Visual and fluorometric determination of telomerase activity by using a cationic conjugated polymer and fluorescence resonance energy transfer

Abstract

The detection of telomerase activity is important in cancer diagnosis and in screening for anti-cancer drugs. This work describes a fluorometric method for the determination of telomerase activity by using a cationic conjugated polymer (CCP). Telomerase substrate primers were labelled with carboxyfluorescein (FAM-TS) which display weak electrostatic interactions with the CCP. Hence, fluorescence resonance energy transfer (FRET) from photoexcited CCP to FAM is weak. However, in the presence of telomerase, telomeric repeats (TTAGGG)x were elongated to the 3′-end of FAM-TS to form multiple G-quadruplexes in the presence of potassium ion (K+). These G-quadruplexes trigger strong electrical interaction between the condensed G-quadruplexes and the CCP, and this results in closer proximity and in more efficient FRET. As a result, strong green fluorescence (peaking at 527 nm) is emitted by FAM. Fluorescence can be visually observed under a UV lamp and be used to quantify the activity of telomerase by using a fluorometer. The assay was applied to the determination of HeLa cells in the 30 to 1000 cells per mL range, with a detection limit as low as 5 cells per mL (at an S/N ratio of 3). The method was applied to the detection of various cancer cell lines in human urine samples. The method is simple, sensitive, selective and accurate.