Construction of an Enzymatically Controlled DNA Nanomachine for One-Step Imaging of Telomerase in Living Cells

Telomerase is a basic reverse transcriptase that maintains the telomere length in cells, and accurate and specific sensing of telomerase in living cells is critical for medical diagnostics and disease therapeutics. Herein, we demonstrate for the first time the construction of an enzymatically contro...

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Published in:Analytical chemistry (Washington) 2024-03, Vol.96 (11), p.4647-4656
Main Authors: Wang, Li-juan, Liu, Wen-jing, Wang, Lu-yao, Ho, Yi-ping, Han, Yun, Li, Dong-ling, Zhang, Chun-yang
Format: Article
Language:English
Subjects:
Cancer
Cells (biology)
Construction
Deoxyribonucleic acid
DNA
DNA - chemistry
DNA probes
DNA-(Apurinic or Apyrimidinic Site) Lyase - metabolism
Endonuclease
Fluorescence
Gold
Gold - chemistry
HeLa Cells
Humans
Imaging techniques
Kinetics
Medical imaging
Metal Nanoparticles - chemistry
Microenvironments
Nanoparticles
Probes
RNA-directed DNA polymerase
Sensors
Substrates
Telomerase
Telomerase - metabolism
Telomerase inhibitors
Telomeres
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Summary:Telomerase is a basic reverse transcriptase that maintains the telomere length in cells, and accurate and specific sensing of telomerase in living cells is critical for medical diagnostics and disease therapeutics. Herein, we demonstrate for the first time the construction of an enzymatically controlled DNA nanomachine with endogenous apurinic/apyrimidinic endonuclease 1 (APE1) as a driving force for one-step imaging of telomerase in living cells. The DNA nanomachine is designed by rational engineering of substrate probes and reporter probes embedded with an enzyme-activatable site (i.e., AP site) and their subsequent assembly on a gold nanoparticle (AuNP). Upon recognition and cleavage of the AP site in the substrate probe by APE1, the loop of the substrate probe unfolds, exposing telomeric primer (TP) with the 3′–OH end. Subsequently, the TP is elongated by telomerase at the 3′–OH end to generate a long telomeric product. The resultant telomeric product acts as a swing arm that can hybridize with a reporter probe to initiate the APE1-powered walking reaction, ultimately generating a significantly enhanced fluorescence signal. Notably, endogenous APE1 is used as the driving force of the DNA nanomachine, avoiding the introduction of exogenous auxiliary cofactors into the cellular microenvironment. Owing to the high kinetics and high amplification efficiency of the APE1-powered DNA nanomachine, this strategy enables one-step sensitive sensing of telomerase in vitro and in vivo. It can successfully discriminate telomerase activity between cancer cells and normal cells, screen telomerase inhibitors, and monitor the variations of telomerase activity in living cells, offering a prospective platform for molecular diagnostics and drug discovery.
ISSN:0003-2700
1520-6882
1520-6882
DOI:10.1021/acs.analchem.3c05795