Mulholland Catherine, Jestřábová Ivana, Sett Arghya, Ondruš Marek, Sýkorová Veronika, Manzanares C Lorena, Šimončík Oliver, Muller Petr, Hocek Michal
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000, Prague 6, Prague, Czech Republic.
Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, Prague 2, Prague, 12843, Czech Republic.
Commun Chem. 2023 Apr 6;6(1):65. doi: 10.1038/s42004-023-00862-0.
Nucleic acids aptamers often fail to efficiently target some proteins because of the hydrophilic character of the natural nucleotides. Here we present hydrophobic 7-phenylbutyl-7-deaadenine-modified DNA aptamers against the Heat Shock Protein 70 that were selected via PEX and magnetic bead-based SELEX. After 9 rounds of selection, the pool was sequenced and a number of candidates were identified. Following initial screening, two modified aptamers were chemically synthesised in-house and their binding affinity analysed by two methods, bio-layer interferometry and fluorescent-plate-based binding assay. The binding affinities of the modified aptamers were compared with that of their natural counterparts. The resulting modified aptamers bound with higher affinity (low nanomolar range) to the Hsp70 than their natural sequence (>5 µM) and hence have potential for applications and further development towards Hsp70 diagnostics or even therapeutics.
由于天然核苷酸的亲水性，核酸适配体常常无法有效地靶向某些蛋白质。在此，我们展示了通过指数富集的配体系统进化（PEX）和基于磁珠的指数富集配体系统进化技术（SELEX）筛选得到的针对热休克蛋白70（Heat Shock Protein 70）的疏水7-苯基丁基-7-脱氮腺嘌呤修饰的DNA适配体。经过9轮筛选后，对文库进行测序并鉴定出多个候选适配体。经过初步筛选，在内部化学合成了两种修饰的适配体，并通过生物层干涉术和基于荧光板的结合测定这两种方法分析了它们的结合亲和力。将修饰适配体的结合亲和力与其天然对应物的结合亲和力进行了比较。所得修饰适配体与热休克蛋白70的结合亲和力更高（低纳摩尔范围），高于其天然序列（>5µM），因此在热休克蛋白70诊断甚至治疗的应用和进一步开发方面具有潜力。
