Mirror-image aptamers made from chirally inverted nucleic acids are nuclease-resistant and exceptionally biostable, opening up opportunities for unique applications. However, the directed evolution and selection of mirror-image aptamers directly from large randomized l-DNA libraries has, to our knowledge, not been demonstrated previously. Here, we developed a ‘mirror-image selection’ scheme for the directed evolution and selection of biostable l-DNA aptamers with a mirror-image DNA polymerase. We performed iterative rounds of enrichment and mirror-image polymerase chain reaction (PCR) amplification of l-DNA sequences that bind native human thrombin, in conjunction with denaturing gradient gel electrophoresis (DGGE) to isolate individual aptamers and l-DNA sequencing-by-synthesis to determine their sequences. Based on the selected l-DNA aptamers, we designed biostable thrombin sensors and inhibitors, which remained functional in physiologically relevant nuclease-rich environments, even in the presence of human serum that rapidly degraded d-DNA aptamers. Mirror-image selection of biostable l-DNA aptamers directly from large randomized l-DNA libraries greatly expands the range of biomolecules that can be targeted, broadening their applications as biostable sensors, therapeutics and basic research tools.

由手性反转核酸制成的镜像适配体具有核酸酶抗性和出色的生物稳定性，为独特的应用开辟了机会。然而，据我们所知，直接从大型随机l- DNA 文库中直接进化和选择镜像适配体之前并未得到证实。在这里，我们开发了一种“镜像选择”方案，用于使用镜像 DNA 聚合酶定向进化和选择生物稳定的l -DNA 适体。我们对结合天然人凝血酶的l- DNA 序列进行了多轮富集和镜像聚合酶链反应 (PCR) 扩增，并结合变性梯度凝胶电泳 (DGGE) 来分离个体适体和l - DNA 合成测序以确定它们的序列。基于选定的l- DNA 适配体，我们设计了生物稳定的凝血酶传感器和抑制剂，即使在存在快速降解d - DNA 适配体的人血清的情况下，它们在生理相关的富含核酸酶的环境中也能保持功能。直接从大型随机l- DNA 文库中镜像选择生物稳定的l- DNA 适体极大地扩展了可靶向的生物分子的范围，拓宽了它们作为生物稳定传感器、治疗学和基础研究工具的应用。