Functional Nucleic Acid Sensors

Liu, Juewen; Cao, Zehui; Lu, Yi

doi:10.1021/cr030183i

Cited by 2,035 publications

(1,569 citation statements)

References 637 publications

(1,598 reference statements)

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“…A number of fluorescent Pb 2+ sensors have been developed, which are based on the fluorescently labeled small organic chelators, 55,56 organic polymers, 57 peptides, 58 and functional nucleic acid. 3,9 The detection limit of our nonnanoparticle-dependent FA approach (100 pM) is lower than that of nanoparticle-dependent FA approaches (LOD, 1 nM,) 18 and other fluorescence intensity-based homogeneous analysis (LOD, 3.7 nM). 38 Our approach for detection of free Pb 2+ has several advantages.…”

Section: ■ Results and Discussionmentioning

confidence: 74%

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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Section: ■ Results and Discussionmentioning

confidence: 74%

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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“…the hammerhead ribozyme, 32 HDV ribozyme, 33 [10][11][12][13][14][15][16][17][18][19][20][21][22][23] DNAzyme, 37 RNase P, 38 Group II intron, 39 ) use pro-Rp for metal binding (mostly Mg 2+ ). This study also indicates that the pro-Rp oxygen is more important for metal binding in the Ce13d DNAzyme.…”

Section: Resultsmentioning

confidence: 99%

Biochemical Characterization of a Lanthanide-Dependent DNAzyme with Normal and Phosphorothioate-Modified Substrates

et al. 2015

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A trivalent lanthanide (Ln 3+ )-dependent RNAcleaving DNAzyme, Ce13d, was recently isolated via in vitro selection. Ce13d is active in the presence of all Ln 3+ ions. Via introduction of a single phosphorothioate (PS) modification at the cleavage site, its activity with Ln 3+ decreases while all thiophilic metals can activate this DNAzyme. This property is unique to Ce13d and is not found in many other tested DNAzymes. This suggests the presence of a well-defined but general metal binding site. Herein, a systematic study of Ce13d with the PO substrate (using Ce 3+ ) and the PS substrate (using Cd 2+ ) is performed. In both the PO and PS systems, the highest activity was with ∼10 μM metal ions. Higher concentrations of Ce 3+ completely inhibit the activity, while Cd 2+ only slows the activity. A comparison of different metal ions suggests that the role of metal is to neutralize the phosphate negative charge. Both systems follow a similar pH−rate profile with a single deprotonation step, indicating similar reaction mechanisms. The activity difference between the R p and S p form of the PS substrate is <10-fold, which is much smaller than most known RNA-cleaving enzymes. Mutation studies identified eight highly conserved purines, among which the two adenines play mainly structural roles, while the guanines are likely to be involved in metal binding. Ce13d can serve as a model system for further understanding of DNAzyme biochemistry and bioinorganic chemistry.

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“…[7][8][9] In particular, DNA-directed assembly of gold nanoparticles (AuNPs) has tremendously fueled the growth of nanobiotechnology. [10][11][12][13][14][15][16] The dense layer of DNA creates a unique colloidal system with many fundamentally interesting physical properties. 10 End-labeled thiol is the main anchor for linking DNA to AuNPs due to strong Au-thiol interactions.…”

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confidence: 99%

Tandem Phosphorothioate Modifications for DNA Adsorption Strength and Polarity Control on Gold Nanoparticles

Zhou

Wang

Ding

et al. 2014

ACS Appl. Mater. Interfaces

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Unmodified DNA was recently used to functionalize gold nanoparticles via DNA base adsorption. Compared to thiolated DNA, however, the application of unmodified DNA is limited by the lack of sequence generality, adsorption polarity control and poor adsorption stability. We report that these problems can be solved using phosphorothioate (PS) DNA. PS DNA binds to gold mainly via the sulfur atom and is thus less sequence dependent. The adsorption affinity is ranked to be thiol > PS > adenine > thymine. Tandem PS improves adsorption strength, allows tunable DNA density, and the resulting conjugates are functional at a low cost.

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Functional Nucleic Acid Sensors

Cited by 2,035 publications

References 637 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

Biochemical Characterization of a Lanthanide-Dependent DNAzyme with Normal and Phosphorothioate-Modified Substrates

Tandem Phosphorothioate Modifications for DNA Adsorption Strength and Polarity Control on Gold Nanoparticles

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