Defining a Stem Length-Dependent Binding Mechanism for the Cocaine-Binding Aptamer. A Combined NMR and Calorimetry Study

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

doi:10.1021/bi100952k

Cited by 96 publications

(213 citation statements)

References 62 publications

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“…The addition of cocaine is predicted to trigger a conformational rearrangement in the aptamer ( Figure 1B), with two adjacent GA base pairs and a dinucleotide bulge (T20 and C21) within the aptamer binding pocket. 27 Upon 50 μM cocaine addition, we observed a competitive target binding to MNS-4.1, resulting in successful displacement of ATMND from the aptamer with a signal gain of 9.6 ( Figure 2A). This fluorescence increase was stable for at least several hours (data not shown).…”

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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“…8 The shorter stem also decreases the aptamer affinity for cocaine to 28-45 µM 7 (K d ), which is 4-5-fold lower than for a version of the aptamer with a longer stem. 7 An examination of the literature from the past three years shows that the vast majority of sensors developed to incorporate the intact cocaine aptamer included the longer S1 stem, 5,[9][10][11][12][13][14][15][16][17][18][19][20][21][22] with occasional comparisons of the short and long S1 aptamers. 5,16 Sensors have also been infrequently reported based on intact short stem cocaine aptamers.…”

Section: Structural Changes In the Cocaine Aptamer Accompanying Cocaimentioning

confidence: 99%

“…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%

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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“…The reason for the continued utilization of the cocaine-binding aptamer likely lies in the ability to engineer into the aptamer a structure switching binding mechanism. The secondary structure of the MN4 aptamer (Figure 1) is pre-formed in the absence of ligand, and upon binding its target there is no observable change in the secondary structure (21). However, if the length of stem one is shortened to three base pairs, such as for MN19 (Figure 1), the aptamer is loosely structured in the absence of target and upon binding the secondary structure rigidifies in a ligand-induced folding process (4,21).…”

Section: Introductionmentioning

confidence: 99%

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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Defining a Stem Length-Dependent Binding Mechanism for the Cocaine-Binding Aptamer. A Combined NMR and Calorimetry Study

Cited by 96 publications

References 62 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

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

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

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