Characterization of a DNA-Cleaving deoxyribozyme

Carmi, Nir; Breaker, Ronald R.

doi:10.1016/s0968-0896(01)00035-9

Cited by 108 publications

(113 citation statements)

References 25 publications

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“…We observed that 0.5 mM Cu 2+ inhibits ribozyme cleavage even in the presence of an equal concentration of Mg 2+ . This is not surprising since copper ions are known to strongly bind to biopolymers and disrupt the functions of structured RNAs at concentrations similar to those used herein (Rifkind et al 1976;Carmi and Breaker 2001;Zivarts et al 2005).…”

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confidence: 64%

Biochemical analysis of pistol self-cleaving ribozymes

Harris

Lünse

et al. 2015

RNA

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Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min −1 under physiological Mg 2+ and pH conditions. The reaction proceeds via the nucleophilic attack of a 2 ′ -oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA.

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Section: +mentioning

confidence: 64%

Biochemical analysis of pistol self-cleaving ribozymes

Harris

Lünse

et al. 2015

RNA

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“…Some in vitro-selected ribozymes have been engineered to generate trans-acting catalysts (26)(27)(28). To test whether 16.min could react in trans, the ribozyme was split in the loop sequence of stem P2 to generate a two-piece molecule (Fig.…”

Section: Resultsmentioning

confidence: 99%

A ribozyme that ligates RNA to protein

Baskerville

Bartel

2002

Proc. Natl. Acad. Sci. U.S.A.

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We have used a combination of in vitro selection and rational design to generate ribozymes that form a stable phosphoamide bond between the 5 terminus of an RNA and a specific polypeptide. This reaction differs from that of previously identified ribozymes, although the product is analogous to the enzymenucleotidyl intermediates isolated during the reactions of certain proteinaceous enzymes, such as guanyltransferase, DNA ligase, and RNA ligase. Comparative sequence analysis of the isolated ribozymes revealed that they share a compact secondary structure containing six stems arranged in a four-helix junction and branched pseudoknot. An optimized version of the ribozyme reacts with substrate-fusion proteins, allowing it to be used to attach RNA tags to proteins both in vitro and within bacterial cells, suggesting a simple way to tag a specific protein with amplifiable information.in vitro selection ͉ nucleic acid tag ͉ phosphoamide ͉ Tat ͉ TAR I n vitro selection has been used in combination with rational design to identify and optimize numerous ribozymes and binding motifs (1-3). The results of these experiments continue to give insight into the capacity of RNA and DNA to catalyze a variety of chemical reactions and the ability of nucleic acids to recognize an array of substrates. Some of these catalysts are being developed into effective tools for use in molecular biology. These include allosteric ribozymes, used as detectors of small molecules and proteins, and DNA enzymes that efficiently cleave target sequences (4-6).Here we use in vitro selection coupled with rational design to produce a new ribozyme that ligates RNA to protein through the formation of a phosphoamide bond. This is similar to a reaction found in naturally occurring proteins such as guanyltransferase, and DNA and RNA ligases that form phosphoamide bonds as enzyme-nucleotidyl intermediates during cap formation and ligation events (7-9). The selection of this new ribozyme expands the repertoire of RNA-catalyzed chemistry and further illustrates the ability of RNA to chemically modify a variety of substrates. Moreover, this ribozyme might be used to synthesize oligonucleotide-tagged proteins in vitro and in vivo. These attached oligonucleotides might be useful as affinity tags or could function as amplifiable identifying markers. For example, this ribozyme might be used to attach mRNAs to the proteins that they encode. A diverse library of mRNA-protein fusions could be used in conjunction with in vitro selection strategies to identify previously undescribed proteins with unique or enhanced properties, as has been shown previously by using phage display, ribosome display, and mRNA display (10-13). Materials and MethodsPool Construction. The double-stranded DNA pool was constructed from two 110-nt DNA fragments, each containing 76 completely randomized positions (14). Each fragment was amplified separately in 110 ml PCR reactions and ligated through BanI sites to generate a final pool with the sequence: TTCTAATACGACTCACTATAG-GACAGCTCCGAGCATTCTCGTGT...

