DNAzymes for sensing, nanobiotechnology and logic gate applications

Willner, Itamar; Shlyahovsky, Bella; Zayats, Maya; Willner, Bilha

doi:10.1039/b718428j

Cited by 739 publications

(537 citation statements)

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“…7 This class of DNAzymes has been applied in nucleic acid detection, 8 metal ions sensing, 3,4,6,7,9−11 and molecular logic gate constructing. 4,12,13 The enzymatic activities play central roles in any extended analytical applications of DNAzymes. A number of biochemical and biophysical methods have been carried out for measuring DNAzyme activities, especially for RNA-cleaving DNAzymes.…”

mentioning

confidence: 99%

Nanoparticles-Free Fluorescence Anisotropy Amplification Assay for Detection of RNA Nucleotide-Cleaving DNAzyme Activity

Zhang

Zhao

et al. 2015

Anal. Chem.

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Fluorescence anisotropy is a homogeneous, sensitive, ratiometric, and real-time analytical technology. However, it is a great challenge to produce a large fluorescence anisotropy change upon the presence of target small molecules without nanoparticles-dependent amplification. This work reports a nanoparticle-free and multiple G-enhanced fluorescence anisotropy assay for detection of DNAzyme activity. A Pb 2+ -dependent GR-5 DNAzyme was used as a model. We hybridized the rA-cleavable substrate strand containing a TMR label at the 5′-end with the DNAzyme strand containing an extended three G bases at the 3′-end. By this design, we demonstrate that both fluorescence quenching and the enhanced DNAzyme activity contribute to a Pb 2+ -induced large fluorescence anisotropy change (|Δr| = 0.168). The limit of detection for Pb 2+ is estimated to be about 100 pM with a dynamic range from 200 pM to 100 nM. The interference from the other nine divalent metal ions of 1000-times excess amount is negligible. Moreover, we show an extended assay for evaluation of the interactions of Pb 2+ with cysteine and glutathione by the detection of GR5 DNAzyme activity. Collectively, we developed a novel fluorescence anisotropy amplification assay, enabling us to detect DNAzyme activity and associated cofactors and inhibitors and to characterize the Pb 2+ -chelation capability of free thiols.O ne class of functional nucleic acids with catalytic activity is DNAzymes, which have potential applications in diagnosis, therapeutics, and bioanalysis. 1−4 Various DNAzymes have been in vitro selected to catalyze a wide range of reactions, such as RNA cleavage, DNA cleavage, RNA ligation, DNA ligation, DNA phosphorylation, cleavage of the phosphoramidate bond, and porphyrin metalation. 5,6 One type of DNAzymes can cleave RNA. These RNA-cleaving DNAzymes are composed of a DNA enzyme strand and a DNA substrate strand containing a single RNA linkage (rA), which serves as the cleavage site. 7 This class of DNAzymes has been applied in nucleic acid detection, 8 metal ions sensing, 3,4,6,7,9−11 and molecular logic gate constructing. 4,12,13 The enzymatic activities play central roles in any extended analytical applications of DNAzymes. A number of biochemical and biophysical methods have been carried out for measuring DNAzyme activities, especially for RNA-cleaving DNAzymes. These methods include polyacrylamide gel electrophoresis (PAGE), 14 MALDI-TOF mass spectrometry, 15 fluorescence resonance energy transfer (FRET), 16,17 and fluorescence anisotropy (FA). 18−22 Among known techniques for detection of DNAzyme activity, FA has a number of advantages. It is a simple, realtime, and truly homogeneous analysis. Moreover, it does not require the separation of substrates and the cleaved products. This technique also presents several other attractive features, such as the requirement of only one fluorescent label and relative insensitivity to the photobleaching of the fluorophore and to instrument parameters due to its ratiometric nature. In fact, F...

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confidence: 99%

Nanoparticles-Free Fluorescence Anisotropy Amplification Assay for Detection of RNA Nucleotide-Cleaving DNAzyme Activity

Zhang

Zhao

et al. 2015

Anal. Chem.

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“…[16][17][18][19][20][21][22] Winfree, Stojanovic, Willner, Katz and their co-workers have, for example, developed optically reported 'AND' , 'OR' and 'SET-RESET' logic gate operations. 7,[23][24][25][26][27][28][29][30][31] Although the above examples serve as promising proofs of principle (see also refs 27-35), it remains necessary to create complex, multicomponent devices on a single biomolecular platform to achieve increased computational complexity and develop realistic DNAbased information processing systems. In this study, we fabricated and tested encoders and decoders based on a multiplex, DNA-based electrochemical biosensor that uses electronic (electrochemical) signals as its readout.…”

