Abstract:
Numerous aptamers against various targets have been identified through the technology of systematic evolution of ligands by exponential enrichment (SELEX), but the affinity of these aptamers are often insufficient due to the limitations of SELEX. Therefore, a more rational in silico screening strategy (ISS) was developed for efficient screening of high affinity aptamers, which took shape complementarity and thermodynamic stability into consideration. Neuron specific enolase (NSE), a tumor marker, was selected as the target molecule. In the screening process, three aptamer candidates with good shape complementarity, lower ΔG values, and higher ZDOCK scores were produced. The dissociation constant (Kd) of these candidates to NSE was determined to be 10.13 nM, 14.82 nM, and 2.76 nM, respectively. Each of them exhibited higher affinity to NSE than the parent aptamer (Kd = 23.83 nM). Finally, an antibody-free fluorescence aptasensor assay, based on the aptamer with the highest affinity, P-5C8G, was conducted, resulting in a limit of detection (LOD) value of 1.8 nM, which was much lower than the parental aptamer (P, LOD = 12.6 nM). The proposed ISS approach provided an efficient and universal strategy to improve the aptamer to have a high affinity and good analytical utility.
摘要:
通过指数富集(SELEX)配体的系统进化技术,已经鉴定了许多针对各种靶标的适体,但是由于SELEX的限制,这些适体的亲和力通常不足。因此,开发了一种更合理的计算机筛选策略(ISS),用于高效筛选高亲和力适体,考虑了形状互补性和热力学稳定性。神经元特异性烯醇化酶(NSE),肿瘤标志物,被选为目标分子。在筛选过程中,三个具有良好形状互补性的适体候选物,较低的ΔG值,并且产生了更高的ZDOCK分数。这些候选物对NSE的解离常数(Kd)测定为10.13nM,14.82nM,和2.76nM,分别。它们中的每一个对NSE表现出比亲本适体更高的亲和力(Kd=23.83nM)。最后,无抗体荧光aptasensor分析,基于具有最高亲和力的适体,P-5C8G,进行了,导致检测限(LOD)值为1.8nM,远低于亲本适体(P,LOD=12.6nM)。所提出的ISS方法提供了一种有效且通用的策略来改善适体以具有高亲和力和良好的分析实用性。