Anal Chem. 2018 Mar 6;90(5):3521-3530. doi: 10.1021/acs.analchem.7b05373. Epub 2018 Feb 22.
Reliable and accurate detection of telomerase activity is crucial to better understand its role in cancer cells and to further explore its function in cancer diagnosis and treatment. Here, we construct a smart DNA tweezer (DT) for detection of telomerase activity. The DT is assembled by three specially designed single-stranded oligonucleotides: a central strand dually labeled with donor/acceptor fluorophores and two arm strands containing overhangs complementary to telomerase reaction products (TRPs). It can get closed through hybridization with TRPs and get reopen through strand displacement reaction by TRPs' complementary sequences. First, under the action of telomerase, telomerase binding substrates (TS) are elongated to generate TRPs ended with telomeric repeats (TTAGGG) . TRPs hybridize with the two arm overhangs cooperatively and strain DT to closed state, inducing an increased fluorescence resonance energy transfer (FRET) efficiency, which is utilized for telomerase activity detection. Second, upon introduction of a removal strand (RS) complementary to TRPs, the closed DT is relaxed to open state via the toehold-mediated strand displacement, inducing a decreased FRET efficiency, which is utilized for determination of TRP length distribution. The detection limit of telomerase activity is equivalent to 141 cells/μL for HeLa cells, and telomerase-active cellular extracts can be differentiated from telomerase-inactive cellular extracts. Furthermore, TRPs owning 1, 2, 3, 4, and ≥5 telomeric repeats are identified to account for 25.6%, 20.5%, 15.7%, 12.5%, and 25.7%, respectively. The proposed strategy will offer a new approach for reliable, accurate detection of telomerase activity and product length distribution for deeper studying its role and function in cancer.
可靠且准确地检测端粒酶活性对于更好地理解其在癌细胞中的作用以及进一步探索其在癌症诊断和治疗中的功能至关重要。在这里，我们构建了一种用于检测端粒酶活性的智能 DNA 镊子 (DT)。该 DT 由三条专门设计的单链寡核苷酸组装而成：一条中央链双重标记供体/受体荧光团，两条臂链包含与端粒酶反应产物 (TRP) 互补的突出端。它可以通过与 TRP 杂交而闭合，并通过 TRP 的互补序列的链置换反应而重新打开。首先，在端粒酶的作用下，端粒酶结合底物 (TS) 被延伸以生成以端粒重复 (TTAGGG) 结尾的 TRP。TRP 与两个臂突出端协同杂交，将 DT 应变至闭合状态，诱导增加的荧光共振能量转移 (FRET) 效率，用于端粒酶活性检测。其次，引入与 TRP 互补的去除链 (RS) 后，通过 toehold 介导的链置换，闭合的 DT 松弛至打开状态，诱导降低的 FRET 效率，用于确定 TRP 长度分布。端粒酶活性的检测限相当于 HeLa 细胞中的 141 个细胞/μL，并且可以区分端粒酶活性细胞提取物和端粒酶非活性细胞提取物。此外，鉴定出具有 1、2、3、4 和≥5 个端粒重复的 TRP 分别占 25.6%、20.5%、15.7%、12.5%和 25.7%。该策略将为可靠、准确地检测端粒酶活性和产物长度分布提供一种新方法，以更深入地研究其在癌症中的作用和功能。