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“…The Cu 2+ -dependent DNA-cleaving deoxyribozyme, which was first isolated by Breaker and co-workers, [22] can catalytically cleave DNA substrates by oxidative modification of DNA, which involves the generation of hydroxyl radicals through metal-ion-mediated redox reactions. [22][23][24] Figure 1. The Cu 2+ -dependent deoxyribozyme and logic gates.…”

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confidence: 99%

“…Note that the triplex-forming stem-loop was essential for DNAzyme_Y (see Supporting Information). [23,24] In the absence of the effector EY, the deoxyribozyme was active ("ON" state) as the triplex was intact. In contrast, in the presence of the effector, EY bound to loop II, which competitively opened stem II and disrupted triplex formation.…”

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confidence: 99%

“…Notably, the gate activities of oligonuclotides with minor sequence differences can vary by as much as several orders of magnitude. Other possible disadvantages are as follows: First, the optimal conditions for the oxidative cleavage reaction of deoxyribozymes are different from physiological conditions, [24] and thus the logic gates described herein are not expected to work in vivo in their present form. Second, deoxyribozymes are known to undergo self-cleavage.…”

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Construction of Molecular Logic Gates with a DNA‐Cleaving Deoxyribozyme

et al. 2006

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Logic gates are devices that perform the basic logic operations AND, NOT, and OR, as well as their combinations. It is well-known that electronic logic gates form the basis of conventional silicon computer microprocessors. By analogy, molecular logic gates are crucial for the development of molecular-scale computers, which have attracted significant research interest.[1] In particular, nucleic acids (DNA and RNA) have proven to be highly useful building blocks for the construction of molecular logic gates and computational devices.[2-8] Herein, we report the construction of molecular logic gates that are made entirely of DNA molecules. As examples, we demonstrate the performance of logic operations including "YES", "NOT", and a three-input gate "AND(A,NOT(B),NOT(C))" through the recognition and catalytic properties of DNA. Notably, the combination of the "NOT" gate and the "AND(A,NOT(B),NOT(C))" gate can, in principle, make a universal operator set.Artificially designed DNA or RNA sequences can fold into well-ordered, three-dimensional structures that either recognize corresponding ligands (aptamers) [9] or catalyze specific chemical reactions (deoxyribozymes or ribozymes). [10][11][12][13][14][15][16][17][18][19][20][21] In their pioneering work, Stojanovic and coworkers constructed molecular logic gates based on in vitro selected RNA-cleaving deoxyribozymes (DNAzymes) by exploiting allosteric regulation. [6][7][8] In their system, the activities of deoxyribozymes were allosterically regulated by specific effectors, which were rationally designed oligonucleotides containing complementary sequences to target deoxyribozymes. Either the presence or the absence of effectors was employed as the input, while intact or cleaved substrates were regarded as outputs. The same group later constructed complex systems based on this elegant strategy, such as a molecular-scale half-adder [7] and a MAYA automaton that could play a tic-tac-toe game with a human adversary. [8] Despite the elegance of this strategy, these logic gates remain to be improved. In particular, as they are based on RNA-cleaving deoxyribozymes, the corresponding substrates must be either RNA or chimeric DNA (a piece of oligodeoxynucleotide containing at least one ribonucleotide base). [6][7][8] This prerequisite leads to high synthesis costs and susceptibility to oligonucleotide degradation. In view of this fact, it is highly desirable to develop ribonucleotide-free molecular logic gates. Herein, we employ a Cu 2+ -dependent DNA-cleaving deoxyribozyme as the building block for the construction of robust molecular logic gates that are based on DNA only. Our inputs (effectors), gates (deoxyribozymes), and outputs (substrates) are all inexpensive, chemically stable DNA oligonucleotides (Figure 1). The Cu 2+ -dependent DNA-cleaving deoxyribozyme, which was first isolated by Breaker and co-workers, [22] can catalytically cleave DNA substrates by oxidative modification of DNA, which involves the generation of hydroxyl radicals through metal-ion-mediated redox re...

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Characterization of a DNA-Cleaving deoxyribozyme

Cited by 108 publications

References 25 publications

Biochemical analysis of pistol self-cleaving ribozymes

Biochemical analysis of pistol self-cleaving ribozymes

A ribozyme that ligates RNA to protein

Construction of Molecular Logic Gates with a DNA‐Cleaving Deoxyribozyme

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