Studying DNA G-Quadruplex Aptamer by 19F NMR

Abstract

In this study, we demonstrated that ¹⁹F NMR can be used to study the thrombin-binding aptamer (TBA) DNA G-quadruplex, widely used as a model structure for studying G-quadruplex aptamers. We systematically examined the structural feature of the TBA G-quadruplex aptamer with fluorine-19 (¹⁹F) labels at all of the thymidine positions. We successfully observed the structural change between the G-quadruplex and the unstructured single strand by ¹⁹F NMR spectroscopy. The thermodynamic parameters of these DNA G-quadruplex aptamers were also determined from the ¹⁹F NMR signals. We further showed that the ¹⁹F NMR method can be used to observe the complex formed by TBA G-quadruplex and thrombin. Our results suggest that ¹⁹F NMR spectroscopy is a useful approach to study the aptamer G-quadruplex structure.

Figure 1

(a) Schematic cartoon of the NMR structure of the G-quadruplex aptamer. (b) Sequences of the ¹⁹F-labeled oligonucleotides synthesized in this study and chemical structure of ¹⁹F-labeled 2′-deoxyuridine having 3,5-bis(trifluoromethyl)phenyl group.

Figure 2

(a) ¹⁹F NMR spectra of ¹⁹F-labeled oligonucleotides in the presence or absence of KCl. Condition: [DNA] = 100 μM, [KCl] = 100 mM, [Tris–HCl buffer (pH 7.0)] = 10 mM. (b) ¹H imino proton NMR of T12 corresponding to ¹⁹F NMR at 25 or 70 °C.

Figure 3

(a) ¹⁹F NMR spectra of ¹⁹F-labeled oligonucleotides at different temperatures. Blue and red spots indicated single strand and G-quadruplex. Temperatures indicated on the right. (b) Melting profiles derived from the relative peak areas of the ¹⁹F signals on different temperatures.

Figure 4

(a) Molecular model of the G-quadruplex formed by T9. (b) Distances between F–H8 and F–H2′ in T9. dG and ¹⁹F-labeled 2′-deoxyuridine are indicated in magenta and yellow, respectively. ¹⁹F is light blue.