Crystal structure of an RNA aptamer bound to thrombin

Abstract

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 Å resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.

Affinity capillary electrophoresis with laser induced fluorescence detection for thrombin analysis using nuclease-resistant RNA aptamers

“…As previous study shows Apt-29 binds with the heparin-binding site [27], the binding site of Toggle-25 on thrombin is possibly close to the heparin-binding site. This result is in agreement with the previous report [31,32,34]. The characterized crystal structure of complex thrombin and Toggle-25 also proves the binding site of Toggle-25 is near the heparin binding site of thrombin [32].…”

“…This result is in agreement with the previous report [31,32,34]. The characterized crystal structure of complex thrombin and Toggle-25 also proves the binding site of Toggle-25 is near the heparin binding site of thrombin [32]. It is noted that the 1000-fold Apt-29 (10 M) added only made the peak area of the complex peak decreased about 49%.…”

“…It suggests that the bound Toggle-25-TMR was completely displaced by unlabeled Toggle-25. Considering the fact that high concentration of Apt-29 (10 M) did not totally displace the bound Toggle-25-TMR from thrombin, it indicates the binding site of Toggle-25 on thrombin is very close to that of Apt-29 on thrombin, but their binding sites are not completely the same, which is in agreement with the results of the characterized crystal structures of Toggl-25-thrombin complex and Apt-29-thrombin complex [32,35].…”

“…1), and the bulge is flanked by two short duplex regions [31]. The crystal structure of the complex of Toggle-25 and thrombin is already available, and it confirms the presence of stem-loop structure and shows an additional base pair formed between C11 and G16 and the existence of extensive -stacking of bases [32]. The unique three-dimensional structure of Toggle-25 presents good shape complementarity to native surface of thrombin, allowing for the specific and tight interaction between thrombin and Toggle-25.…”

“…MgCl 2 and CaCl 2 may help the aptamer to form a right structure for binding to thrombin [31], and the difference effects of MgCl 2 and CaCl 2 may be due to the properties of the divalent metal ions. The crystal structure of the complex of Toggle-25 and thrombin also shows the existence of Mg 2+ coordinating nonbridging oxygen atoms of phosphates on two nucleotides of the aptamer, and it is expected that Ca 2+ can play similar function [32].…”