Enzyme-Linked Small-Molecule Detection Using Split Aptamer Ligation

Sharma, Ashwani; Kent, Alexandra D.; Heemstra, Jennifer M.

doi:10.1021/ac300997q

Cited by 103 publications

(87 citation statements)

References 28 publications

(47 reference statements)

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“…1,2 Recently, this aptamer's specificity has been extended to include norcocaine and the off-target ligand, quinine. [3][4][5][6] Since the cocaine-binding activity of the original MNS-4.1 aptamer was reported, 1 many functional variants of this aptamer have been reported. Here we report on the specificity, ligand binding and structure of three sequence variants of the cocaine aptamer 2,7 and some of their 2-aminopurine (2AP) substituted variants.…”

Section: Introductionmentioning

confidence: 99%

Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl

et al. 2016

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The cocaine aptamer has been seen as a good candidate for development as a probe for cocaine in many contexts. Here, we demonstrate that the aptamer binds cocaine, norcocaine, and cocaethylene with similar affinities and aminoglycosides with similar or higher affinities in a mutually exclusive manner with cocaine. Analysis of its affinities for a series of cocaine derivatives shows that the aptamer specificity is the consequence of its interaction with all faces of the cocaine molecule. Circular dichroism spectroscopy and 2-aminopurine (2AP) fluorescence studies show no evidence of large structural rearrangement of the cocaine aptamer upon ligand binding, which is contrary to the general view of this aptamer. The aptamer's affinity for cocaine and neomycin-B decreases with the inclusion of physiological NaCl. The substitution of 2AP for A in position 6 (2AP6) of the aptamer sequence eliminated the effect of NaCl on its affinities for cocaine and analogues, but not for neomycin-B, showing a selective effect of 2AP substitution on cocaine binding. The affinity for cocaine also decreased with increasing concentrations of serum or urine, with the 2AP6 substitution blunting the effect of urine. Its low affinities for cocaine and metabolites and its ability to bind irrelevant compounds limit the opportunities for application of this aptamer in its current form as a selective and reliable sensor for cocaine. However, these studies also show that a small structural adjustment to the aptamer (2AP exchanged for adenine) can increase its specificity for cocaine in physiological NaCl relative to an off-target ligand.

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Section: Introductionmentioning

confidence: 99%

Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl

et al. 2016

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“…The use of aptamers as alternatives to antibodies allows the development of enzyme-linked aptamer assays [33][34][35][36][37]. In many enzyme linked aptamer assays, enzyme molecules (e.g., horseradish peroxidase and alkaline phosphatase) were usually attached on the aptamer or antibodies through antibody-antigen interaction, biotin-streptavidin (avidin) interaction, or chemical cross linking [33][34][35][36][37]. Rolling circle amplification (RCA) was also combined with the enzyme linked aptamer assay to further improve sensitivity [38].…”

Section: Introductionmentioning

confidence: 99%

Thrombin-linked aptamer assay for detection of platelet derived growth factor BB on magnetic beads in a sandwich format

Guo

Zhao

2016

Talanta

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a b s t r a c tHere we describe a thrombin-linked aptamer assay (TLAA) for protein by using thrombin as an enzyme label, harnessing enzyme activity of thrombin and aptamer affinity binding. TLAA converts detection of specific target proteins to the detection of thrombin by using a DNA sequence that consists of two aptamers with the first aptamer binding to the specific target protein and the second aptamer binding to thrombin. Through the affinity binding, the thrombin enzyme is labeled on the protein target, and thrombin catalyzes the hydrolysis of small peptide substrate into product, generating signals for quantification. As a proof of principle, we show a sandwich TLAA for platelet derived growth factor BB (PDGF-BB) by using anti-PDGF-BB antibody coated on magnetic beads and an oligonucleotide containing the aptamer for PDGF-BB and the aptamer for thrombin. The binding of PDGF-BB to both the antibody and the aptamer results in labeling the complex with thrombin. We achieved detection of PDGF-BB at 16 pM. This TLAA contributes a new application of thrombin and its aptamer in bioanalysis, and shows potentials in assay developments.

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“…As an alternative class of MRE, aptamers display several attractive features. They are produced in vitro in a short time and are characterized by a smaller size (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). They usually possess high affinity (with dissociation constants ranging from the pico-to the micromolar range) and high selectivity for their target.…”

Section: Introductionmentioning

confidence: 99%

“…They exploited the ability of nucleic acid aptamers to be split into two pieces which are able to specifically form a ternary assembly in the presence of the ligand. This elegant approach has been extensively used in the small target sensing field using different transduction techniques [7][8][9][10][11]. However, the main drawback of the split aptamer approach is related to the decrease in the target binding affinity of the functional oligonucleotide upon splitting [9], leading to a low-sensitivity assay in most cases.…”

Section: Introductionmentioning

confidence: 99%

An improved design of the kissing complex-based aptasensor for the detection of adenosine

et al. 2015

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We very recently reported a novel aptamer biosensing concept based on a dual recognition mechanism originating from the small target-induced formation of a functional nucleic acid assembly. This assembly is constituted of a hairpin aptamer (named aptaswitch) for which the apical loop of the parent aptamer is substituted by a short RNA sequence prone to loop-loop interactions. It can switch between folded and unfolded states in the presence and in the absence of targets, respectively. The apical loop of the folded aptaswitch is then recognized by a second hairpin (called aptakiss), forming a kissing complex that signals the presence of the target. In the present work, we focus on the design improvement of this biosensing platform by using a previously described adenosine-adenoswitch couple as a model system and a fluorophore-labeled aptakiss as a reporting probe for fluorescence anisotropy (FA) detection. In the first step, the initially described adenoswitch was re-engineered to optimally convert the unfolded structure into the active stem-loop form upon adenosine binding. To further improve the assay performance, a blocking DNA oligonucleotide of the adenoswitch sequence was subsequently introduced into the assay scheme. This blocking strategy led to a significant increase in the FA response by reducing the background signal generated by the undesired binding of the free adenoswitch to the aptakiss probe. We obtained a detection limit which is fivefold lower than that observed with the previously reported kissing complex-based sensor. Finally, the optimized biosensing platform was successfully applied under biologically relevant conditions, i.e., diluted human serum, suggesting the potential practical applicability of the kissing sensing approach.

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Enzyme-Linked Small-Molecule Detection Using Split Aptamer Ligation

Cited by 103 publications

References 28 publications

Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl

Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl

Thrombin-linked aptamer assay for detection of platelet derived growth factor BB on magnetic beads in a sandwich format

An improved design of the kissing complex-based aptasensor for the detection of adenosine

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