Abraham Kevin M, Roueinfar Mina, Ponce Alex T, Lussier Mia E, Benson Danica B, Hong Ka Lok
Department of Pharmaceutical Sciences, Nesbitt School of Pharmacy, and Department of Biology, College of Science and Engineering, Wilkes University, 84 W. South Street, Wilkes-Barre, Pennsylvania 18766, United States.
ACS Omega. 2018 Oct 31;3(10):13576-13583. doi: 10.1021/acsomega.8b01859. Epub 2018 Oct 19.
Atrazine is an herbicide that is widely used in crop production at about 70 million pounds per year in the United States. Its widespread use has led to contamination of groundwater and other aquatic systems. It has resulted in many serious environmental and human health issues. This study focuses on the identification and characterization of a single-stranded DNA (ssDNA) aptamer that binds to atrazine. In this study, a variation of the systematic evolution of ligands by exponential enrichment (SELEX) process was used to identify an aptamer, which binds to atrazine with high affinity and specificity. This SELEX focused on inducing the aptamer's ability to change conformation upon binding to atrazine, and stringent negative target selections. After 12 rounds of in vitro selection, the ssDNA aptamer candidate R12.45 was chosen and truncated to obtain a 46-base sequence. The binding affinity, specificity, and structural characteristics of this truncated candidate was investigated by using isothermal titration calorimetry, circular dichroism (CD) analysis, SYBR Green I (SG) fluorescence displacement assays, and gold nanoparticles (AuNPs) colorimetric assays. The truncated R12.45 candidate aptamer bound to atrazine with high affinity ( = 3.7 nM) and displayed low cross-binding activities on structurally related herbicides. In addition, CD analysis data indicated a target induced structural stabilization. Finally, SG assays and AuNPs assays showed nonconventional binding activities between the truncated R12.45 aptamer candidate and atrazine, which warrants future studies.
莠去津是一种除草剂，在美国每年约有7000万磅广泛用于作物生产。其广泛使用导致了地下水和其他水生系统的污染。它引发了许多严重的环境和人类健康问题。本研究聚焦于一种与莠去津结合的单链DNA（ssDNA）适配体的鉴定与表征。在本研究中，采用指数富集配体系统进化（SELEX）过程的一种变体来鉴定一种适配体，该适配体与莠去津具有高亲和力和特异性。这种SELEX着重诱导适配体在与莠去津结合时改变构象的能力，以及严格的阴性靶标筛选。经过12轮体外筛选，选择了单链DNA适配体候选物R12.45并进行截短以获得一个46个碱基的序列。通过等温滴定量热法、圆二色性（CD）分析、SYBR Green I（SG）荧光位移测定法和金纳米颗粒（AuNP）比色测定法研究了这种截短候选物的结合亲和力、特异性和结构特征。截短的R12.45候选适配体与莠去津具有高亲和力（Kd = 3.7 nM），并且对结构相关的除草剂表现出低交叉结合活性。此外，CD分析数据表明靶标诱导的结构稳定。最后，SG测定法和AuNP测定法显示截短的R12.45适配体候选物与莠去津之间存在非常规的结合活性，这值得未来进一步研究。
