DNA walker-based strategies have gained significant attention in nucleic acid analysis. However, they face challenges related to balancing design complexity, sequence dependence, and amplification efficiency. Furthermore, most existing DNA walkers rely on walking and lock probes, requiring optimization of various parameters like DNA probe sequence, walking-to-lock probe ratio, lock probe length, etc. to achieve optimal performance. This optimization process is time-consuming and adds complexity to experiments. To enhance the performance and reliability of DNA walker nanomachines, there is a need for a simpler, highly sensitive, and selective alternative strategy. A sensitive and rapid miRNA analysis strategy named hairpin-shaped DNA aligner and nicking endonuclease-fueled DNA walker (HDA-NE DNA walker) was developed. The HDA-NE DNA walker was constructed by modifying hairpin-shaped DNA aligner (HDA) probe and substrate report (SR) probe on the surface of AuNPs. Under normal conditions, HDA and SR remained stable. However, in the presence of miR-373, HDA underwent a conformational transition to an activated structure to continuously cleave the SR probe on the AuNPs with the assistance of Nt.AlwI nicking endonuclease, resulting in sensitive miRNA detection with a detection limit as low as 0.23 pM. Additionally, the proposed HDA-NE DNA walker exhibited high selectivity in distinguishing miRNAs with single base differences and can effectively analyze miR-373 levels in both normal and breast cancer patient serums. The proposed HDA-NE DNA walker system was activated by a conformational change of HDA probe only in the presence of the target miRNA, eliminating the need for a lock probe and without sequence dependence for SR probe. This strategy demonstrated a rapid reaction rate of only 30 min, minimal background noise, and a high signal-to-noise ratio (S/B) compared to capture/lock-based DNA walker. The method is expected to become a powerful tool and play an important role in disease diagnosis and precision therapy.

中文翻译：

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一种基于发夹形 DNA 对准器并由切口核酸内切酶驱动的 DNA 步行器，用于灵敏、快速的 miRNA 分析


基于 DNA walker 的策略在核酸分析中受到了广泛关注。然而，他们面临着平衡设计复杂性、序列依赖性和扩增效率相关的挑战。此外，大多数现有的DNA步行器依赖于步行和锁定探针，需要优化各种参数，如DNA探针序列、步行与锁定探针的比率、锁定探针长度等，以实现最佳性能。这种优化过程非常耗时并且增加了实验的复杂性。为了提高 DNA walker 纳米机器的性能和可靠性，需要一种更简单、高度灵敏和选择性的替代策略。开发了一种灵敏且快速的 miRNA 分析策略，称为发夹形 DNA 对齐器和切口核酸内切酶驱动的 DNA 步行器（HDA-NE DNA 步行器）。 HDA-NE DNA walker是通过修饰AuNPs表面的发夹形DNA对准器(HDA)探针和底物报告(SR)探针构建的。正常情况下，HDA和SR保持稳定。然而，在 miR-373 存在的情况下，HDA 经历构象转变为激活结构，在 Nt.AlwI 切口核酸内切酶的帮助下连续切割 AuNP 上的 SR 探针，从而实现灵敏的 miRNA 检测，检测限低至下午 0.23 点。此外，所提出的HDA-NE DNA walker在区分具有单碱基差异的miRNA方面表现出高选择性，并且可以有效分析正常和乳腺癌患者血清中的miR-373水平。所提出的 HDA-NE DNA walker 系统仅在目标 miRNA 存在的情况下通过 HDA 探针的构象变化激活，从而消除了对锁探针的需要，并且不依赖于 SR 探针的序列。 与基于捕获/锁定的 DNA walker 相比，该策略表现出仅 30 分钟的快速反应速率、最小的背景噪音和高信噪比 (S/B)。该方法有望成为有力的工具，在疾病诊断和精准治疗中发挥重要作用。