Ferreira C S M, Matthews C S, Missailidis S
Chemistry Department, The Open University, Milton Keynes, MK7 6AA, UK.
Tumour Biol. 2006;27(6):289-301. doi: 10.1159/000096085. Epub 2006 Oct 9.
Agents able to bind tightly and selectively to disease markers can greatly benefit disease diagnosis and therapy. Aptamers are functional molecules, usually DNA or RNA oligonucleotides, with the appropriate sequence and structure to form a complex with a target molecule. MUC1 is a well-known tumour marker present in a variety of malignant tumours and it has been a target of interest for many years. In this work we report the selection of DNA aptamers that bind with high affinity and selectivity to the MUC1 peptides. Combinatorial chemistry techniques based on the SELEX methodology were used for the identification of the specific aptamers. These were selected from an initial library containing a 25-base-long variable region, resulting in 4(25) random sequences of single-stranded DNA molecules, for their ability to bind to synthetic forms of MUC1. Ten rounds of in vitro selection were performed enriching for MUC1 binding. By round ten more than 90% of the pool of sequences consisted of MUC1-binding molecules. Selected aptamer families were cloned, sequenced and found to be unique, sharing no sequence consensus. The binding properties of these aptamers were quantitated by enzyme-linked immunosorbent assay and surface plasmon resonance, whereas their specificity for MUC1-expressing cancer cells has been validated using fluorescent microscopy. Aptamers offer significant advantages over existing antibody-based recognition procedures in that they offer higher binding affinity (higher retention/reduced dissociation) and specificity to the target (ability to determine variations on the protein target down to single amino acid changes), higher selectivity against mutated protein epitopes and potentially reduced immunogenicity and increased tumour penetration associated with their size.
能够紧密且选择性地结合疾病标志物的试剂对疾病诊断和治疗大有裨益。适体是功能性分子，通常为DNA或RNA寡核苷酸，具有合适的序列和结构以与靶分子形成复合物。MUC1是一种在多种恶性肿瘤中存在的著名肿瘤标志物，多年来一直是人们感兴趣的靶点。在这项工作中，我们报告了与MUC1肽具有高亲和力和选择性结合的DNA适体的筛选。基于SELEX方法的组合化学技术用于鉴定特异性适体。这些适体是从一个包含25个碱基长可变区的初始文库中筛选出来的，该文库产生了4(25)个单链DNA分子的随机序列，筛选依据它们与合成形式的MUC1结合的能力。进行了十轮体外筛选以富集与MUC1结合的分子。到第十轮时，超过90%的序列库由与MUC1结合的分子组成。对选定的适体家族进行克隆、测序，发现它们是独特的，没有序列共识。通过酶联免疫吸附测定和表面等离子体共振对这些适体的结合特性进行了定量，而它们对表达MUC1的癌细胞的特异性已通过荧光显微镜验证。与现有的基于抗体的识别方法相比，适体具有显著优势，因为它们对靶标具有更高的结合亲和力（更高的保留率/更低的解离率）和特异性（能够确定蛋白质靶标上直至单个氨基酸变化的变异），对突变的蛋白质表位具有更高的选择性，并且可能具有更低的免疫原性以及因其尺寸而增加的肿瘤穿透性。
