In vitro selection of self-cleaving DNAs

Carmi, Nir; Shultz, Lisa A.; Breaker, Ronald R.

doi:10.1016/s1074-5521(96)90170-2

Cited by 247 publications

(185 citation statements)

References 25 publications

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“…Finally, DNA is easily synthesized with a variety of useful modifications and its biocompatibility makes DNAzyme-based sensors excellent tools for live-cell imaging of metal ions. As a result, several metal-specific DNAzymes have been isolated and converted into sensors for their respective metal ion cofactors, including Pb 2+ (35,42,43), Cu 2+ (44,45) However, in contrast to the previously reported DNAzymes with divalent metal ion selectivity, no DNAzymes have been reported to have high selectivity toward a specific monovalent metal ion. Although DNAzymes that are independent of divalent metal ions have been obtained (53)(54)(55), including those using modified nucleosides with protein-like functionalities (i.e., guanidinium and imidazole) (56)(57)(58), no DNAzyme has been found to be selective for a specific monovalent metal ion over other monovalent metal ions.…”

Section: Significancementioning

confidence: 99%

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

Torabi¹,

Wu²,

McGhee³

et al. 2015

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

316

286

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Over the past two decades, enormous progress has been made in designing fluorescent sensors or probes for divalent metal ions. In contrast, the development of fluorescent sensors for monovalent metal ions, such as sodium (Na + ), has remained underdeveloped, even though Na + is one the most abundant metal ions in biological systems and plays a critical role in many biological processes. Here, we report the in vitro selection of the first (to our knowledge) Na + -specific, RNA-cleaving deoxyribozyme (DNAzyme) with a fast catalytic rate [observed rate constant (k obs ) ∼0.1 min −1 ], and the transformation of this DNAzyme into a fluorescent sensor for Na + by labeling the enzyme strand with a quencher at the 3′ end, and the DNA substrate strand with a fluorophore and a quencher at the 5′ and 3′ ends, respectively. The presence of Na + catalyzed cleavage of the substrate strand at an internal ribonucleotide adenosine (rA) site, resulting in release of the fluorophore from its quenchers and thus a significant increase in fluorescence signal. The sensor displays a remarkable selectivity (>10,000-fold) for Na + over competing metal ions and has a detection limit of 135 μM (3.1 ppm). Furthermore, we demonstrate that this DNAzyme-based sensor can readily enter cells with the aid of α-helical cationic polypeptides. Finally, by protecting the cleavage site of the Na + -specific DNAzyme with a photolabile o-nitrobenzyl group, we achieved controlled activation of the sensor after DNAzyme delivery into cells. Together, these results demonstrate that such a DNAzyme-based sensor provides a promising platform for detection and quantification of Na + in living cells.M etal ions play crucial roles in a variety of biochemical processes. As a result, the concentrations of cellular metal ions have to be highly regulated in different parts of cells, as both deficiency and surplus of metal ions can disrupt normal functions (1-4). To better understand the functions of metal ions in biology, it is important to detect metal ions selectively in living cells; such an endeavor will not only result in better understanding of cellular processes but also novel ways to reprogram these processes to achieve novel functions for biotechnological applications.Among the metal ions in cells, sodium (Na + ) serves particularly important functions, as changes in its concentrations influence the cellular processes of numerous living organisms and cells (5-8), such as epithelial and other excitable cells (9). As one of the most abundant metal ions in intracellular fluid (10), Na + affects cellular processes by triggering the activation of many signal transduction pathways, as well as influencing the actions of hormones (11). Therefore, it is important to carefully monitor the concentrations of Na + in cells. Toward this goal, instrumental analyses by atomic absorption spectroscopy (12), Xray fluorescence microscopy (13), and 23 Na NMR (14) have been used to detect the concentration of intracellular Na + . However, it is difficult to use these methods to o...

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Section: Significancementioning

confidence: 99%

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

Torabi¹,

Wu²,

McGhee³

et al. 2015

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

316

286

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“…For example, a number of DNA ''aptamers'' (1) can be made that function as ligands for proteins or as highly specific receptors for small organic molecules (2)(3)(4). In addition, certain single-stranded DNAs act as artificial enzymes (4)(5)(6), catalyzing such chemical reactions as phosphoester transfer (7)(8)(9)(10)(11)(12), phosphoester formation (13), porphyrin metalation (14), phosphoramidate cleavage (15), and DNA cleavage (16). Most likely, DNA can be made to perform a much broader repertoire of catalytic activities (6).…”

