Defining the secondary structural requirements of a cocaine-binding aptamer by a thermodynamic and mutation study

Neves, Miguel A. D.; Reinstein, Oren; Saad, Makbul; Johnson, Phyllis E.

doi:10.1016/j.bpc.2010.09.009

Cited by 103 publications

(125 citation statements)

References 31 publications

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“…This is consistent with previous studies showing that mutations to nucleotides that contribute to formation of this hydrophobic pocket, including the unpaired T20 and C21, can profoundly alter the aptamer's binding affinity for cocaine. 33 Our mutation experiments indicate that ATMND binding is also heavily dependent on C21 and T20. Nakatani et al 34,35 used 15 N NMR experiments to reveal that 1,8-naphthridine selectively binds to cytosine or thymine via a three-point hydrogen bond 36 and that the binding affinity of ATMND to various target nucleotides is as follows: cytosine > thymine > adenine > guanine.…”

Section: ■ Results and Discussionmentioning

confidence: 94%

A Label-Free Aptamer-Fluorophore Assembly for Rapid and Specific Detection of Cocaine in Biofluids

et al. 2014

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We report a rapid and specific aptamer-based method for one-step cocaine detection with minimal reagent requirements. The feasibility of aptamer-based detection has been demonstrated with sensors that operate via target-induced conformational change mechanisms, but these have generally exhibited limited target sensitivity. We have discovered that the cocaine-binding aptamer MNS-4.1 can also bind the fluorescent molecule 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) and thereby quench its fluorescence. We subsequently introduced sequence changes into MNS-4.1 to engineer a new cocaine-binding aptamer (38-GC) that exhibits higher affinity to both ligands, with reduced background signal and increased signal gain. Using this aptamer, we have developed a new sensor platform that relies on the cocaine-mediated displacement of ATMND from 38-GC as a result of competitive binding. We demonstrate that our sensor can detect cocaine within seconds at concentrations as low as 200 nM, which is 50-fold lower than existing assays based on target-induced conformational change. More importantly, our assay achieves successful cocaine detection in body fluids, with a limit of detection of 10.4, 18.4, and 36 μM in undiluted saliva, urine, and serum samples, respectively.

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Section: ■ Results and Discussionmentioning

confidence: 94%

A Label-Free Aptamer-Fluorophore Assembly for Rapid and Specific Detection of Cocaine in Biofluids

et al. 2014

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“…For the previously reported signaling aptamers based on such structural transition, the target-induced switch is commonly achieved through the destabilization of the stems [20][21][22]. However, the instability created in the aptamer structure typically generates a loss of the target binding affinity [22,23]. …”

Section: Resultsmentioning

confidence: 98%

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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“…de-los-Santos-Álvarez et al [104,105] opened up the way to use the modified-RNA aptamer-based sensor for competitive impedimetric assay and SPR assay of neomycin B, respectively, which demonstrated the feasibility of detecting small molecules using RNA aptamers with high sensitivity and stability. In addition, many research studies have been performed to explore the binding mechanism and interaction between aptamer and target and the changes of secondary structure in different conditions, such as ion intensity, temperature, and acidity-alkalinity [62,129,130]. Therefore, it is possible to enhance the stability and improve other molecular characteristics of aptamers by developing their resistance to ion intensity, temperature, and acidity-alkalinity.…”

Section: Prospectsmentioning

confidence: 98%

Aptamer-Based Technology for Food Analysis

Liu

Zhang

2014

Appl Biochem Biotechnol

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Aptamers are short and functional single-stranded oligonucleotide sequences selected from systematic evolution of ligands by exponential enrichment (SELEX) process, which have the capacity to recognize various classes of target molecules with high affinity and specificity. Various analytical aptamers acquired by SELEX are widely used in many research fields, such as medicine, biology, and chemistry. However, the application of this innovative and emerging technology to food safety is just in infant stage. Food safety plays a very important role in our daily lives because varieties of poisonous and harmful substances in food affect human health. Aptamer technique is promising, which can overcome many disadvantages of existing detection methods in food safety, such as long detection time, low sensitivity, difficult, and expensive antibody preparation. This review provides an overview of various aptamer screening technologies and summarizes the recent applications of aptamers in food safety, and future prospects are also discussed.

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Defining the secondary structural requirements of a cocaine-binding aptamer by a thermodynamic and mutation study

Cited by 103 publications

References 31 publications

A Label-Free Aptamer-Fluorophore Assembly for Rapid and Specific Detection of Cocaine in Biofluids

A Label-Free Aptamer-Fluorophore Assembly for Rapid and Specific Detection of Cocaine in Biofluids

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

Aptamer-Based Technology for Food Analysis

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