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镜像DNA聚合酶实现生物稳定L-DNA适配体的定向进化和选择
2022-06-12 22:46

近日,清华大学朱听及其研究组用镜像DNA聚合酶实现生物稳定L-DNA适配体的定向进化和选择。2022年6月6日,《自然—生物技术》杂志在线发表了这项成果。

研究人员开发了一个"镜像选择"方案,用镜像DNA聚合酶定向进化和选择生物稳定的L-DNA适配体。研究人员对结合人类凝血酶的L-DNA序列进行了反复的富集和镜像聚合酶链式反应(PCR)扩增,并结合变性梯度凝胶电泳(DGGE)分离出单个适配体和L-DNA合成测序以确定其序列。基于所选择的L-DNA诱导体,研究人员设计了生物稳定的凝血酶传感器和抑制剂,它们在生理上相关的核酸酶丰富的环境中保持功能,甚至在存在快速降解D-DNA诱导体的人类血清中也是如此。直接从大型随机L-DNA库中镜像选择生物稳定的L-DNA诱导体极大地扩展了可被靶向的生物分子的范围,并扩大了其作为生物稳定的传感器、治疗剂和基础研究工具的应用。

据了解,由手性颠倒的核酸制成的镜像适配体具有核酸酶抗性和特别的生物稳定性,为独特的应用提供了机会。然而,直接从大型随机L-DNA文库中定向进化和选择镜像适配体的做法以前还没有被证明。

附:英文原文

Title: Directed evolution and selection of biostable l-DNA aptamers with a mirror-image DNA polymerase

Author: Chen, Ji, Chen, Mengyin, Zhu, Ting F.

Issue&Volume: 2022-06-06

Abstract: 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.

DOI: 10.1038/s41587-022-01337-8

Source: https://www.nature.com/articles/s41587-022-01337-8

Nature Biotechnology:《自然—生物技术》,创刊于1996年。隶属于施普林格·自然出版集团,最新IF:68.164
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex


本期文章:《自然—生物技术》:Online/在线发表

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