Abstract：
Nucleic acid aptamers are of great potentials in diagnostic and therapeutic applications because of their unique molecular recognition capabilities. However, satisfactory aptamers with high affinity and specificity are still in short supply. Herein, we have developed new selection methods allowing the free interactions between the targets and potential aptamers in solution. In our selection system, the protein targets (biotinylated randomly or site-specifically) were first incubated with the random DNA library, followed by the pull-down with the streptavidin magnetic beads or biolayer-interferometry (BLI) sensors. By comparing the two biotinylation strategies (random or site-specific) and two states of the targets (free or immobilized), we have found that the combination of the site-specific biotinylation and free-target strategies was most successful. Based on these highly-efficient selection strategies, HPV L1 aptamers were obtained. By designing the sandwich aptasensor assisted with RCA and CRISPR/Cas12a, we have diagnosed various HPV subtypes in clinical samples, such as easily-collected urine samples. In summary, our new strategy can allow efficient selection of aptamers with high affinity and specificity for clinical applications.
摘要：
核酸适体由于其独特的分子识别能力而在诊断和治疗应用中具有巨大的潜力。然而,具有高亲和力和特异性的令人满意的适体仍然短缺。在这里，我们已经开发了新的选择方法，允许靶标和溶液中潜在的适体之间的自由相互作用。在我们的选拔系统中，首先将蛋白质靶标（随机或位点特异性生物素化）与随机DNA文库一起孵育，然后用链霉亲和素磁珠或生物层干涉(BLI)传感器进行下拉。通过比较两种生物素化策略（随机或位点特异性）和靶标的两种状态（游离或固定），我们发现位点特异性生物素化和无靶点策略的组合是最成功的.基于这些高效的选择策略，获得HPVL1适体。通过设计RCA和CRISPR/Cas12a辅助的三明治aptasensor，我们在临床样本中诊断出各种HPV亚型，例如容易收集的尿液样本。总之,我们的新策略可以有效选择具有高亲和力和特异性的适体，用于临床应用。
