Probing the Nitroindole-Modified Central Loop of Thrombin Aptamer HD1 as a Recognition Site.

Abstract

Thrombin-binding aptamer HD1 is a DNA-based thrombin inhibitor that features an antiparallel G-quadruplex (GQ) structure. We recently reported a single-nucleotide G8 to 5-nitroindole (NI) modification of HD1 (N8) that notably improves the anticoagulant properties and binding affinity of the aptamer. Based on molecular modeling and binding studies, it was originally proposed that N8 may acquire the ability to bind thrombin by a modified central loop. To verify this possibility, in this study, we report new variations of the N8 aptamer with intact or damaged TT loops. Anomeric alpha-thymidine was used as a "damaging" residue to disable the primary recognition site of N8. Biophysical characterization of modified aptamers supports the formation of HD1-like antiparallel GQs with varying stability by all studied variants. Binding experiments showed that N8 variants with impaired TT loops lost the ability to bind thrombin, suggesting the primary role of thymines in TT loops for the thrombin-N8 interaction. Aptamer N8α(7/9) bearing NI at position 8 and damaged thymidines 7 and 9 retained thrombin affinity, which was intermediate between N8 and HD1. Fluorescence polarization studies suggest 1:1 stoichiometry for thrombin complexes with either HD1, N8, or N8α(7/9). Further molecular dynamics (MD) study of complexes formed by these three aptamers with thrombin disproves the idea of direct interaction between central loop residues and the protein. Based on MD results, the origin of the NI tuning effect is associated with its ability to promote the formation of compact and rigid structures through hydrophobic interactions with the GQ core and loop thymines.