mentioning

confidence: 99%

“…In a previous study (16), we reported the isolation of two distinct types of deoxyribozymes (classes I and II) that undergo oxidative self-cleavage in the presence of copper ions. By using in vitro selection (17), class II self-cleaving DNAs have been further optimized for catalytic function, and the most active structure obtained from this process has been engineered to act as a ''restriction endonuclease'' for single-stranded DNA substrates.…”

mentioning

confidence: 99%

Cleaving DNA with DNA

Carmi

Balkhi

Breaker

1998

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

Self Cite

244

165

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A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ''deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a k obs of 0.2 min ؊1 , whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ''restriction enzymes'' for the site-specific cleavage of single-stranded DNA.DNA in biological systems exists primarily in duplex form where it serves almost exclusively as a storage system for genetic information. Outside the confines of cells, DNA in its single-stranded form can be made to perform both molecular recognition and catalysis-biochemical operations that were until recently thought to be possible only with macromolecules made of protein or RNA. For example, a number of DNA ''aptamers'' (1) can be made that function as ligands for proteins or as highly specific receptors for small organic molecules (2-4). In addition, certain single-stranded DNAs act as artificial enzymes (4-6), catalyzing such chemical reactions as phosphoester transfer (7-12), phosphoester formation (13), porphyrin metalation (14), phosphoramidate cleavage (15), and DNA cleavage (16). Most likely, DNA can be made to perform a much broader repertoire of catalytic activities (6).These capabilities of DNA conceivably can be exploited to create a variety of structured DNAs that perform useful tasks, either in vitro or in vivo, involving molecular recognition and catalysis. Such functional DNAs offer several advantages, including ease of synthesis and chemical stability, that might make attractive properties for polymers that serve as artificial receptors or as biocatalysts for various applications. Characteristics such as thermostability and solvent͞solute preferences could be conferred upon deoxyribozymes by using both rational and combinatorial methods of molecular design. Catalytic DNAs may offer distinct advantages over natural protein enzymes for operation under nonbiological conditions.In a previous study (16), we reported the isolation of two distinct types of deoxyribozymes (classes I and II) that undergo oxidative self-cleavage in the presence of copper ions. By using in vitro selection (17), class II self-cleaving DNAs have been further optimized for catalytic function, and the most active structure obtained from this process has been engineered to act as a ''restriction endonuclease'' for single-stranded DNA substrates. MATERIALS AND METHODSOligonucleotides. Synthetic DNAs were prepared by automated chemical synthesis (Keck Biotechnology Resource Laboratory, Yale University) and were purified by denaturing (8 M urea) PAGE prior to use. Double-stran...

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“…The latter have been shown to discriminate against Mg 2+ , while selecting for Ca 2+ [89][90][91], Cu 2+ [92][93][94], Co 2+ [95], Zn 2+ [96], Mn 2+ [94], Pb 2+ [97], Ni 2+ [98] or a small subgroup of transition metal ions [99]. By the same means extraordinarily selective DNA biosensors for various metal ions have been engineered [100,101] (see also Chapter 8).…”

Section: Expanding the Natural Metal Repertoire Of Catalytic Rnas Andmentioning

confidence: 99%

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

Pechlaner

Sigel

2011

Metal Ions in Life Sciences

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Metal ions are inextricably involved with nucleic acids due to their polyanionic nature. In order to understand the structure and function of RNAs and DNAs, one needs to have detailed pictures on the structural, thermodynamic, and kinetic properties of metal ion interactions with these biomacromolecules. In this review we first compile the physicochemical properties of metal ions found and used in combination with nucleic acids in solution. The main part then describes the various methods developed over the past decades to investigate metal ion binding by nucleic acids in solution. This includes for example hydrolytic and radical cleavage experiments, mutational approaches, as well as kinetic isotope effects. In addition, spectroscopic techniques like EPR, lanthanide(III) luminescence, IR and Raman as well as various NMR methods are summarized. Aside from gaining knowledge about the thermodynamic properties on the metal ion-nucleic acid interactions, especially NMR can be used to extract information on the kinetics of ligand exchange rates of the metal ions applied. The final section deals with the influence of anions, buffers, and the solvent permittivity on the binding equilibria between metal ions and nucleic acids. Little is known on some of these aspects, but it is clear that these three factors have a large influence on the interaction between metal ions and nucleic acids.

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In vitro selection of self-cleaving DNAs

Abstract: Despite the absence of 2' hydroxyls, single-stranded DNA can adopt structures that promote divalent-metal-dependent self-cleavage via an oxidative mechanism. These results suggest that an efficient DNA enzyme might be made to cleave DNA in a biological context.

Cited by 247 publications

References 25 publications

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

Cleaving DNA with DNA

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

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