Jain Priyamvada, Chakma Babina, Singh Naveen Kumar, Patra Sanjukta, Goswami Pranab
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India.
Mol Biotechnol. 2016 Jul;58(7):497-508. doi: 10.1007/s12033-016-9946-x.
A novel ssDNA aptamer (P38), with a 40 mer random region flanked by primer-binding sites on both sides, targeting Plasmodium falciparum lactate dehydrogenase (PfLDH) has been developed through systematic evolution of ligands by exponential enrichment (SELEX), including counter SELEX against human lactate dehydrogenase A and B (hLDH A and B). The 2D structure of P38 shows the presence of three stem loops with a δG of -9.18 kcal/mol. EMSA studies on P38 complexes with the increasing concentration of PfLDH revealed a dissociation constant of 0.35 µM. P38 has been exploited for the quantitative detection of PfLDH using cationic surfactant-mediated aggregation of gold nanoparticles (16-nm diameter) as an optical probe. Among the three different cationic surfactants, characterized by different hydrocarbon tail groups, benzalkonium chloride (BCK) was found to be most efficient with a limit of detection of 281 ± 11 pM. This BCK-based approach with the novel highly selective aptamer provides simple and sensitive detection of PfLDH in the clinically relevant range.
一种新型单链DNA适配体（P38）已通过指数富集配体系统进化技术（SELEX）开发出来，它两侧的引物结合位点之间有一个40个碱基的随机区域，靶向恶性疟原虫乳酸脱氢酶（PfLDH），包括针对人乳酸脱氢酶A和B（hLDH A和B）的反SELEX。P38的二维结构显示存在三个茎环，自由能变化为-9.18千卡/摩尔。对P38与浓度不断增加的PfLDH形成的复合物进行的电泳迁移率变动分析（EMSA）研究显示解离常数为0.35微摩尔。P38已被用于利用阳离子表面活性剂介导的16纳米直径金纳米颗粒聚集作为光学探针来定量检测PfLDH。在三种以不同烃基尾基团为特征的不同阳离子表面活性剂中，发现苯扎氯铵（BCK）效率最高，检测限为281±11皮摩尔。这种基于BCK的方法与新型高选择性适配体相结合，可在临床相关范围内对PfLDH进行简单而灵敏的检测。
