Cocaine-binding aptamer

Aptamers are oligonucleic acid or peptide molecules that can selectively bind a specific target molecule. Recently, for the determination of a small-molecule drug, a highly sensitive ECL aptamer-based biosensor has been constructed based on two components a cocaine-binding aptamer as molecular recognition element and as a model analyte and a Ru(II) complex as an ECL label [153]. 

Fig. 1. Scheme for RNA aptamer identification targeting cocaine-binding sites on the nicotinic acetylcholine receptor (nAchR). 

DNA- binding aptamers have been designed to bind to mRNA, g-quad-ruplexes, Tenascin-C, a protein found in the tumor matrix, and to thrombin. Aptamers have been selected as inhibitors of HIV-1 integrase, human RNase Hl, human pro-urokinase " and for the design of molecular beacons. An allosteric aptamer has been designed for binding as a colorimetric probe for cocaine. ... 

FIGURE 8.22 Cocaine-sensing aptamer binding to cocaine. Reproduced with permission from Reference 69. Copyright 2011 Institute of Organic Chemistty and Biochemistry AS CR. 

A simple protocol detailed in this chapter was established to develop RNA aptamers that bind to the electric organ nAChR and that are displaced by cocaine (8) (see Fig. 1 for a scheme). This protocol can be easily transferred to SELEX applications with other receptors or cell-surface epitopes, given that these are enriched in membrane preparations. 

Selection of Aptamers that Bind to the nAChR and Displace Cocaine... 

For the elution of receptor-bound, cocaine-displaceable RNA aptamers, incubate each of these filter pieces in 100 pi of 1 mM cocaine (or 100 pi of 1 mM MK-801 that binds to the same site as cocaine on the nAChR) in incubation buffer for 20 min at room temperature. 

Determination of unspecific binding reaction by competition of the RNA aptamers with cocaine 79 pi incubation buffer, 10 pi nAChR-enriched plasma membranes (800 pg/ml protein 1.6 pM receptor), 1 pi P-RNA dilution containing 10 mg/ml t-RNA anti-RNAse (40 U/pl), and 10 pi cocaine (10 mM). The percentage of binding of the ( P) RNA aptamers to the... 

Fig. 43 Left The split aptamer concept applied to the detection of cocaine. The binding constant of the cocaine-aptamer complex is approximately 100 pM [162]. The line indicates the split of the aptamer resulting in two oligos. Right Histograms display the results performed in pure buffo-solution top), with 1 mM cocaine in the buffer solution middle), and after rinsing eoeaine away with pure buffer bottom). Each set of data was fitted with a Gaussian distribution. Data adapted from the PhD thesis of Huong Nguyen [163]...

FIGURE 16.5 (a) Cocaine 13 binding DNA aptamer bearing fluorescein and dabcyl units at 5 and 3 ends. (b) MAB for thrombin 14 binding. (c) thrombin beacon design. (Sources Copyright (2005), American Chemical Society and copyright (2002), Elsevier. With permission.)... 

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