Title
Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity
Author
Rafal Dolot, Curtis H Lam, Malgorzata Sierant, Qiang Zhao, Feng-Wu Liu, Barbara Nawrot, Martin Egli, Xianbin Yang
Year
2018
Journal
Nucleic Acids Research
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.
Instrument
J-815
Keywords
Circular dichroism, DNA structure, Biochemistry