Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity.
Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity.
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DOI：
10.1093/nar/gky268
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发表时间：
2018-05-18
影响因子：
14.9
通讯作者：
Yang X
中科院分区：
文献类型：
--
作者：
Dolot R;Lam CH;Sierant M;Zhao Q;Liu FW;Nawrot B;Egli M;Yang X
凝血酶结合适体（Thrombin-binding aptamer，TBA）是一种由5′-GGT TGG TGT GGT TGG-3′组成的15聚体DNA，它折叠成G-四链体结构，由位于一侧的两个T-T环和位于另一侧的T-G-T环连接。这些环对于后SELEX修饰以改善TBA靶亲和力是关键的。考虑到这一目标，我们合成了一个T类似物，5-（吲哚-3-乙酰基-3-氨基-1-丙烯基）-2′-脱氧尿苷（W），以取代一个T或一对T。随后，通过生物层干涉测量法测定对每种类似物的亲和力。与天然TBA相比，在位置4处具有W的适体表现出约3倍增加的结合亲和力，并且用W替换T4和T12两者提供了几乎10倍的增强。为了更好地理解取代基的芳香族部分的作用，还合成了用5-（甲基-3-乙酰基-3-氨基-1-丙烯基）-2′-脱氧尿苷（K; W，没有吲哚部分）代替T4的适体。发现该K4适体相对于天然TBA将亲和力提高7倍。用与凝血酶结合的W或K替换T4的适体的晶体结构提供了对增加的亲和力的起源的洞察。我们的工作表明，一个简单的DNA适体的简单的化学修饰可以用来显着提高其结合亲和力的一个完善的药理学靶蛋白。
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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影响因子：
3.5
作者：
Coppola, Teresa;Varra, Michela;Borbone, Nicola
通讯作者：
Borbone, Nicola
影响因子：
56.9
作者：
AGGARWAL, AK;RODGERS, DW;HARRISON, SC
通讯作者：
HARRISON, SC
DOI：
10.1073/pnas.1615475114
发表时间：
2017-03-14
影响因子：
11.1
作者：
Gawande BN;Rohloff JC;Carter JD;von Carlowitz I;Zhang C;Schneider DJ;Janjic N
通讯作者：
Janjic N
DOI：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Chen VB;Arendall WB 3rd;Headd JJ;Keedy DA;Immormino RM;Kapral GJ;Murray LW;Richardson JS;Richardson DC
通讯作者：
Richardson DC
影响因子：
2.5
作者：
Cianci M;Bourenkov G;Pompidor G;Karpics I;Kallio J;Bento I;Roessle M;Cipriani F;Fiedler S;Schneider TR
通讯作者：
Schneider TR