Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding

Abstract

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.

Figure 1.

Schematic representation of the structure of TBA (A) and of the organization proposed on the basis of sequence for HD22–27mer (B) and RE31 (C).

Figure 2.

(A) Surface/cartoon representation of thrombin–RE31 complex. Thrombin heavy and light chain are coloured dark and light magenta, respectively, the aptamer is coloured cyan and the potassium ion orange. (B) 2F_o − F_c electron density maps of the RE31 aptamer contoured at 1.5σ level.

Figure 3.

The duplex/quadruplex junction in RE31 aptamer. On the left a cartoon representation of the aptamer with different motifs marked in different colours (duplex segment in pink, quadruplex segment in yellow, the junction in green), on the right stick representation of the three base pairs composing the junction.

Figure 4.

Ribbon representation of RE31 highlighting the continuous stacking of bases from the duplex to the quadruplex region.

Figure 5.

Comparison of interaction within thrombin–RE31 (protein molecule depicted in magenta and aptamer molecule in teal) and thrombin–TBA (colored in gray) complexes. Polar contacts between Arg H75, Arg H77A, Thy12 (Thy4 in TBA) and Gua13 (Gua5 in TBA) are highlighted.