Optimization and modifications of aptamers selected from live cancer cell lines
Optimization and modifications of aptamers selected from live cancer cell lines
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DOI：
10.1002/cbic.200600532
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发表时间：
2007-04-16
期刊：
影响因子：
3.2
通讯作者：
Tan, Weihong
中科院分区：
文献类型：
--
作者：
Shangguan, Dihua;Tang, Zhiwen;Tan, Weihong
适体是源自称为SELEX（通过指数富集的配体系统进化）的体外进化过程的单链寡核苷酸。[1，2]选择的适体可以通过折叠成明确定义的三维形状以高亲和力和特异性识别其靶分子。[3，4]最近，使用人T细胞急性淋巴细胞白血病细胞系作为靶标，我们产生了一组用于特异性识别白血病细胞的适体。适体具有nM至pM范围内的平衡解离常数（Kd）。它们可以特异性识别与正常人骨髓穿刺液混合的靶白血病细胞，可以识别临床标本中与靶细胞系密切相关的癌细胞，也可以用于富集血液样本中掺入的靶细胞。[5，6]这些适体在各种应用中非常有希望用于分子识别。与抗体不同，适体具有低分子量，快速组织渗透和低毒性，并且可以用DNA合成仪重复生产。[7-9]它们可以很容易地用放射性、荧光或其他报告物标记。此外，适体在长期储存期间保持稳定并维持可逆变性。[10]这些优点使得适体特别适合作为诊断的分子探针或作为靶向癌症治疗的药物。尽管常规DNA分子在体外比抗体更坚固，但体内稳定性仍然是一个严重的问题。为了作为有效的治疗和诊断工具，适体必须抵抗核酸外切酶和核酸内切酶的快速降解。在本文中，我们提出的实验结果设计的适体具有良好的生物稳定性，亲和力和特异性的疾病的研究。通过SELEX方法产生的全长适体含有70-100个核苷酸，其在每个末端包括两个用于PCR扩增的固定引物序列。通常，并非所有核苷酸都是与靶标直接相互作用或折叠成促进靶标结合的结构所必需的。[10]毫无疑问，较长的序列导致合成中较低的产率和较高的成本。不必要的nu-
Aptamers are single-stranded oligonucleotides derived from an in vitro evolution process called SELEX (systematic evolution of ligands by exponential enrichment).[1, 2] The selected aptamers can recognize their target molecules with high affinity and specificity by folding into well-defined three-dimensional shapes.[3, 4] Recently, using a human T-cell acute lymphoblastic leukemia cell line as target, we generated a group of aptamers for the specific recognition of leukemia cells. The aptamers have an equilibrium dissociation constant (Kd) in the nM to pM range. They can specifically recognize target leukemia cells that have been mixed with normal human bone marrow aspirates, can identify cancer cells closely related to the target cell line in clinical specimens, and can also be used to enrich target cells spiked in blood samples.[5, 6] These aptamers are very promising for molecular recognition in a variety of applications.Unlike antibodies, aptamers have low molecular weight, fast tissue penetration, and low toxicity, and can be reproducibly produced with a DNA synthesizer.[7–9] They can be easily labeled with radioscopic, fluorescent, or other reporters. Moreover, aptamers remain stable during long-term storage and sustain reversible denaturation.[10] These advantages make aptamers uniquely suitable as molecular probes for diagnosis or as drugs for targeted cancer therapy. Even though regular DNA molecules are sturdier than antibodies in vitro, in vivo stability is still a serious problem. To serve as effective therapeutic and diagnostic tools, aptamers must resist rapid degradation by exo-and endonucleases. In this paper, we present the results of experiments for the design of aptamers with excellent biostability, affinity, and specificity for the study of diseases. Full-length aptamers generated by the SELEX process contain 70–100 nucleotides, which include two fixed primer sequences at each terminus for PCR amplification. Generally, not all nucleotides are necessary for direct interaction with the target or for folding into the structure that facilitates target binding.[10] There is no doubt that longer sequences result in lower yield and higher cost in synthesis. The unnecessary nu-