Zasedateleva Olga A, Vasiliskov Vadim A, Surzhikov Sergey A, Sazykin Alexey Y, Putlyaeva Lidiya V, Schwarz Anton M, Kuprash Dmitry V, Rubina Alla Y, Barsky Victor E, Zasedatelev Alexander S
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
Biotechnol J. 2014 Aug;9(8):1074-80. doi: 10.1002/biot.201300556. Epub 2014 Jul 9.
Microarrays allow for the simultaneous monitoring of protein interactions with different nucleic acid (NA) sequences immobilized in microarray elements. Either fluorescently labeled proteins or specific fluorescently labeled antibodies are used to study protein-NA complexes. We suggest that protein-NA interactions on microarrays can be analyzed by ultraviolet (UV) fluorescence of tryptophan residues in the studied proteins, and this approach may eliminate the protein-labeling step. A specialized UV microscope was developed to obtain fluorescent images of microarrays in the UV wavelengths and to measure the fluorescence intensity of individual microarray elements. UV fluorescence intensity of BSA immobilized in microarray gel elements increased linearly with increased BSA amount with sensitivity of 0.6 ng. Real-time interaction curves between the DNA-binding domain of the NFATc1 transcription factor (NFATc1-DBD) and synthetic hairpin-forming oligodeoxyribonucleotides immobilized within 0.2 nL microarray gel elements at a concentration 5 × 10(-5) M and higher were obtained. The UV fluorescence intensities of microarray gel elements containing NFATc1-DBD-DNA complexes at equilibrium allowed the estimation of the equilibrium binding constant for complex formation. The developed method allows the protein-NA binding to be monitored in real time and can be applied to assess the sequence-specific affinity of NA-binding proteins in parallel studies involving many NA sequences.
微阵列可同时监测蛋白质与固定在微阵列元件中的不同核酸（NA）序列的相互作用。使用荧光标记的蛋白质或特异性荧光标记的抗体来研究蛋白质-NA复合物。我们认为，微阵列上的蛋白质-NA相互作用可以通过所研究蛋白质中色氨酸残基的紫外（UV）荧光进行分析，这种方法可能省去蛋白质标记步骤。开发了一种专门的紫外显微镜，用于获取微阵列在紫外波长下的荧光图像，并测量各个微阵列元件的荧光强度。固定在微阵列凝胶元件中的牛血清白蛋白（BSA）的紫外荧光强度随BSA量的增加呈线性增加，灵敏度为0.6 ng。获得了NFATc1转录因子的DNA结合结构域（NFATc1-DBD）与固定在0.2 nL微阵列凝胶元件中、浓度为5×10⁻⁵ M及更高的合成发夹状寡脱氧核糖核苷酸之间的实时相互作用曲线。平衡时含有NFATc1-DBD-DNA复合物的微阵列凝胶元件的紫外荧光强度可用于估算复合物形成的平衡结合常数。所开发的方法能够实时监测蛋白质-NA结合，可应用于在涉及许多NA序列的平行研究中评估NA结合蛋白的序列特异性亲和力。
