From Molecular Diagnostics to Therapeutics with Aptamers

生物分析化学和生物分离领域最苛刻和最具挑战性的任务之一是用人工竞争对手替代动物和杂交瘤制造的抗体，这些抗体生产成本高，保质期短，可以很容易地选择和以低成本以高纯度化学合成。适配体作为“合成抗体”具有巨大的潜力。这些小的单链DNA或RNA寡核苷酸可以以高亲和力和特异性结合各种不同的靶标，从小分子到蛋白质。它们是具有显著潜力的亲和配体，可以改变亲和探针领域，并取代抗体作为诊断和分离应用。我建议发展三个相关的研究主题：1)可转换适配体对细胞的亲和纯化，2)利用适配体对稀有细胞的检测和成像，以及3)利用适配体和质谱法发现稀有细胞生物标志物。***目前基于抗体的细胞分离和纯化方法不是最佳的，因为抗体对细胞活力和纯度有影响。在第一个研究主题中，我们将开发两种类型的DNA适体，它们以高亲和力特异性结合免疫受体和癌症干细胞受体。第一种适体是EDTA可转换的DNA适体，它在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后释放出纯净完整的细胞。第二种适体将是“冷”可切换的DNA适体，它在室温下选择性地与受体阳性细胞结合，并在0°C时释放它们，以产生无适体的细胞。***在第二个主题中，从肺腺癌术后组织中选择的DNA适配体将用于检测和计数罕见的癌细胞(*该项目在未来5年的联合活动将对新的核酸适配体体外进化技术的发展，以及新的生物检测和生物传感器的设计做出重大贡献

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 (*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.**