Research Center for Molecular Oncology and Functional Nucleic Acids, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, 453003, Henan Province, China.
Department of Clinical Laboratory, West China Second Hospital, Sichuan University, Chengdu, 610041, Sichuan Province, China.
Anal Chim Acta. 2020 Aug 1;1123:28-35. doi: 10.1016/j.aca.2020.04.069. Epub 2020 Apr 30.
One of the major challenges facing the early diagnosis of chronic myelogenous leukemia (CML) patients today is enhancing the simplicity, rapidness, sensitivity and specificity of detection assay for easy clinical implementation. RNA-cleaving fluorogenic DNAzymes (RFDs) are single-stranded DNA molecules with catalytic activity and can produce fluorescent signals when combined with specific targets. As K562 cells were the first established human immortalized myelogenous leukemia line, we try to screen several RFDs using the crude extracellular mixture of K562 cells through the SELEX process. We obtained an RFD probe A1-3 that is able to distinguish K562 cells from other tumor cell lines. 10 nM of A1-3 can induce an increase of detectable fluorescence signal. Moreover, the RFD assay system can work well for target detection in complex serum matrix. The optimized RFD assay system with low cost also has a desirable ability to exactly distinguish K562 cells after truncation of 20 bases in the 5'end of A1-3. This study is the first report to investigate the RFD system for detection of K562 cells using cell culture supernatants as the complex target. This RFD assay system could potentially be applied for the diagnosis of CML.
目前，慢性髓细胞白血病（CML）患者早期诊断面临的主要挑战之一是提高检测方法的简便性、快速性、灵敏度和特异性，以便于临床应用。RNA 切割荧光 DNA 酶（RFD）是具有催化活性的单链 DNA 分子，与特定靶标结合时会产生荧光信号。由于 K562 细胞是最早建立的人永生化髓系白血病细胞系，我们试图通过 SELEX 过程筛选几种 RFD 与 K562 细胞的粗细胞外混合物结合。我们获得了一种 RFD 探针 A1-3，它能够将 K562 细胞与其他肿瘤细胞系区分开来。10 nM 的 A1-3 可以诱导可检测荧光信号的增加。此外，该 RFD 检测系统在复杂的血清基质中也能很好地进行靶标检测。优化后的 RFD 检测系统成本低廉，还具有在 A1-3 的 5'端截断 20 个碱基后准确区分 K562 细胞的理想能力。本研究首次报道了使用细胞培养上清液作为复杂靶标，用 RFD 系统检测 K562 细胞。该 RFD 检测系统有望应用于 CML 的诊断。
