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

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.

SUBMITTER: Dolot R

PROVIDER: S-EPMC5961234 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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

Dolot Rafal R Lam Curtis H CH Sierant Malgorzata M Zhao Qiang Q Liu Feng-Wu FW Nawrot Barbara B Egli Martin M Yang Xianbin X

Nucleic acids research 20180501 9

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 biolay ...[more]

PMID: 29684204

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Project description:This work presents a comprehensive analysis of the biodegradation of polyhydroxybutyrate (PHB) and chemically modified PHB with different chemical and crystal structures in a soil environment. A polymer modification reaction was performed during preparation of the chemically modified PHB films, utilizing 2,5-dimethyl-2,5-di(tert-butylperoxy)-hexane as a free-radical initiator and maleic anhydride. Films of neat PHB and chemically modified PHB were prepared by extrusion and thermocompression. The biological agent employed was natural mixed microflora in the form of garden soil. The course and extent of biodegradation of the films was investigated by applying various techniques, as follows: a respirometry test to determine the production of carbon dioxide through microbial degradation; scanning electron microscopy (SEM); optical microscopy; fluorescence microscopy; differential scanning calorimetry (DSC); and X-ray diffraction (XRD). Next-generation sequencing was carried out to study the microbial community involved in biodegradation of the films. Findings from the respirometry test indicated that biodegradation of the extruded and chemically modified PHB followed a multistage (2-3) course, which varied according to the spatial distribution of amorphous and crystalline regions and their spherulitic morphology. SEM and polarized optical microscopy (POM) confirmed that the rate of biodegradation depended on the availability of the amorphous phase in the interspherulitic region and the width of the interlamellar region in the first stage, while dependence on the size of spherulites and thickness of spherulitic lamellae was evident in the second stage. X-ray diffraction revealed that orthorhombic α-form crystals with helical chain conformation degraded concurrently with β-form crystals with planar zigzag conformation. The nucleation of PHB crystals after 90 days of biodegradation was identified by DSC and POM, a phenomenon which impeded biodegradation. Fluorescence microscopy evidenced that the crystal structure of PHB affected the physiological behavior of soil microorganisms in contact with the surfaces of the films.

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

Publications

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

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