Unraveling the binding mode of a methamphetamine aptamer: A spectroscopic and calorimetric study

Abstract

Nucleic-acid aptamers are bio-molecular recognition agents that bind to their targets with high specificity and affinity and hold promise in a range of biosensor and therapeutic applications. In the case of small-molecule targets, their small size and limited number of functional groups constitute challenges for their detection by aptamer-based biosensors because bio-recognition events may both be weak and produce poorly transduced signals. The binding affinity is principally used to characterize aptamer-ligand interactions; however, a structural understanding of bio-recognition is arguably more valuable in order to design a strong response in biosensor applications. Using a combination of nuclear magnetic resonance, circular dichroism, and isothermal titration calorimetry, we propose a binding model for a new methamphetamine aptamer and determine the main interactions driving complex formation. These measurements reveal only modest structural changes to the aptamer upon binding and are consistent with a conformational-selection binding model. The aptamer-methamphetamine complex formation was observed to be entropically driven, apparently involving hydrophobic and electrostatic interactions. Taken together, our results exemplify a means of elucidating small molecule-aptamer binding interactions, which may be decisive in the development of aptasensors and therapeutics and may contribute to a deeper understanding of interactions driving aptamer selection.

Graphical abstract

Figure 1

Binding models of bioreceptors in the context of aptamer-ligand binding. (A–C) Lock and key (LAK) (A), conformational-selection (CS) (B), and induced-fit (C) binding models. Representation relates to the interaction of the Aptamer-2-40mer with methamphetamine. Binding constant equations are found in Eq. S1. To see this figure in color, go online.

Figure 2

Skeletal formula of protonated methamphetamine (Meth) as dominant at pH 7.5. To see this figure in color, go online.

Figure 3

CD experiments for Aptamer-2 upon Meth addition. The positive ellipticity peak at 277 nm and the negative ellipticity peak at 249 nm are characteristic of a B-form structure. To see this figure in color, go online.

Figure 4

Representation of the Mfold-calculated lowest-energy Aptamer-2-40mer structure with the hairpin structure circled. To see this figure in color, go online.

Figure 5

Watson-Crick basepairs A-T and G-C. To see this figure in color, go online.

Figure 6

(A) Expansion of 1H NMR spectrum of Aptamer-2-40mer showing three thymine imino-proton peaks. (B) 2D NOESY correlations between the three imino protons and the three adenine C2 protons showing A-T base pairing. To see this figure in color, go online.

Figure 7

1H NMR spectra of Aptamer 2-40mer with molar loadings of Meth 0% and 100%. To see this figure in color, go online.

Figure 8

ITC results for the Aptamer-2-40mer and the mutated Aptamer-2-40mer. (A and B) The heat profiles for (A) Aptamer-2-40mer and (B) mutated Aptamer-2-40mer are represented. (C and D) The integrated heat profiles are represented for (C) Aptamer-2-40mer and (D) mutated Aptamer-2-40mer. The heat profiles presented have been corrected for the heat of dilution of the titrant and for the heat of dilution of the aptamer. To see this figure in color, go online.

Figure 9

Molecular docking of Meth with Aptamer-2-40mer. (A) All 100 docking poses of Meth in the hydrophobic pocket are shown in stick form, and the aptamer is shown in cartoon form with a transparent surface rendered. (B) A violin plot of the docking scores of each cluster. (C) The 12 poses of cluster_1 C1 (Meth in stick form) in the pocket, with surrounding nucleotides shown in wire form. (D) The 88 poses of cluster_2 C2 (Meth in stick form) in the pocket with surrounding nucleotides shown in wire form. To see this figure in color, go online.