Construction, stability, and activity of multivalent circular anticoagulant aptamers
Construction, stability, and activity of multivalent circular anticoagulant aptamers
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DOI：
10.1074/jbc.m408037200
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发表时间：
2004-11-05
影响因子：
4.8
通讯作者：
King, GC
中科院分区：
文献类型：
--
作者：
Di Giusto, DA;King, GC
在这里，我们描述了多价环状DNA适体的设计和构建。四个适体结合基序针对血液传播的目标被用作一个模型集，从其中较大的，多结构域的适体构建在一个简单的方式。前体寡核苷酸的分子内或分子间连接提供了一种稳定机制，以抵抗血液制品的主要外切核酸酶活性的降解，而不需要选择后化学修饰。在许多情况下，环状DNA适体在血清和血浆中的半衰期延长超过10小时，使得这样的构建体对于治疗和诊断应用是可行的。双链体和三通接头用作支架结构，围绕其可以以结构上限定的方式排列两个、三个或四个适体基序，通过稳定性和亲合力增益产生改善的结合特性。靶向凝血酶的环状适体显示出改善的抗凝效力，其EC 50值比典型GS-522凝血酶DNA适体的EC 50值好2-3倍。固有双链体区域提供了掺入额外的转录因子结合基序的机会，而辅助环可用于提供进一步的功能。这些抗凝适体提供了一个起点，融合的原则，DNA纳米技术与适体功能。
Here we describe the design and construction of multivalent circular DNA aptamers. Four aptameric binding motifs directed at blood-borne targets are used as a model set from which larger, multidomain aptamers are constructed in a straightforward manner. Intra- or intermolecular ligation of precursor oligonucleotides provides a stabilizing mechanism against degradation by the predominant exonuclease activity of blood products without the need for post-selection chemical modification. In many cases, circular DNA aptamer half-lives are extended beyond 10 h in serum and plasma, making such constructs viable for therapeutic and diagnostic applications. Duplexes and three-way junctions are used as scaffold architectures around which two, three, or four aptameric motifs can be arranged in a structurally defined manner, giving rise to improved binding characteristics through stability and avidity gains. Circular aptamers targeted against thrombin display improved anticoagulant potency with EC50 values 2-3-fold better than those of the canonical GS-522 thrombin DNA aptamer. Intrinsic duplex regions provide an opportunity to incorporate additional transcription factor binding motifs, whereas ancillary loops can be used to provide further functionality. These anticoagulant aptamers provide a starting point for merging the principles of DNA nanotechnology with aptameric functions.