branched nucleic acids for experiments in biotechnology and biochemistry. Here, we report in vitro selection experiments to identify improved deoxyribozymes for synthesis of branched DNA and RNA. Each of the new deoxyribozymes requires Mn2+ as a cofactor, rather than Mg2+ as used by our previous branch-forming deoxyribozymes, and each has an initially random region of 40 rather than 22 or fewer combined nucleotides. The deoxyribozymes all function by forming a three-helix-junction (3HJ) complex with their two oligonucleotide substrates. For synthesis of branched DNA, the best new deoxyribozyme, 8LV13, has kobs on the order of 0.1 min−1, which is about two orders of magnitude faster than our previously identified 15HA9 deoxyribozyme. 8LV13 also functions at closer-to-neutral pH than does 15HA9 (pH 7.5 versus 9.0) and has useful tolerance for many DNA substrate sequences. For synthesis of branched RNA, two new deoxyribozymes, 8LX1 and 8LX6, were identified with broad sequence tolerances and substantial activity at pH 7.5, versus pH 9.0 for many of our previous deoxyribozymes that form branched RNA. These experiments provide new, and in key aspects improved, practical catalysts for preparation of synthetic branched DNA and RNA.  相似文献   

8.
A deoxyribozyme is a catalytic DNA that catalyzes a site-specific RNA cleavage activity and requires various divalent cations. Earlier we have reported that by downsizing the catalytic loop of a deoxyribozyme from 15-mer to 11-mer it resulted in a short and novel Ca2+-dependent deoxyribozyme. In this paper, we investigate the complex formation of deoxyribozymes with their RNA substrates by using surface plasmon resonance (SPR) in order to determine quantitatively the effect of Ca2+ or Mg2+ on the recognition step between a deoxyribozyme and its RNA substrate. The results indicate that both the association and dissociation rate constants (k(a) and k(d)) for the deoxyribozyme-RNA complex depends on metal ions as well as the loop size of the deoxyribozyme. Metal ions with high RNA cleavage activity induced an increase in k(a) and a decrease in k(d). On the basis of the results, we propose that Ca2+ ions may play a role in the rearrangement of the 11-mer catalytic loop of the short Ca2+-dependent deoxyribozyme.  相似文献   

9.
具有RNA裂解活性的DNA分子称为脱氧核酶。它是经过体外选择技术经多次筛选获得的。脱氧核酶在切割与其互补的RNA底物分子时具有极高的特异性和切割效率,有望成为新的RNA灭活工具。  相似文献   

10.
Capping DNA with DNA   总被引:13,自引:0,他引:13  
Li Y  Liu Y  Breaker RR 《Biochemistry》2000,39(11):3106-3114
Twelve classes of deoxyribozymes that promote an ATP-dependent "self-capping" reaction were isolated by in vitro selection from a random-sequence pool of DNA. Each deoxyribozyme catalyzes the transfer of the AMP moiety of ATP to its 5'-terminal phosphate group, thereby forming a 5',5'-pyrophosphate linkage. An identical DNA adenylate structure is generated by the T4 DNA ligase during enzymatic DNA ligation. A 41-nucleotide class 1 deoxyribozyme requires Cu(2+) as a cofactor and adopts a structure that recognizes both the adenine and triphosphate moieties of ATP or dATP. The catalytic efficiency for this DNA, measured at 10(4) M(-1) x min(-1) using either ATP or dATP as substrate, is similar to other catalytic nucleic acids that use small substrates. Chemical probing and site-directed mutagenesis implicate the formation of guanine quartets as critical components of the active structure. The observation of ATP-dependent "self-charging" by DNA suggests that DNA could be made to perform the reactions typically associated with DNA cloning, but without the assistance of protein enzymes.  相似文献   

11.
Schlosser K  Gu J  Lam JC  Li Y 《Nucleic acids research》2008,36(14):4768-4777
Herein, we sought new or improved endoribonucleases based on catalytic DNA molecules known as deoxyribozymes. The current repertoire of RNA-cleaving deoxyribozymes can cleave nearly all of the 16 possible dinucleotide junctions with rates of at least 0.1/min, with the exception of pyrimidine–pyrimidine (pyr–pyr) junctions, which are cleaved 1–3 orders of magnitude slower. We conducted four separate in vitro selection experiments to target each pyr–pyr dinucleotide combination (i.e. CC, UC, CT and UT) within a chimeric RNA/DNA substrate. We used a library of DNA molecules containing only 20 random-sequence nucleotides, so that all possible sequence permutations could be sampled in each experiment. From a total of 245 clones, we identified 22 different sequence families, of which 21 represented novel deoxyribozyme motifs. The fastest deoxyribozymes exhibited kobs values (single-turnover, intermolecular format) of 0.12/min, 0.04/min, 0.13/min and 0.15/min against CC, UC, CT and UT junctions, respectively. These values represent a 6- to 8-fold improvement for CC and UC junctions, and a 1000- to 1600-fold improvement for CT and UT junctions, compared to the best rates reported previously under identical reaction conditions. The same deoxyribozymes exhibited ~1000-fold lower activity against all RNA substrates, but could potentially be improved through further in vitro evolution and engineering.  相似文献   

