A self-cleaving DNA enzyme modified with amines, guanidines and imidazoles operates independently of divalent metal cations (M 2+ )

Hollenstein, Marcel; Hipolito, Christopher J.; Lam, Curtis H.; Perrin, David M.

doi:10.1093/nar/gkn1070

Cited by 122 publications

(113 citation statements)

References 92 publications

(97 reference statements)

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“…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. For example, the DNAzyme with the highest reported selectivity for Na + still binds Na + over K + with only 1.3-fold selectivity (54).…”

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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“…Nucleobase modifications that include polypeptide functional groups involve the use of pendant imidazoles, amines, or guanidinium groups, which mimic the amino acids lysine, histidine and arginine, respectively [8]. In addition to those shown in Figure 3 for modified uridine, there are also the modifications at the 8-position of adenosine and guanosine shown in Figure 5.…”

Section: Modification Of Aptamers and (Deoxy)ribozymes With Hydrogen-mentioning

confidence: 99%

“…RNA and DNA aptamers have been obtained through in vitro selection using both native nucleobases and modifications in the 5-position of uracil [7][8][9][10] or the 8-position and 7-deaza positions of adenine [8,10,11]. In both of these cases, the ability of polymerases to tolerate the modifications is crucial to the development of novel technologies.…”

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Thermodynamics of Nucleic Acid Structural Modifications for Biotechnology Applications

Franzen¹

2011

Biotechnology of Biopolymers

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IntroductionThe repertoire of chemistry available to native nucleotides in DNA and RNA is limited to the purine and pyrimidine functional groups, along with the special role of the 2'-hydroxyl of RNA. The nucleobases have exocyclic amino groups and imines, neither of which is highly reactive or a good candidate for catalytic function. One might argue that the limited range of chemical reactivity is an evolutionary advantage in molecules whose functions are primarily to store (DNA) and process (RNA) genetic information. The most important attribute of both DNA and RNA is the molecular recognition through base pairing. The hydrogen bond pattern combined with the conformation of the ribose sugar gives rise to the specific B-form double helix in DNA and A-form helix with a range of additional standard folds in RNA, loops, bulges and pseudoknots. These structures are well known for their ability to interact with proteins to provide a scaffold for transcription (DNA → RNA) to create messenger RNA (mRNA), and processing of mRNA by enzymes that have active components composed of RNA molecules. The chemical action of RNA on other RNAs by means of the 2'-hydroxyl is an exception to the lack or reactivity of DNA and RNA. Selfsplicing by action of the 2'-hydroxyl as a nucleophile was the process that broke the dogma that RNA is always a passive molecule that simply transmits information [1]. Indeed, RNA i s v e r y a c t i v e i n p r o c e s s i n g o t h e r R N A s u s i n g t h e 2 ' -h y d r o x y l a s a n u c l e o p h i l e f o r hydrolysis of the phosphodiester bond leading to cleavage or to rearrangements of structure such as RNA splicing. Outside of this reactivity, the components of the purine and pyrimidine rings are largely inert. Aromatic amines are poor nucleophiles, and imines are even less reactive. The purine and pyrimidine rings are not particularly electrophilic because of the nitrogen heteroatoms and carbonyls. Using in vitro selection (also known as SELEX) [2][3][4], and appropriate modification, RNA and DNA have been developed for numerous applications that transcend their biological functions. In this chapter we will consider the modifications of the nucleobase as a means to expand upon the native function. The extensive literature on modifications of the phosphodiester backbone and the ribose sugar will not be considered in this chapter due to space limitations. Base modifications may consist of expansions of the purine/pyrimidine ring, appended functional group or chemical modification to increase the stability of the backbone with respect to hydrolysis. Expanded DNA or xDNA has been developed as mimics of native DNA with potential biotechnology applications [5,6]. In these molecules, the purine and pyrimidine rings are fused to phenyl or naphthyl rings to give rise to

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“…Beyond representing a convenient vector for the functionalization of nucleic acids, modified dNTPs can be engaged in SELEX and other related combinatorial methods of in vitro selection for the generation of modified catalytic nucleic acids [21][22][23][24][25][26][27][28][29][30] and aptamers for various practical applications 10,[31][32][33][34][35][36] . The additional side-chains that are introduced by the polymerization of the modified dNTPs are thought to increase the chemical space that can be explored during a selection experiment and supplement the rather poor functional arsenal of nucleic acids 37 .…”

Section: Introductionmentioning

confidence: 99%

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

Hollenstein

Smith

Räz

2014

JoVE

Self Cite

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The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology. Video LinkThe video component of this article can be found at

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A self-cleaving DNA enzyme modified with amines, guanidines and imidazoles operates independently of divalent metal cations (M 2+ )

Cited by 122 publications

References 92 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

Thermodynamics of Nucleic Acid Structural Modifications for Biotechnology Applications

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

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