School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Wenhua West Road, 2#, Weihai, 264209, People's Republic of China.
School of Environment, Harbin Institute of Technology, Harbin, 150001, People's Republic of China.
Mar Biotechnol (NY). 2023 Dec;25(6):935-950. doi: 10.1007/s10126-023-10251-2. Epub 2023 Sep 25.
Among all the paralytic shellfish toxins (PSTs)-producing algae, Alexandrium tamarense is one of the most widespread harmful species posing a serious threat to marine resources and human health. Therefore, it is extremely important to establish a rapid and accurate monitoring method for A. tamarense that can provide early warnings of harmful algal blooms (HABs) caused by this alga and limit the contamination due to PSTs. In this study, an ssDNA library was first obtained by whole cell systematic evolution of ligands by exponential enrichment after 18 consecutive rounds of iterative screening. After sequencing in combination with subsequent multiple alignment of sequences and secondary structure simulation, the library could be classified into 2 families, namely, Family1 and Family2, according to sequence similarity. Flow cytometry was used to test the affinity and cross-reactivity of Ata19, Ata6, Ata25 and Ata29 belonging to Family2. Ata19 was selected to be modified by truncation, through which a new resultant aptamer named as Ata19-1-1 was obtained. Ata19-1-1 with a K of 75.16 ± 11.10 nM displayed a much higher affinity than Ata19. The specificity test showed that Ata19-1-1 has the same discrimination ability as Ata19 and can at least distinguish the target microalga from other microalgae. The observation under a fluorescence microscopy showed that the A. tamarense cells labeled with Ata19-1-1 are exhibiting bright green fluorescence and could be easily identified, factually confirming the binding of the aptamer with target cells. In summary, the aptamer Ata19-1-1 produced in this study may serve as an ideal molecular recognition element for A. tamarense, which has the potential to be developed into a novel detection method for this harmful alga in the future.
在所有产生麻痹性贝类毒素（PSTs）的藻类中，塔玛亚历山大藻是分布最广泛的有害物种之一，对海洋资源和人类健康构成严重威胁。因此，建立一种快速、准确的塔玛亚历山大藻监测方法极为重要，该方法可以对该藻类引起的有害藻华（HABs）进行早期预警，并限制 PSTs 的污染。本研究中，通过对细胞进行 18 轮连续的指数富集配体系统进化（SELEX）筛选，首次获得了 ssDNA 文库。经过测序，并对序列进行多次比对和二级结构模拟，根据序列相似性，文库可分为 2 个家族，即 Family1 和 Family2。流式细胞术用于测试属于 Family2 的 Ata19、Ata6、Ata25 和 Ata29 的亲和力和交叉反应性。通过对 Ata19 进行截断修饰，得到了一个新的适体，命名为 Ata19-1-1。Ata19-1-1 的 K 值为 75.16 ± 11.10 nM，显示出比 Ata19 更高的亲和力。特异性测试表明，Ata19-1-1 具有与 Ata19 相同的识别能力，至少可以区分目标微藻和其他微藻。荧光显微镜观察表明，用 Ata19-1-1 标记的塔玛亚历山大藻细胞呈现出明亮的绿色荧光，很容易识别，实际上证实了适体与靶细胞的结合。综上所述，本研究中产生的适体 Ata19-1-1 可能成为塔玛亚历山大藻的理想分子识别元件，有望成为未来该有害藻类的一种新型检测方法。
