Ultrasensitive Detection of Protein Using an Aptamer-Based Exonuclease Protection Assay

Wang, Xiaoli; Li, Fang; Su, Yuping; Sun, Xi; Li, Xiaobo; Schluesener, Hermann J.; Tang, Fei; Xu, Shunqing

doi:10.1021/ac0494228

Cited by 109 publications

(60 citation statements)

References 21 publications

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“…5B). 71 In this design, exonuclease I was used to digest unbound anti-thrombin aptamers, while the bound aptamers were protected from degradation. Then the bound anti-thrombin aptamer was released, and formed a duplex with two connector sequences which were complementary to half of the anti-thrombin aptamer.…”

Section: Dna Backbone As Binding Agentmentioning

confidence: 99%

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Fluorescent DNA-based enzyme sensors

2011

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Fluorescent sensors that make use of DNA structures have become widely useful in monitoring enzymatic activities. Early studies focused primarily on enzymes that naturally use DNA or RNA as the substrate. However, recent advances in molecular design have enabled the development of nucleic acid sensors for a wider range of functions, including enzymes that do not normally bind DNA or RNA. Nucleic acid sensors present some potential advantages over classical small-molecule sensors, including water solubility and ease of synthesis. An overview of the multiple strategies under recent development is presented in this critical review, and expected future developments in microarrays, single molecule analysis, and in vivo sensing are discussed (160 references).

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Section: Dna Backbone As Binding Agentmentioning

confidence: 99%

“…(B) Scheme for thrombin detection involving exonuclease protection. 71 (1) Anti-thrombin aptamers bound to thrombin. (2) Exonuclease I degraded unbound aptamers.…”

Section: Figmentioning

confidence: 99%

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Fluorescent DNA-based enzyme sensors

2011

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“…Aptamers can be chemically conjugated with various signal-generating components for acoustic [26–28] , electrochemical [29–32] , and optical [33–35] sensing. In addition, by taking advantage of adaptive recognition, aptamers can be designed into analyte-responsive biosensors and thus improve detection sensitivity by reducing background [36–40] or amplifying signals [41–44] . The term DNAzyme refers to nucleic acids with certain sequences and structures that give them catalytic ability towards various chemical reactions, such as RNA or DNA cleavage, ligation and porphyrin metalation [45, 46] .…”

Section: Introductionmentioning

confidence: 99%

Responsive DNA‐Based Hydrogels and Their Applications

Xiong

Zhou

et al. 2013

Macromol. Rapid Commun.

133

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The term hydrogel describes a type of soft and wet material formed by crosslinked hydrophilic polymers. The distinct feature of hydrogels is their ability to absorb a large amount of water and swell. The properties of a hydrogel are usually determined by the type of polymer and crosslinker, the degree of crosslinking, and the water content. However, a group of hydrogels, called “smart hydrogels”, changes properties in response to environmental changes or external stimuli. Recently, DNA or DNA-inspired responsive hydrogels have attracted considerable attention in construction of smart hydrogels because of the intrinsic advantages of DNA. As a biological polymer, DNA is hydrophilic, biocompatible, and highly programmable by Watson-Crick base pairing. DNA can form a hydrogel by itself under certain conditions, and it can also be incorporated into synthetic polymers to form DNA-hybrid hydrogels. Functional DNAs, such as aptamers and DNAzymes, provide additional molecular recognition capabilities and versatility. In this review, we discuss DNA-based hydrogels in terms of their stimulus response, as well as their applications.

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“…Examples include molecular beacon assays [6, 7], proximity ligation-based assays [8, 9] and exonuclease protection assays [10, 11]. …”

Section: Introductionmentioning

confidence: 99%

Single-Pair Fluorescence Resonance Energy Transfer (spFRET) for the High Sensitivity Analysis of Low-Abundance Proteins Using Aptamers as Molecular Recognition Elements

et al. 2009

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We have developed a strategy for the detection of single protein molecules, which uses single-pair fluorescence resonance energy transfer (spFRET) as the readout modality and provides exquisite analytical sensitivity and reduced assay turn-around-time by eliminating various sample pre-processing steps. The single-protein detection assay uses two independent aptamer recognition events to form an assembly conducive to intramolecular hybridization of oligonucleotide complements that are tethered to the aptamers. This hybridization brings a donor-acceptor pair within the Förster distance to create a fluorescence signature indicative of the presence of the protein-aptamer(s) association complex. As an example of spFRET, we demonstrate the technique for the analysis of serum thrombin. The assay requires co-association of two distinct epitope-binding aptamers, each of which is labeled with a donor or acceptor fluorescent dye (Cy3 or Cy5, respectively) to produce a FRET response. The FRET response between Cy3 and Cy5 was monitored by single-molecule photon-burst detection, which provides high analytical sensitivity when the number of single-molecule events is plotted versus the target concentration. We are able to identify thrombin with high efficiency based on photon burst events transduced in the Cy5 detection channel. We also demonstrate that the technique can discriminate thrombin molecules from its analogue prothrombin. The analytical sensitivity was >200-fold better than an ensemble measurement.

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Ultrasensitive Detection of Protein Using an Aptamer-Based Exonuclease Protection Assay

Cited by 109 publications

References 21 publications

Fluorescent DNA-based enzyme sensors

Fluorescent DNA-based enzyme sensors

Responsive DNA‐Based Hydrogels and Their Applications

Single-Pair Fluorescence Resonance Energy Transfer (spFRET) for the High Sensitivity Analysis of Low-Abundance Proteins Using Aptamers as Molecular Recognition Elements

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