Autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of long noncoding RNA in human lung tissues,Talanta

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Autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of long noncoding RNA in human lung tissues
Talanta ( IF 5.6 ) Pub Date : 2024-04-01 , DOI: 10.1016/j.talanta.2024.126030
Yun Han ₁ , Su Jiang ₂ , Peng-Yu Wang ₂ , Juan Hu ₁ , Chun-Yang Zhang ₁

Affiliation

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.

中文翻译：

功能性核酸的自主酶促合成用于灵敏测量人肺组织中的长非编码RNA

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，我们展示了基于多引物生成介导的滚环扩增（mPG-RCA）的功能性核酸的自主酶促合成，用于灵敏测量人肺组织中的lncRNA。该测定涉及两个挂锁探针，它们既充当识别靶 lncRNA 的检测探针，又充当生成互补 DNAzyme 的结构域。两个挂锁探针可以在不同位点与目标lncRNA杂交，然后在RNA连接酶的帮助下连接形成环状模板。环状模板可以启动mPG-RCA产生丰富的Mg依赖性DNAzymes，这些DNAzymes可以特异性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脱氧核糖核酸酶的固有特性赋予该测定具有优异的特异性，并且多个挂锁探针的引入赋予该测定高灵敏度。该策略可以在1.5 h内快速、灵敏地测量lncRNA，线性范围为1 fM − 1 nM，检测限为678 aM，并且无需精确的温度控制和繁琐的操作，显示出简单和免固定化的明显优势。纳米材料的制备过程。此外，它还能够准确测量正常细胞和恶性肿瘤细胞中的lncRNA水平，以及区分非小细胞肺癌（NSCLC）患者和正常个体组织中的lncRNA表达，在生物医学研究和疾病诊断中具有广阔的应用前景。

更新日期：2024-04-01

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