Structure–affinity relationship of the cocaine-binding aptamer with quinine derivatives

Slavkovic, Sladjana; Altunisik, Merve; Reinstein, Oren; Johnson, Phyllis E.

doi:10.1016/j.bmc.2015.02.052

Cited by 45 publications

(72 citation statements)

References 32 publications

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“…The other potential binding models of identical independent and identical cooperative binding (32) were not considered as it is not possible to have two identical binding sites within the monomeric cocaine-binding aptamer. The high affinity site is slightly tighter with no added NaCl than previously reported in 140 mM NaCl (5.5 ± 0.4 μM) at the same pH value and temperature (19). This higher affinity in the absence of NaCl is consistent with electrostatic interactions playing a role in the affinity of the positively charged ligand to the polyanionic DNA (18,23) as reducing the salt concentration will decrease shielding of the charge-charge interactions, increasing the electrostatic attractions and yielding higher affinity.…”

Section: Resultsmentioning

confidence: 41%

Salt-mediated two-site ligand binding by the cocaine-binding aptamer

et al. 2016

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Multisite ligand binding by proteins is commonly utilized in the regulation of biological systems and exploited in a range of biochemical technologies. Aptamers, although widely utilized in many rationally designed biochemical systems, are rarely capable of multisite ligand binding. The cocaine-binding aptamer is often used for studying and developing sensor and aptamer-based technologies. Here, we use isothermal titration calorimetry (ITC) and NMR spectroscopy to demonstrate that the cocaine-binding aptamer switches from one-site to two-site ligand binding, dependent on NaCl concentration. The high-affinity site functions at all buffer conditions studied, the low-affinity site only at low NaCl concentrations. ITC experiments show the two ligand-binding sites operate independently of one another with different affinities and enthalpies. NMR spectroscopy shows the second binding site is located in stem 2 near the three-way junction. This ability to control ligand binding at the second site by adjusting the concentration of NaCl is rare among aptamers and may prove a useful in biotechnology applications. This work also demonstrates that in vitro selected biomolecules can have functions as complex as those found in nature.

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Section: Resultsmentioning

confidence: 41%

Salt-mediated two-site ligand binding by the cocaine-binding aptamer

et al. 2016

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“…43 Originally reported to bind cocaine and neither benzoylecgonine nor ecgonine methyl ester, 2 the aptamer was later shown to also bind norcocaine. 4, 6 We show here that it also binds cocaethylene with similar affinity as for cocaine and more tightly with norcocaine. Broadening the specificity of the cocaine aptamer to cocaethylene, a cocaine metabolite associated with ethanol consumption, 44,45 is noteworthy because drugs are often consumed in combination with alcohols.…”

Section: Specificity Of the Cocaine Aptamer For Cocaine And Related Cmentioning

confidence: 87%

“…Despite this evidence that the cocaine is buried in the binding pocket, there are several troublesome off-target ligands that bind with similar or 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 16 higher affinities (cocaethylene, norcocaine, aminoglycosides and quinine), [3][4][5][6] which complicate the application of this aptamer in a cocaine sensor.…”

Section: Discussionmentioning

confidence: 98%

“…These results are consistent with the reports of others. 5,7,8 To test for independent effects of cocaine on 2AP fluorescence, the structures of the 38COC1-2AP derivatives were distorted by annealing them with a complementary 20 nt oligonucleotide (cDNA (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)) that covers the presumptive cocaine binding pocket. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 These hybrid molecules showed no change in 2AP fluorescence upon the addition of cocaine, cocaine metabolites, or neomycin-B ( Figure S5).…”

Section: Affinity Of the Cocaine Aptamer And 2ap Derivatives For Cocainementioning

confidence: 99%

“…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%

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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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Development of Aptamer‐Based Non‐labeling Methods

Hao

Wang

2018

Aptamers for Analytical Applications

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Structure–affinity relationship of the cocaine-binding aptamer with quinine derivatives

Cited by 45 publications

References 32 publications

Salt-mediated two-site ligand binding by the cocaine-binding aptamer

Salt-mediated two-site ligand binding by the cocaine-binding aptamer

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

Development of Aptamer‐Based Non‐labeling Methods

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