From Molecular Diagnostics to Therapeutics with Aptamers

生物分析化学和生物分离最困难和最具挑战性的任务之一是用人工竞争对手取代生产成本高、保质期短的动物和杂交瘤制造的抗体，这些竞争对手可以很容易地选择并以低成本高纯度化学合成抗体。适配子作为“合成抗体”具有巨大的潜力。这些小的单链DNA或RNA寡核苷酸可以高亲和力和特异性与从小分子到蛋白质的各种不同的靶标结合。它们是具有改变亲和探针领域和取代抗体作为诊断和分离应用的巨大潜力的亲和配体。我建议开展三个相关的研究主题：1)可切换适配子对细胞的亲和纯化，2)适配子对稀有细胞的检测和成像，3)利用适配子和质谱学发现稀有细胞生物标记物。在第一个研究主题中，我们将开发两种类型的DNA适配子，它们与免疫受体和癌症干细胞受体具有高亲和力。第一类适配子是EDTA可切换的DNA适配子，在Ca(II)或Mg(II)离子存在的情况下与细胞结合，在溶液中加入EDTA或EGTA后释放纯净和完整的细胞。第二类适体将是“冷”的可切换的DNA适体，在室温下选择性地与受体阳性细胞结合，并在0°C的温度下释放它们，产生无适配子的细胞。*在第二类主题中，从术后组织中为肺腺癌细胞选择的DNA适体将用于检测和计数血液中罕见的癌细胞(&lt；100个/毫升)。肿瘤循环肿瘤细胞(CTCs)、肿瘤干细胞(CSCs)等稀有细胞在生物体液中的计数和分子谱分析是检测和治疗癌症的一种新方法。这种肿瘤特异性适配子可以为个别患者生产，并在抗癌治疗中多次合成，从而为个性化诊断打开了可能性。*第三个主题将使用前两项技术的发现，如稀有细胞的适体，以及通过质谱仪识别新的蛋白质生物标记物的细胞分离程序。这一主题的重要特征是在发现生物标记物的同时产生合成亲和探针(适配子)。*该计划在未来5年的联合活动将对发展新的核酸适体体外进化技术、设计新的生物检测和生物传感器做出重大贡献。**

One of the most demanding and challenging tasks of bioanalytical chemistry and bioseparation is the replacement of animal- and hybridoma-made antibodies that have high production cost and short shelf life with artificial competitors that can be easily selected and chemically synthesized in high purity at low cost. Aptamers as "synthetic antibodies" have enormous potential. These small single-stranded DNA or RNA oligonucleotides can bind with high affinity and specificity to a variety of different targets ranging from small molecules to proteins. They are affinity ligands with significant potential to change the field of affinity probes and to replace antibodies as diagnostic and separation applications. I propose the development of three related research themes: 1) affinity purification of cells by switchable aptamers, 2) detection and imaging of rare cells by aptamers, and 3) discovery of rare cell biomarker by aptamers and mass spectrometry.***Current antibody-based approaches for cell isolation and purification are not optimal because of the antibody's effect on cell viability and purity. In the first research theme, we will develop two types of DNA aptamers that bind specifically with high affinity to immune receptors and cancer stem cell receptors. The first type of aptamers will be EDTA-switchable DNA aptamers that bind to cells in the presence of Ca(II) or Mg(II) ions, and release the pure and intact cells upon addition of EDTA or EGTA in solution. The second type of aptamers will be "cold" switchable DNA aptamers that selectively bind to receptor positive cells at room temperature and release them at 0°C to yield aptamer-free cells.***In the second theme, DNA aptamers selected for lung adenocarcinoma cells from postoperative tissues will be used for sensing and counting rare cancer cells (<100 cells/ml) in blood. The enumeration and molecular profiling of rare cells, such as cancer circulating tumour cells (CTCs) and cancer stem cell (CSCs) in biological fluids, is an emergent methodology for the detection and treatment of cancer. Such tumour specific aptamers can be produced for individual patients and synthesized many times during anticancer therapy, thereby opening up the possibility of personalized diagnostics. ***The third theme will use the findings of the first two technologies such as aptamers to rare cells, and a cell isolation procedure for identification of new protein biomarkers by mass spectrometry. The important feature of this theme is that it yields synthetic affinity probes (aptamers) simultaneously with biomarker discovery. ***The combined activities of this program over the next 5 years will make a significant contribution to the development of new in vitro evolution techniques for nucleic acid aptamers, and the design of new bioassays and biosensors.**