12.
Lam JC  Kwan SO  Li Y 《Molecular bioSystems》2011,7(7):2139-2146
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.  相似文献   

13.
We describe a rapid and inexpensive method to monitor the kinetics of small RNA-cleaving deoxyribozymes, based on the exogenous fluorophore ethidium bromide. Ethidium binds preferentially to double-stranded nucleic acids, and its fluorescence emission increases dramatically upon intercalation. Thus, ethidium can be used in single-turnover experiments to measure both annealing of the deoxyribozyme to its substrate and release of the products. Under conditions in which dissociation of the product is fast compared with cleavage, the apparent rate of product release reflects the cleavage step. The method was developed for characterizing the so-called 8-17 catalytic DNA, but its general applicability in the deoxyribozyme field was verified using the 10-23 RNA-cleaving construct. Catalysis by both deoxyribozymes was not inhibited in the presence of substoichiometric amounts of ethidium, and the rates obtained through the ethidium assay were virtually identical to the rates determined using radiolabeled substrates. In contrast, the assay cannot be applied to the large, structured ribozymes, and its use to study the kinetics of the small hammerhead ribozyme was hampered by the presence on the catalyst of at least one high-affinity ethidium binding site.  相似文献   

14.
Dk1 and Dk2 are two catalytically proficient, manganese-dependent, guanine-rich deoxyribozymes previously isolated for DNA phosphorylation. In this study, we carried out a series of experiments that aimed to understand the structural properties of Dk1 and Dk2 and compare the structural similarities or differences of these two distinct deoxyribozymes that carry out similar catalytic functions. First, we performed reselections from two partially randomized DNA libraries on the basis of the original Dk1 and Dk2 sequences to isolate catalytically active sequence variants and identify nucleotides that are invariable, well-conserved, or highly mutagenized. Sequence analysis of these variants assisted by secondary-structure predictions led to the identification of possible Watson-Crick base-pairing regions within each deoxyribozyme. Sequence truncation and base-pair partner exchange experiments were conducted to confirm, or rule out, the existence of the predicted secondary-structure elements. Finally, methylation interference experiments were applied to identify nucleotides that are potentially important for the tertiary structure folding of the deoxyribozymes. Our data suggest that Dk1 and Dk2, despite the differences in their primary sequences and NTP requirements, use an analogous stem-loop element to anchor a structural domain of substantial tertiary interactions to execute their catalytic functions.  相似文献   

15.
Eukaryotic DNA topoisomerase I introduces transient single-stranded breaks on double-stranded DNA and spontaneously breaks down single-stranded DNA. The cleavage sites on both single and double-stranded SV40 DNA have been determined by DNA sequencing. Consistent with other reports, the eukaryotic enzymes, in contrast to prokaryotic type I topoisomerases, links to the 3'-end of the cleaved DNA and generates a free 5'-hydroxyl end on the other half of the broken DNA strand. Both human and calf enzymes cleave SV40 DNA at the identical and specific sites. From 827 nucleotides sequenced, 68 cleavage sites were mapped. The majority of the cleavage sites were present on both double and single-stranded DNA at exactly the same nucleotide positions, suggesting that the DNA sequence is essential for enzyme recognition. By analyzing all the cleavage sequences, certain nucleotides are found to be less favored at the cleavage sites. There is a high probability to exclude G from positions -4, -2, -1 and +1, T from position -3, and A from position -1. These five positions (-4 to +1 oriented in the 5' to 3' direction) around the cleavage sites must interact intimately with topo I and thus are essential for enzyme recognition. One topo I cleavage site which shows atypical cleavage sequence maps in the middle of a palindromic sequence near the origin of SV40 DNA replication. It occurs only on single-stranded SV40 DNA, suggesting that the DNA hairpin can alter the cleavage specificity. The strongest cleavage site maps near the origin of SV40 DNA replication at nucleotide 31-32 and has a pentanucleotide sequence of 5'-TGACT-3'.  相似文献   

