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. 2018 May 18;46(9):4819-4830.
doi: 10.1093/nar/gky268.

Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity

Rafal Dolot  1 , Curtis H Lam  2 , Malgorzata Sierant  1 , Qiang Zhao  3 , Feng-Wu Liu  4 , Barbara Nawrot  1 , Martin Egli  5 , Xianbin Yang  2
Affiliations

Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity

Rafal Dolot et al. Nucleic Acids Res. .

Abstract

Thrombin-binding aptamer (TBA) is a DNA 15-mer of sequence 5'-GGT TGG TGT GGT TGG-3' that folds into a G-quadruplex structure linked by two T-T loops located on one side and a T-G-T loop on the other. These loops are critical for post-SELEX modification to improve TBA target affinity. With this goal in mind we synthesized a T analog, 5-(indolyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (W) to substitute one T or a pair of Ts. Subsequently, the affinity for each analog was determined by biolayer interferometry. An aptamer with W at position 4 exhibited about 3-fold increased binding affinity, and replacing both T4 and T12 with W afforded an almost 10-fold enhancement compared to native TBA. To better understand the role of the substituent's aromatic moiety, an aptamer with 5-(methyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (K; W without the indole moiety) in place of T4 was also synthesized. This K4 aptamer was found to improve affinity 7-fold relative to native TBA. Crystal structures of aptamers with T4 replaced by either W or K bound to thrombin provide insight into the origins of the increased affinities. Our work demonstrates that facile chemical modification of a simple DNA aptamer can be used to significantly improve its binding affinity for a well-established pharmacological target protein.

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Figures

Figure 1.
Figure 1.
(A) Guanine quadruplex structure adopted by TBA. In this study, T4 was substituted by W or K (X4). (B) Chemical structure of 5-(indolyl-3-acetyl-3-amino-1-propenyl)-2′-deoxyuridine (W). (C) Chemical structure of 5-(methyl-3-acetyl-3-amino-1-propenyl)-2′-deoxyuridine (K).
Figure 2.
Figure 2.
Synthesis of W phosphoramidite building block 3.
Figure 3.
Figure 3.
CD spectra of TBA (solid line), T4K (dashed line) and T4W (dotted line) at ca. 4 μM concentration in a 10 mM potassium phosphate buffer (KH2PO4/K2HPO4, pH 7.0) with 70 mM KCl.
Figure 4.
Figure 4.
Overall structures of the α-thrombin complexes with (A) T4W (PDB ID: 6EO6; this work), (B) T4K (PDB ID: 6EO7; this work) and (C) TBA (PDB ID: 1hao). Only a portion of the thrombin molecule (gray ribbon) is shown in the panels, and aptamer molecules are colored by atom, with carbon atoms of T4W, T4K and TBA colored in tan, light-blue and pink, respectively. The modified nucleotide W is highlighted in yellow, the modified nucleotide K is highlighted in magenta and selected residues are labeled.
Figure 5.
Figure 5.
Fourier 2Fo–Fc sum electron density contoured at the 1.0σ level around the potassium ion (indicated as a purple sphere) coordinated between aptamer G-quartets, and around the closest water molecules (indicated as red spheres) in (A) the α-thrombin:T4W and (B) the α-thrombin:T4K complex structures. Potassium coordination and water H bonds are depicted as dashed lines in black and gray, respectively.
Figure 6.
Figure 6.
Comparison between the T4W- and T4K-thrombin interfaces. Close-up view of the overlaid T4W:thrombin (W4 carbon atoms are colored in yellow, remaining residues and thrombin backbone ribbon and side chain carbons are tan) and T4K:thrombin complex binding regions (carbon atoms are colored in magenta, remaining residues and thrombin backbone ribbon and side chain carbons are light-blue). Important side chains that interact with the modified residue in one (Arg-436…K4 H-bond) or both structures (Tyr-434 and Ile-441 engage in stacking with T3 and W4) are highlighted in green. Arrows indicate key interactions between W4 or K4 and thrombin side chains, and H-bonds that occur in both the T4W and T4K complex structures are depicted as thin black lines.
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