Quinine Binding by the Cocaine-Binding Aptamer. Thermodynamic and Hydrodynamic Analysis of High-Affinity Binding of an Off-Target Ligand

Abstract

The cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We find that the affinity of the cocaine-binding aptamer for quinine is 30-40 times stronger than it is for cocaine. Competitive-binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more negative change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle X-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism.

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

“…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.…”

“…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. 23,24 To understand the extent of structural unfolding that occurs with the cocaine aptamer, we first examined the most frequently employed aptamer with a longer S1 (38COC1A) that has been proposed to form a helix in the absence of cocaine.…”

“…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).…”

“…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.…”

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

“…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). This ligand-induced folding mechanism is retained with quinine binding and when the sequence is altered to allow for binding of the steroid deoxycholic acid (DCA) (18,22). The cocaine-binding aptamer has also been shown to function when separated into two strands, referred to as a split-aptamer (3,13,23).…”