Song Juan, Li Shan, Zhou Jie, Yu Qiao, Yang Xue-Jiao, Chen Hong-Yuan, Xu Jing-Juan
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China.
School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211816, China.
Anal Chim Acta. 2023 May 22;1256:341145. doi: 10.1016/j.aca.2023.341145. Epub 2023 Mar 28.
Abnormal upregulation of telomerase RNA (TR) is a hallmark event at various stages of tumor progression, providing a universal marker for early diagnosis of cancer. Here, we have developed a triple signal amplification strategy for in situ visualization of TR in living cells, which sequentially incorporated the target-initiated strand displacement circuit, multidirectional rolling circle amplification (RCA), and Mg DNAzyme-mediated amplification. All oligonucleotide probes and cofactors were transfected into cells in one go, and then escaped from lysosomes successfully. Owing to the specific base pairing, the amplification cascades could only be triggered by TR and performed as programmed, resulting in a satisfactory signal-to-background ratio. Especially, the netlike DNA structure generated by RCA encapsulated high concentrations of DNAzyme and substrates (FQS) in a local region, thereby improving the reaction efficiency and kinetics of the third amplification cycle. Under optimal conditions, the proposed method exhibited ultrasensitive detection of TR mimic with a detection limit at pM level. Most importantly, after transfection with the proposed sensing platform, tumor cells can be easily distinguished from normal cells based on TR abundance-related fluorescence signal, providing a new insight into initial cancer screening.
端粒酶RNA（TR）的异常上调是肿瘤进展各个阶段的标志性事件，为癌症早期诊断提供了通用标志物。在此，我们开发了一种用于在活细胞中原位可视化TR的三重信号放大策略，该策略依次整合了靶标引发的链置换电路、多向滚环扩增（RCA）和Mg脱氧核酶介导的扩增。所有寡核苷酸探针和辅助因子一次性转染到细胞中，然后成功从溶酶体中逃逸。由于特定的碱基配对，扩增级联反应只能由TR触发并按程序进行，从而产生令人满意的信噪比。特别是，RCA产生的网状DNA结构在局部区域封装了高浓度的脱氧核酶和底物（FQS），从而提高了第三个扩增循环的反应效率和动力学。在最佳条件下，所提出的方法对TR模拟物表现出超灵敏检测，检测限为pM水平。最重要的是，用所提出的传感平台转染后，基于TR丰度相关的荧光信号可以很容易地将肿瘤细胞与正常细胞区分开来，为癌症早期筛查提供了新的见解。
