Abstract：
BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：基于DNAwalker的策略在核酸分析中获得了极大的关注。然而,他们面临着平衡设计复杂性的挑战，序列依赖，和放大效率。此外，大多数现有的DNA行走者都依赖于行走和锁定探针，需要优化各种参数，如DNA探针序列，行走与锁定探针的比率，锁定探头长度,等。以达到最佳性能。这种优化过程是耗时的并且增加了实验的复杂性。为了提高DNAwalker纳米机器的性能和可靠性，需要一个更简单的，高度敏感,和选择性替代策略。
结果：开发了一种灵敏而快速的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DNAwalker（HDA-NEDNAwalker）。HDA-NEDNA助行器是通过在AuNP表面修饰发夹形DNA比对（HDA）探针和底物报告（SR）探针来构建的。在正常情况下,HDA和SR保持稳定。然而,在miR-373的存在下,HDA经历了向活化结构的构象转变,从而在Nt的帮助下连续切割AuNP上的SR探针.尽管刻痕核酸内切酶，导致灵敏的miRNA检测，检测限低至0.23pM。此外,提出的HDA-NEDNAwalker在区分具有单碱基差异的miRNA方面表现出高度选择性,并且可以有效分析正常和乳腺癌患者血清中的miR-373水平.
结论：提出的HDA-NEDNAwalker系统仅在靶miRNA存在下通过HDA探针的构象变化被激活，消除了对锁定探针的需要并且对SR探针没有序列依赖性。该策略显示出只有30分钟的快速反应速率，最小的背景噪声，与基于捕获/锁定的DNA步行器相比，具有较高的信噪比（S/B）。该方法有望成为一种强大的工具，并在疾病诊断和精确治疗中发挥重要作用。
结果：开发了一种灵敏而快速的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DNAwalker（HDA-NEDNAwalker）。HDA-NEDNA助行器是通过在AuNP表面修饰发夹形DNA比对（HDA）探针和底物报告（SR）探针来构建的。在正常情况下,HDA和SR保持稳定。然而,在miR-373的存在下,HDA经历了向活化结构的构象转变,从而在Nt的帮助下连续切割AuNP上的SR探针.尽管刻痕核酸内切酶，导致灵敏的miRNA检测，检测限低至0.23pM。此外,提出的HDA-NEDNAwalker在区分具有单碱基差异的miRNA方面表现出高度选择性,并且可以有效分析正常和乳腺癌患者血清中的miR-373水平.
结论：提出的HDA-NEDNAwalker系统仅在靶miRNA存在下通过HDA探针的构象变化被激活，消除了对锁定探针的需要并且对SR探针没有序列依赖性。该策略显示出只有30分钟的快速反应速率，最小的背景噪声，与基于捕获/锁定的DNA步行器相比，具有较高的信噪比（S/B）。该方法有望成为一种强大的工具，并在疾病诊断和精确治疗中发挥重要作用。
