title: PCR-free and label-free fluorescent detection of telomerase activity at single-cell level based on triple amplification.
abstract: As a universal biomarker for cancer diagnostics and cancer therapeutics, telomerase has attracted extensive attention concerning its detection and discovery of its inhibitors. Herein, we developed a PCR-free and label-free fluorescent strategy for facile, reliable and highly sensitive assay of human telomerase activity from crude cancer cell extracts. A G-quadruplex-selective fluorescent dye, N-methyl mesoporphyrin IX (NMM), was utilized as signal probe. Two hairpin probes with hidden G-quadruplex strand in their stem were designed as assembly components of strand displacement reaction (SDR). In this strategy, one telomerase elongation product contains several hexamer repeats which can hybridize with numerous assistant DNA to release a lot of trigger DNA (T-DNA) of SDR for achieving first step amplification. Then, strand displacement reaction led to the formation of G-quadruplex at the both end of two hairpin DNA probes for realizing second step amplification. Finally, the re-released T-DNA initiated another cycle of SDR, resulting in a significant increase in the fluorescence intensity of NMM. By taking advantage of triple signal amplification, the telomerase activity in the HeLa extracts equivalent to 1-3000 cells was detected in homogeneous solution. Telomerase activities of different cell lines, including cancer cells and normal cell, were also successfully evaluated. Meanwhile, the inhibition effect of 3'-azido-3'-deoxythymidine (AZT) was also investigated. Therefore, it offers a simple and reliable method for detecting telomerase activity at single-cell level without complex pre-modification of probe and enzyme auxiliary signal amplification, which has the merits of simplicity, rapid response, low cost and high reliability.
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Baoxin
authors.initials: B
authors.lastname: Li
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China. Electronic address: jinyan@snnu.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Jin
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D054856:G-Quadruplexes; D006367:HeLa Cells; D006801:Humans; D008652:Mesoporphyrins / Q000737:chemistry*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods; D019098:Telomerase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015215:Zidovudine / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008652:Mesoporphyrins; C031468:mesoporphyrin IX; D015215:Zidovudine; D004247:DNA; D019098:Telomerase
keywords: Fluorescence; Label-free; Telomerase activity; Triple amplification