Amplification-free and direct fluorometric determination of telomerase activity in cell lysates using chimeric DNA-templated silver nanoclusters

Lee, Shi Ting; Rahman, Ruman; Muthoosamy, Kasturi; Mohamed, Nur Aliana Hidayah; Mohamed, Hidayah; Su, Xiao Di; Tayyab, Saad; New, Siu Yee

Abstract

A fluorogenic probe has been developed for determination of telomerase activity using chimeric DNA-templated silver nanoclusters (AgNCs). The formation of AgNCs was investigated before (route A) and after (route B) telomerase elongation reaction. Both routes caused selective quenching of the yellow emission of the AgNCs (best measured at excitation/emission wavelength of 470/557 nm) in telomerase-positive samples. The quenching mechanism was studied using synthetically elongated DNA to mimic the telomerase-catalyzed elongation. The findings show that quenching is due to the formation of parallel G-quadruplexes with a –TTA– loop in the telomerase elongated products. The assay was validated using different cancer cell extracts, with intra- and interassay coefficients of variations of <9.8%. The limits of detection for MCF7, RPMI 2650 and HT29 cell lines are 15, 22 and 39 cells/μL. This represents a distinct improvement over the existing telomeric repeat amplification protocol (TRAP) assay in terms of time, sensitivity and cost.

Citation

Lee, S. T., Rahman, R., Muthoosamy, K., Mohamed, N. A. H., Mohamed, H., Su, X. D., Tayyab, S., & New, S. Y. (2019). Amplification-free and direct fluorometric determination of telomerase activity in cell lysates using chimeric DNA-templated silver nanoclusters. Microchimica Acta, 186(2), Article 81. https://doi.org/10.1007/s00604-018-3194-7

• Journal Article Type: Article
• Acceptance Date: Dec 20, 2018
• Online Publication Date: Jan 9, 2019
• Publication Date: Feb 1, 2019
• Deposit Date: Jun 3, 2019
• Journal: Microchimica Acta
• Print ISSN: 0026-3672
• Electronic ISSN: 1436-5073
• Publisher: Springer Verlag
• Peer Reviewed: Peer Reviewed
• Volume: 186
• Issue: 2
• Article Number: 81
• DOI: https://doi.org/10.1007/s00604-018-3194-7
• Public URL: https://nottingham-repository.worktribe.com/output/2130357
• Publisher URL: https://link.springer.com/article/10.1007%2Fs00604-018-3194-7