The effect on quadruplex stability of North-nucleoside derivatives in the loops of the thrombin-binding aptamer

Abstract

Modified thrombin-binding aptamers (TBAs) carrying uridine (U), 2'-deoxy-2'-fluorouridine (FU) and North-methanocarbathymidine (NT) residues in the loop regions were synthesized and analyzed by UV thermal denaturation experiments and CD spectroscopy. The replacement of thymidines in the TGT loop by U and FU results in an increased stability of the antiparallel quadruplex structure described for the TBA while the presence of NT residues in the same positions destabilizes the antiparallel structure. The substitution of the thymidines in the TT loops for U, FU and NT induce a destabilization of the antiparallel quadruplex, indicating the crucial role of these positions. NMR studies on TBAs modified with uridines at the TGT loop also confirm the presence of the antiparallel quadruplex structure. Nevertheless, replacement of two Ts in the TT loops by uridine gives a more complex scenario in which the antiparallel quadruplex structure is present along with other partially unfolded species or aggregates.

Figure 1

(A) North-Methanocarbathymidine, uridine and 2′-deoxy-2′-fluorouridine residues used for the synthesis of modified thrombin-binding aptamers (TBAs). (B) Scheme of the modified TBAs showing the changes in the TGT loop. (C) Scheme of the modified TBAs showing the changes in the TT loops.

Figure 2

CD spectra of the modified thrombin-binding aptamers (TBAs). A) TBAs modified in the central TGT loop. B) TBAs modified in the TT loops.

Figure 3

Selected region of monodimensional ¹H-NMR (top) and 2D-NOESY spectrum (below) of TBA-U7,U9 acquired at 5°C

Figure 4

Imino regions of the ¹H-NMR spectra of TBA-U7,U9 (left) and of TBA-U4,U13 (right) at a range of temperatures.