Bolotnikov Grigory, Kissmann Ann-Kathrin, Gruber Daniel, Bellmann Andreas, Hasler Roger, Kleber Christoph, Knoll Wolfgang, Rosenau Frank
Institute of Pharmaceutical Biotechnology, Ulm University, 89081 Ulm, Germany.
Faculty of Medicine and Dentistry, Danube Private University, Steiner Landstraße 124, 3500 Krems an der Donau, Austria.
Biosensors (Basel). 2025 Jul 12;15(7):448. doi: 10.3390/bios15070448.
Shaking off the forgetfulness towards the methodological power of inosine-mediated error-prone PCR (epPCR), this study reintroduces an often-underappreciated method as a considerably powerful approach for generating aptamer libraries from a single decameric ATCG-repeat-oligonucleotide. The aim was to demonstrate that this simple way of creating sequence diversity was suitable for delivering functional starting libraries for a set of ten whole-cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) processes. This epPCR method uses inosine to introduce targeted mutations, avoiding the need for commercial oligo pools or large-scale synthesis. We applied this method to a "universal aptamer" and subjected the three resulting libraries to two rounds of selection against ten diverse targets including probiotic and pathogenic bacteria (Gram-negative and -positive) as well as human cell lines. The enriched aptamers exhibited new binding specificities, demonstrating that the approach supports functional selection. Much like dusting off an old tool and finding it perfectly suited for a modern task, this work shows that revisiting established techniques can address current challenges in aptamer development. Our main finding is that epPCR provides a robust, cost-effective strategy for generating starting libraries and lowers the barrier for initiating successful SELEX campaigns.
摆脱对肌苷介导的易错PCR（epPCR）方法学力量的遗忘，本研究重新引入了一种常被低估的方法，作为从单个十聚体ATCG重复寡核苷酸生成适体文库的相当强大的方法。目的是证明这种创建序列多样性的简单方法适用于为一组十个全细胞SELEX（指数富集配体系统进化）过程提供功能性起始文库。这种epPCR方法使用肌苷引入靶向突变，无需商业寡核苷酸池或大规模合成。我们将此方法应用于一个“通用适体”，并将所得的三个文库针对包括益生菌和病原菌（革兰氏阴性和阳性）以及人类细胞系在内的十个不同靶标进行两轮筛选。富集的适体表现出新的结合特异性，表明该方法支持功能筛选。就像拂去旧工具上的灰尘并发现它非常适合现代任务一样，这项工作表明重新审视既定技术可以应对适体开发中的当前挑战。我们的主要发现是，epPCR为生成起始文库提供了一种强大、经济高效的策略，并降低了启动成功的SELEX活动的障碍。
