Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, National & Local Joint Biomedical Engineering Research Center on Photodynamic Technologies, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350002, China; Key Laboratory of Laboratory Medicine, Ministry of Education of China, and Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, China.
Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, National & Local Joint Biomedical Engineering Research Center on Photodynamic Technologies, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350002, China.
Talanta. 2024 Dec 1;280:126735. doi: 10.1016/j.talanta.2024.126735. Epub 2024 Aug 21.
While the intracellular imaging of miRNA biomarkers is of significant importance for the diagnosis and treatment of human cancers, DNA assembled nanoprobe has recently attracted considerable attention for imaging intracellular biomolecules. However, the complex construction process, intrinsic vulnerability to nuclease degradation and the limited signal transduction efficiency hamper its widespread application. In this contribution, based on persistent autonomous molecular motion of DNAzyme walker along a nano-substrate track, a DNA nanosphere probe (PNLD) is developed for the sensitive intracellular miR-21 imaging. Specifically, DNA nanosphere (called PN, single-molecule nano-track) is assembled from only one palindromic substrate, into which the locking strand-silenced DNAzymes (LD) are installed in a controlled manner. PNLD (made of PN and LD) can protect all DNA components against nuclease attack and maintain its structural integrity in serum solution over 24 h. Upon the activation by target miRNA, DNAzyme walker can move on the substrate scattered within PNLD (or on the surface) and between different PNLD objects and cleave many DNA substrates, generating an amplified signal. As a result, miR-21 can be detected down to 6.83 pM without the detectable interference from co-existing nontarget miRNAs. Moreover, PNLD system can accurately screen the different expression levels of miR-21 within the same type of cells and different types of cells, which is consistent with gold standard polymerase chain reaction (PCR) assay. Via changing the target recognition sequence, the PNLD system can be suitable for the intracellular imaging of miR-155, exhibiting the desirable universality. In addition, the DNAzyme walker-based PNLD system can be used to distinguish cancer cells from healthy cells, implying the potential application in cancer diagnosis and prognosis.
虽然 miRNA 生物标志物的细胞内成像对于人类癌症的诊断和治疗非常重要，但 DNA 组装纳米探针最近因其可用于细胞内生物分子成像而受到广泛关注。然而，复杂的构建过程、对核酸酶降解的固有脆弱性以及有限的信号转导效率限制了其广泛应用。在本研究中，基于 DNA 酶 walker 在纳米基质轨道上的持续自主分子运动，开发了一种 DNA 纳米球探针 (PNLD)，用于灵敏的细胞内 miR-21 成像。具体来说，DNA 纳米球（称为 PN，单分子纳米轨道）由仅一个回文底物组装而成，其中锁定链沉默的 DNA 酶（LD）以受控的方式安装。PNLD（由 PN 和 LD 组成）可以保护所有 DNA 成分免受核酸酶攻击，并在血清溶液中保持其结构完整性超过 24 小时。在靶 miRNA 的激活下，DNA 酶 walker 可以在 PNLD 内（或表面上）以及不同 PNLD 物体之间的基质上移动，并切割许多 DNA 底物，产生放大信号。结果，miR-21 可以在没有共存非靶 miRNA 可检测干扰的情况下检测到低至 6.83 pM 的浓度。此外，PNLD 系统可以准确地筛选同一类型细胞和不同类型细胞中 miR-21 的不同表达水平，与金标准聚合酶链反应 (PCR) 检测结果一致。通过改变目标识别序列，PNLD 系统可适用于 miR-155 的细胞内成像，表现出良好的通用性。此外，基于 DNA 酶 walker 的 PNLD 系统可用于区分癌细胞和健康细胞，这意味着其在癌症诊断和预后中的潜在应用。
