Figure 6.

Telomerase activity assays using in vitro reconstituted recombinant Tetrahymena telomerase and G-quadruplexes formed from Tetrahymena oligonucleotides. For all panels, LC=loading control (³²P-labelled 100-mer), ^* represents the unextended 5′ ³²P-labelled gel-purified G-quadruplex, GP and UP refer to gel-purified and unpurified G-quadruplexes, respectively. All assays were conducted at 25°C for 60 min (A–C), 10 min (D) or 15 min (E) using ∼2 nM enzyme. (A) Telomerase extension of ³²P-labelled intramolecular 21GG (‘GP Intra'; 1.8 μM, 85% purity) and unlabelled 21GG (1.8 μM) in 50 mM NaGlu. (B) Telomerase extension of ³²P-labelled intramolecular 21GG (1.1 μM, 78% purity) and unlabelled 21GG (1.1 μM) in 150 mM KGlu. (C) Telomerase extension of ³²P-labelled intermolecular 21GG (0.7 μM, 66% purity) and unlabelled 21GG (0.7 μM) in 150 mM KGlu. (D) Telomerase extension of ³²P-labelled intermolecular 21GG (0.09–1.5 μM, 63% purity) in 50 mM KGlu. The control is unlabelled linear 21GG over the same concentration range, in the absence of KGlu. (E) Telomerase extension of intermolecular 12GT (0.06–8 μM, 99% purity) in 100 mM NaCl. Linear 12GT (−NaCl) and denatured 12GT (+NaCl) G-quadruplex over the same concentration range were used as controls.