Primer-Modified G-Quadruplex-Au Nanoparticles for Colorimetric Assay of Human Telomerase Activity and Initial Screening
G-quadruplexes formed by 3′-overhang of guanine-rich human telomeric DNA at the end of chromosome have important implication in inhibiting the telomerase activity. Telomerase catalyzes the elongation of telomeres by adding telomeric repeats sequence TTAGG
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Introduction Guanine-rich DNA sequences can form G-quadruplex through π-π stacking of the G-tetrads in which four guanines are arranged in a plane by Hoogsteen hydrogen bonds with the assistance of a specific metal cation [1]. Telomere is located at the ends of chromosomes and protects the chromosome ends from deterioration and fusion [2]. Human telomeres play a significant role in genome stability, cancer, and aging. Human telomeric DNA consists of a duplex region composed of TTAGGG hexamer repeats and an extended 30 -overhang of the G-rich single strand [3, 4]. The single-stranded G-rich overhang containing four contiguous TTAGGG repeats can fold into a G-quadruplex structure in the presence of Na+ or K+ ions [5].
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_21, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Telomerase, a cellular reverse transcriptase, is composed of telomerase integral RNA and catalytic subunit [6]. It catalyzes the addition of multiple telomeric repeats TTAGGG onto the end of the chromosomes using telomerase RNA as templates. It may cause genetically abnormal situations. Telomerase is activated in 80–90% of human tumors, while is low or undetectable in most normal somatic cells. Therefore, telomerase is recognized not only as a biomarker of cancer diagnosis but also as a specific target for cancer therapy [7]. Owing to the significance of telomerase in the biomedical filed, it is necessary to develop a reliable and sensitive analytical method for monitoring telomerase activity [8]. The classic telomeric repeat amplification protocol (TRAP) assay based on polymerase chain reaction (PCR) is the most widely used method [9, 10]. However, it suffers from some drawbacks including timeconsuming, sophisticated experimental procedure, and harsh condition. Moreover, the assay is not easily quantitated and subject to PCR-related artifacts [11]. Some PCR-free methods for telomerase activity have also been constructed [12–14]. However, the shortcomings such as low sensitivity, complicated operation, and requirement of elaborate equipment or expensive fluorescent labels still need to be solved. Telomeric DNA that folds into G-quadruplexes can’t be elongated by telomerase, thus inhibiting the telomerase activity. Small molecules, which can stabilize the G-quadruplex structure formed by the human telomeric DNA, have been considered as efficient telomerase inhibitor [15]. Therefore, the design, synthesis, and screening of them can provide a viable strategy toward anticancer drugs development. Gold nanoparticles (AuNPs) have been extensively used in analytical methods since AuNPs possess strong size-and distancedependent optical properties and extremely high extinction coefficient [16]. AuNP-based assays present color changes in response to analytes, which can be observed by the naked eye. A visual method for measuring telomerase activi
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