Department of Chemistry, University of Idaho, Moscow, ID-83844, USA.
Org Biomol Chem. 2021 Nov 3;19(42):9276-9290. doi: 10.1039/d1ob01888d.
Development of molecules capable of binding to specific sequences of double-stranded (ds) DNA continues to attract considerable interest, as this may yield useful tools for applications in life science, biotechnology, and medicine. We have previously demonstrated sequence-unrestricted of dsDNA using Invader probes, , DNA duplexes that are energetically activated through incorporation of +1 interstrand zipper arrangements of O2'-intercalator-functionalized RNA monomers. Nonetheless, recognition of extended dsDNA target regions remains challenging due to the high stability of the corresponding probes. To address this, we introduce toehold Invader probes, , Invader probes with 5'-single-stranded overhangs. This design provides access to probes with shortened double-stranded segments, which facilitates probe denaturation. The single-stranded overhangs can, furthermore, be modified with affinity-enhancing modifications like LNA (locked nucleic acid) monomers to additionally increase target affinity. Herein, we report the biophysical and dsDNA-targeting properties of different toehold Invader designs and compare them to conventional Invader probes. LNA-modified toehold Invader probes display promising recognition characteristics, including greatly improved affinity to dsDNA, excellent binding specificity, and fast recognition kinetics, which enabled recognition of chromosomal DNA targets that have proven refractory to recognition by conventional Invader probes. Thus, toehold Invader probes represent another step toward a robust, oligonucleotide-based approach for sequence-unrestricted dsDNA-recognition.
开发能够与双链 DNA （ds）特定序列结合的分子继续引起极大的兴趣，因为这可能为生命科学、生物技术和医学中的应用提供有用的工具。我们之前已经使用 Invader 探针展示了 dsDNA 的序列无限制识别， ，这些 DNA 双链体通过整合 O2'- 嵌入功能性 RNA 单体的 +1 链间拉链排列而在能量上被激活。尽管如此，由于相应探针的高稳定性，识别扩展的 dsDNA 靶区仍然具有挑战性。为了解决这个问题，我们引入了固位 Invader 探针， ，带有 5' - 单链突出端的 Invader 探针。这种设计提供了具有缩短双链段的探针的途径，这有利于探针变性。此外，单链突出端可以用亲和力增强修饰（如 LNA 单体）进行修饰，以进一步增加靶亲和力。在此，我们报告了不同固位 Invader 设计的生物物理和 dsDNA 靶向特性，并将其与传统的 Invader 探针进行了比较。LNA 修饰的固位 Invader 探针显示出有希望的识别特性，包括对 dsDNA 的极大改善亲和力、优异的结合特异性和快速识别动力学，这使得能够识别常规 Invader 探针难以识别的染色体 DNA 靶标。因此，固位 Invader 探针代表了朝着基于寡核苷酸的、对序列无限制的 dsDNA 识别的稳健方法又迈进了一步。
