Liu Mengqin, Li Angda, An Ran, Liang Xingguo
State Key Laboratory of Marine Food Processing & Safety Control, College of Food Science and Engineering, Ocean University of China, No. 1299 Sansha Road, Qingdao 266404, China.
Laboratory for Marine Drugs and Bioproducts, Qingdao Marine Science and Technology Center, No. 1 Wenhai Road, Qingdao 266237, China.
ACS Chem Biol. 2025 Jun 20;20(6):1371-1381. doi: 10.1021/acschembio.5c00185. Epub 2025 May 21.
Z-DNA, a left-handed DNA conformation, plays critical roles in transcriptional regulation, genetic recombination, genomic instability, immunity, and human diseases. In 2019, a stable LR-chimera containing Z-DNA (Lk = 0) under physiological ionic conditions was prepared by hybridizing two complementary circular ssDNAs. However, the difficulty in preparing circular ssDNA precursors and the excessively long Z-DNA segment in the chimera limit its applications. In this study, using a splint-free circularization method, we prepared two circular ssDNAs (each with a hairpin structure). Hybridization of these two circles whose loops are complementary (but not the two hairpins) yielded a Stem-LR chimera containing short Z-DNA and B-DNA and two hairpins that could not hybridize with each other. Stability analysis revealed that the 18-34 bp Z-DNA segment with only unmodified nucleotides in the Stem-LR chimera remained stable under physiological conditions (10 mM Mg, 37 °C). When hairpins were far apart (180°), multiple Stem-LR chimera isomers (varying in B-Z junction numbers and Z-DNA lengths) formed. Intriguingly, higher hybridization temperatures (60 °C) favored continuous B-DNA and Z-DNA segments (minimal B-Z junctions). When hairpins were adjacent (0° orientation), exclusively continuous B-DNA/Z-DNA was obtained, even for hybridization at 10 °C. As expected, Stem-LR chimeras exhibited enhanced resistance to topoisomerase I compared to chimeras without hairpins. This approach holds promise for delivery into cells or organisms to investigate the impact of Z-DNA and its biological functions under physiological conditions.
Z-DNA是一种左手DNA构象，在转录调控、基因重组、基因组不稳定性、免疫及人类疾病中发挥着关键作用。2019年，通过杂交两条互补的环状单链DNA，制备出了在生理离子条件下含有Z-DNA（连环数Lk = 0）的稳定LR嵌合体。然而，制备环状单链DNA前体的困难以及嵌合体中过长的Z-DNA片段限制了其应用。在本研究中，我们采用无夹板环化方法制备了两条环状单链DNA（每条都带有一个发夹结构）。将这两个环杂交，其环互补（但两个发夹不互补），产生了一种包含短Z-DNA和B-DNA以及两个无法相互杂交的发夹的茎-LR嵌合体。稳定性分析表明，茎-LR嵌合体中仅含有未修饰核苷酸的18 - 34 bp Z-DNA片段在生理条件下（10 mM Mg，37 °C）保持稳定。当发夹相距较远（180°）时，会形成多种茎-LR嵌合体异构体（B-Z连接数和Z-DNA长度不同）。有趣的是，较高的杂交温度（60 °C）有利于形成连续的B-DNA和Z-DNA片段（B-Z连接最少）。当发夹相邻（0°方向）时，即使在10 °C杂交也能得到完全连续的B-DNA/Z-DNA。正如预期的那样，与没有发夹的嵌合体相比，茎-LR嵌合体对拓扑异构酶I的抗性增强。这种方法有望用于导入细胞或生物体，以研究Z-DNA在生理条件下的影响及其生物学功能。
