School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing, 211189, China.
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China.
Talanta. 2024 Jul 1;274:126030. doi: 10.1016/j.talanta.2024.126030. Epub 2024 Apr 1.
Aberrant long noncoding RNA (lncRNA) expression is linked to varied pathological processes and malignant tumors, and lncRNA can serve as potential disease biomarkers. Herein, we demonstrate the autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of lncRNA in human lung tissues on the basis of multiple primer generation-mediated rolling circle amplification (mPG-RCA). This assay involves two padlock probes that act as both a detection probe for recognizing target lncRNA and a domain for producing complementary DNAzyme. Two padlock probes can hybridize with target lncRNA at different sites, followed by ligation to form a circular template with the aid of RNA ligase. The circular template can initiate mPG-RCA to generate abundant Mg-dependent DNAzymes that can specifically cleave signal probes to induce the recovery of Cy3 fluorescence. The inherent characteristics of ligase-based ligation reaction and DNAzymes endow this assay with excellent specificity, and the introduction of multiple padlock probes endows this assay with high sensitivity. This strategy can rapidly and sensitively measure lncRNA with a wide linear range of 1 fM - 1 nM and a detection limit of 678 aM within 1.5 h, and it shows distinct advantages of simplicity and immobilization-free without the need of precise temperature control and tedious procedures of nanomaterial preparation. Moreover, it enables accurate measurement of lncRNA level in normal cells and malignant tumor cells as well as differentiation of lncRNA expressions in tissues of non-small cell lung cancer (NSCLC) patients and normal individuals, with promising applications in biomedical studies and disease diagnosis.
异常的长非编码 RNA（lncRNA）表达与多种病理过程和恶性肿瘤有关，lncRNA 可以作为潜在的疾病生物标志物。在此，我们基于多引物生成介导的滚环扩增（mPG-RCA），展示了用于在人肺组织中灵敏测量 lncRNA 的功能性核酸的自主酶促合成。该测定法涉及两个发夹探针，它们既可以作为识别靶 lncRNA 的检测探针，也可以作为产生互补 DNA 酶的结构域。两个发夹探针可以在不同的位点与靶 lncRNA 杂交，然后在 RNA 连接酶的帮助下连接形成带有圆形模板。圆形模板可以启动 mPG-RCA，生成大量依赖于 Mg 的 DNA 酶，该酶可以特异性地切割信号探针以诱导 Cy3 荧光的恢复。基于连接酶的连接反应和 DNA 酶的固有特性赋予了该测定法极好的特异性，并且引入多个发夹探针赋予了该测定法高灵敏度。该策略可以快速灵敏地测量 lncRNA，线性范围为 1 fM - 1 nM，检测限为 678 aM，在 1.5 小时内，具有无需精确温度控制和繁琐的纳米材料制备程序的简单和免固定化的明显优势。此外，它能够准确测量正常细胞和恶性肿瘤细胞中的 lncRNA 水平，并区分非小细胞肺癌（NSCLC）患者和正常人组织中 lncRNA 的表达，在生物医学研究和疾病诊断中有很好的应用前景。