16.
Characterization of a DNA-cleaving deoxyribozyme   总被引:1,自引:0,他引:1  
A copper-dependent self-cleaving DNA that was isolated by in vitro selection has been minimized to its smallest active domain using both in vitro selection and rational design methods. The minimized 46-nucleotide deoxyribozyme forms duplex and triplex substructures that flank a highly conserved catalytic core. This self-cleaving construct can be converted into a bimolecular complex that comprises separate substrate and enzyme domains. Substrate cleavage is directed at one of two adjacent nucleotides and proceeds via an oxidative cleavage mechanism that is unique to the position cleaved. The structural, kinetic and mechanistic characteristics of this DNA-cleaving deoxyribozyme are reported.  相似文献   

17.
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   

18.
DNA-hydrolyzing DNAs represent an attractive type of DNA-processing catalysts distinctive from the protein-based restriction enzymes. The innate DNA property has enabled them to readily join DNA-based manipulations to promote the development of DNA biotechnology. A major in vitro selection strategy to identify these DNA catalysts relies tightly on the isolation of linear DNAs processed from a circular single-stranded (ss) DNA sequence library by self-hydrolysis. Herein, we report that by programming a terminal hybridization stem in the library, other than the previously reported classes (I & II) of deoxyribozymes, two new classes (III & IV) were identified with the old selection strategy to site-specifically hydrolyze DNA in the presence of Zn2+. Their representatives own a catalytic core consisting of ∼20 conserved nucleotides and a half-life of ∼15 min at neutral pH. In a bimolecular construct, class III exhibits unique broad generality on the enzyme strand, which can be potentially harnessed to engineer DNA-responsive DNA hydrolyzers for detection of any target ssDNA sequence. Besides the new findings, this work should also provide an improved approach to select for DNA-hydrolyzing deoxyribozymes that use various molecules and ions as cofactors.  相似文献   

19.
The minimal DNA duplex requirements for topoisomerase I-mediated cleavage at a specific binding sequence were determined by analyzing the interaction of the enzyme with sets of DNA substrates varying successively by single nucleotides at the 5'- or 3' end of either strand. Topoisomerase I cleavage experiments showed a minimal region of nine nucleotides on the scissile strand and five nucleotides on the noncleaved strand. On the scissile strand, seven of the nine nucleotides were situated upstream to the cleavage site, while all five nucleotides required on the non-cleaved strand were located to this side. The results suggested that topoisomerase I bound tightly to this region, stabilizing the DNA duplex extensively. On minimal substrates which were partially single-stranded downstream to the cleavage site, cleavage was suicidal, that is, the enzyme was able to cleave the substrates, but unable to perform the final religation.  相似文献   

20.
Zn2+-dependent deoxyribozymes that form natural and unnatural RNA linkages   总被引:1,自引:0,他引:1  
We report Zn(2+)-dependent deoxyribozymes that ligate RNA. The DNA enzymes were identified by in vitro selection and ligate RNA with k(obs) up to 0.5 min(-)(1) at 1 mM Zn(2+) and 23 degrees C, pH 7.9, which is substantially faster than our previously reported Mg(2+)-dependent deoxyribozymes. Each new Zn(2+)-dependent deoxyribozyme mediates the reaction of a specific nucleophile on one RNA substrate with a 2',3'-cyclic phosphate on a second RNA substrate. Some of the Zn(2+)-dependent deoxyribozymes create native 3'-5' RNA linkages (with k(obs) up to 0.02 min(-)(1)), whereas all of our previous Mg(2+)-dependent deoxyribozymes that use a 2',3'-cyclic phosphate create non-native 2'-5' RNA linkages. On this basis, Zn(2+)-dependent deoxyribozymes have promise for synthesis of native 3'-5'-linked RNA using 2',3'-cyclic phosphate RNA substrates, although these particular Zn(2+)-dependent deoxyribozymes are likely not useful for this practical application. Some of the new Zn(2+)-dependent deoxyribozymes instead create non-native 2'-5' linkages, just like their Mg(2+) counterparts. Unexpectedly, other Zn(2+)-dependent deoxyribozymes synthesize one of three unnatural linkages that are formed upon the reaction of an RNA nucleophile other than a 5'-hydroxyl group. Two of these unnatural linkages are the 3'-2' and 2'-2' linear junctions created when the 2'-hydroxyl of the 5'-terminal guanosine of one RNA substrate attacks the 2',3'-cyclic phosphate of the second RNA substrate. The third unnatural linkage is a branched RNA that results from attack of a specific internal 2'-hydroxyl of one RNA substrate at the 2',3'-cyclic phosphate. When compared with the consistent creation of 2'-5' linkages by Mg(2+)-dependent ligation, formation of this variety of RNA ligation products by Zn(2+)-dependent deoxyribozymes highlights the versatility of transition metals such as Zn(2+) for mediating nucleic acid catalysis.  相似文献   

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