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confidence: 99%

DNA biomolecular-electronic encoder and decoder devices constructed by multiplex biosensors

et al. 2012

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We fabricated and tested encoders and decoders based on a multiplex, DNA-based electrochemical biosensor that uses electronic (electrochemical) signals as its readout. These devices use two or more sequence-specific DNA probes, with each being modified with a distinct redox reporter. These probes, when interrogated together, serve as encoders and decoders, converting patterns that are encoded and decoded by the presence or absence of specific DNA sequences into specific electronic outputs. We demonstrated these multifunctional, bio-electrochemical devices, for example, 4-to-2 and 8-to-3 encoders and 1-to-2 and 2-to-3 decoders. Accordingly, these devices bridge the division between DNA-based devices and silicon-based electronics. NPG Asia Materials (2012) 4, e1; doi:10.1038/am.2012.1; published online 18 January 2012Keywords: biosensors; decoder; DNA-based; encoder; multiplex INTRODUCTIONIn recent decades, serious attempts have been made to implement computational approaches, models and paradigms that utilize the information transfer and processing abilities of naturally occurring and modified biomolecules. 1-11 A decade ago, for example, Adleman performed a now-classic experiment in which computations were conducted using DNA molecules, demonstrating that biology can provide new computing substrates. [12][13][14][15] In the decade since Adleman's invention, reports have been made of a wide and diverse range of DNA logic devices. [16][17][18][19][20][21][22] Winfree, Stojanovic, Willner, Katz and their co-workers have, for example, developed optically reported 'AND' , 'OR' and 'SET-RESET' logic gate operations. 7,[23][24][25][26][27][28][29][30][31] Although the above examples serve as promising proofs of principle (see also refs 27-35), it remains necessary to create complex, multicomponent devices on a single biomolecular platform to achieve increased computational complexity and develop realistic DNAbased information processing systems. In this study, we fabricated and tested encoders and decoders based on a multiplex, DNA-based electrochemical biosensor that uses electronic (electrochemical) signals as its readout. These devices uses two or more sequence-specific DNA probes that, when interrogated together, serve as encoders, converting patterns that are encoded and decoded by the presence or absence of specific DNA sequences into specific electronic outputs.

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“…[8][9][10][11] DNAzymes are DNA-based catalysts that often require divalent metal ions for activity. [12][13][14] In vitro selection has been intentionally carried out in the presence of specific metal ions to isolate sensor DNAzymes.…”

Section: Introductionmentioning

confidence: 99%

An Ultrasensitive Light-up Cu²⁺ Biosensor Using a New DNAzyme Cleaving a Phosphorothioate-Modified Substrate

Huang

Liu

2016

Anal. Chem.

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Cu 2+ is a very important metal ion in biology, environmental science, and industry. Developing biosensors for Cu 2+ is a key topic in analytical chemistry. DNAzyme-based sensors are highly attractive for their excellent sensitivity, stability, and programmability. In the past decade, a few Cu 2+ biosensors were reported using DNAzymes with DNA cleavage or DNA ligation activity.However, they require unstable ascorbate or imidazole activation. So far, no RNA-cleaving DNAzymes specific for Cu 2+ are known. In this work, a phosphorothioate (PS) RNA containing library was used for in vitro selection, and a few new Cu 2+ -specific RNA-cleaving DNAzymes were isolated. Among them, a DNAzyme named PSCu10 was studied further. It has only eight nucleotides in the enzyme loop with a cleavage rate of 0.1 min -1 in the presence of 1 µM Cu 2+ at pH 6.0 (its optimal pH). Between the two diastereomers of the PS RNA chiral center, the Rp isomer is 37 times more active than the Sp one. Among the other divalent metal ions, only Hg 2+ can cleave the substrate due to its extremely high thiophilicity. A catalytic beacon sensor was designed with a detection limit of 1.6 nM Cu 2+ and extremely high selectivity. PSCu10 is specific for Cu 2+ and it has no cleavage in the presence of ascorbate, which reduces Cu 2+ to Cu + .3

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DNAzymes for sensing, nanobiotechnology and logic gate applications

Cited by 739 publications

References 61 publications

Nanoparticles-Free Fluorescence Anisotropy Amplification Assay for Detection of RNA Nucleotide-Cleaving DNAzyme Activity

Nanoparticles-Free Fluorescence Anisotropy Amplification Assay for Detection of RNA Nucleotide-Cleaving DNAzyme Activity

DNA biomolecular-electronic encoder and decoder devices constructed by multiplex biosensors

An Ultrasensitive Light-up Cu²⁺ Biosensor Using a New DNAzyme Cleaving a Phosphorothioate-Modified Substrate

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DNAzymes for sensing, nanobiotechnology and logic gate applications

Cited by 739 publications

References 61 publications

Nanoparticles-Free Fluorescence Anisotropy Amplification Assay for Detection of RNA Nucleotide-Cleaving DNAzyme Activity

Nanoparticles-Free Fluorescence Anisotropy Amplification Assay for Detection of RNA Nucleotide-Cleaving DNAzyme Activity

DNA biomolecular-electronic encoder and decoder devices constructed by multiplex biosensors

An Ultrasensitive Light-up Cu2+ Biosensor Using a New DNAzyme Cleaving a Phosphorothioate-Modified Substrate

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An Ultrasensitive Light-up Cu²⁺ Biosensor Using a New DNAzyme Cleaving a Phosphorothioate-Modified Substrate