凝血酶
化学
等温滴定量热法
胸腺嘧啶
寡核苷酸
适体
碱基
G-四倍体
立体化学
DNA
生物化学
生物物理学
分子生物学
生物
血小板
免疫学
作者
Satoru Nagatoishi,Noburu Isono,Kouhei Tsumoto,Naoki Sugimoto
出处
期刊:Biochimie
[Elsevier]
日期:2011-08-01
卷期号:93 (8): 1231-1238
被引量:83
标识
DOI:10.1016/j.biochi.2011.03.013
摘要
The thrombin-binding DNA aptamer (TBA) 5′-d(GGTTGGTGTGGTTGG)-3′ forms a G-quadruplex that is necessary for binding to the coagulation factor thrombin. The stability of the G-quadruplex of TBA when bound to thrombin and potassium ion (K+) were investigated for the wild-type oligonucleotide and for mutants in which thymine residues were substituted by adenine. In the presence of thrombin, G-quadruplexes formed by oligonucleotides in which the fourth or thirteenth residues were changed (T4A and T13A, respectively) were more unstable than that of wild-type, whereas T3A, T7A, T9A and T12A were more stable. The opposite effect was observed in the presence of 100 mM K+: the G-quadruplexes formed by T4A and T13A were more stable and T3A, T7A, T9A and T12A were more unstable than that of wild-type. Isothermal titration calorimetry measurements indicated that the binding constant of the interaction between T3A, T7A, T9A and T12A mutants and thrombin at 25 °C were close to that of wild-type, whereas T13A was significantly lower and T4A did not appear to bind to thrombin. Therefore, the stabilization of the G-quadruplex structure of TBA by thrombin appears to be due to an interaction between certain thymine nucleobases rather than to the quadruplex structure. The present study demonstrates that thrombin stabilizes the G-quadruplex via the interaction with residues in the loops but not via direct stabilization of G-quartets.
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