基于单链DNA浓度法的哈维氏弧菌核酸适配体亲和特异性研究

本研究采用核酸适配体的单链DNA浓度来表征其亲和力,通过测定核酸适配体与靶细菌哈维氏弧菌(Vibrio harveyi)结合后ssDNA的浓度,来研究该核酸适配体的亲和特异性和亲和常数,并通过荧光显微镜法对其亲和特异性进行验证。结果显示,采用单链DNA浓度法测得该核酸适配体对靶细菌哈维氏弧菌的亲和力是非目标菌的15.2倍以上;荧光显微镜直接观察发现只有靶细菌能较好结合有荧光标记的核酸适配体,并呈现明显荧光;荧光阻断法发现靶细菌和非靶细菌都未呈现明显荧光,证明了该适配体有较好的亲和特异性,也进一步验证了单链DNA法的测定结果。在亲和常数的测定方面,利用单链DNA浓度法测得该核酸适配体的亲和常数K_d=(33.70±7.83) nmol/L,相应的拟合系数为R~2=0.960,有较好的准确性和可靠性,说明采用单链DNA浓度法来测定适配体的亲和力和亲和常数是可行的。

Affinity and specificity of aptamer against Vibrio harveyi based on its ssDNA concentration

Aptamers are oligonucleotide molecules obtained by systematic evolution of ligands by exponential enrichment, which have a high affinity and specificity towards their target. Aptamers are applied in many fields, such as life science research, drug screening, target identification, and environmental monitoring. Affinity determination of an aptamer and its specificity are necessary for the research and development of the aptamer. Therefore, a novel affinity determination of aptamer, called ssDNA concentration method, was developed to study the specificity and affinity constant of an aptamer against . The affinity and affinity constant of the aptamer were determined by measuring the ssDNA concentration of the aptamer binding to the V. harveyi detected by the ssDNA concentration method, was 15.2 times higher than that of the non-target bacteria. The affinity and specificity of the aptamer were also verified by fluorescence microscopy. There are two main methods of fluorescence microscopy, the direct observation method and the fluorescence blocking method. The direct observation method is observing the binding of aptamer and target bacteria under fluorescent microscope after bacteria were incubated with fluorescent labeled aptamer. The results verified by the direct observation method under fluorescence microscopy showed that the target bacteria emitted significant yellow-red fluorescence after binding with the FAM-labeled aptamer, but little fluorescence was found in other four kinds of non-target bacteria (Escherichia coli), proving that the bacteria have no spontaneous fluorescence. Furthermore, the fluorescence blocking method involves incubating bacteria with no fluorescent label aptamer first, followed by incubating with fluorescently labeled aptamer, and observing whether the fluorescence is blocked under the microscope. We found that all bacteria including the target bacteria had no significant fluorescence. These results of the fluorescence blocking method proved that the aptamer had an excellent affinity and specificity towards the target bacteria , which also verified the determination of the ssDNA concentration method. The affinity constant () of the aptamer measured by the ssDNA concentration method is equal to 33.70±7.83 nmol/L, and its corresponding fitting coefficient is equal to 0.960, which showed that the ssDNA concentration method is accurate, reliable, and feasible in the determination of affinity and affinity constant of an aptamer. The ssDNA concentration method used in this study is practical due to the simple equipment, short measurement time, and convenient operation.