FIGURE 5:
Imetelstat inhibits the formation of long-static telomerase–telomere interactions. (A) Experimental rationale. If long-static telomerase–telomere interactions require the base pairing of TR to the chromosome end, then imetelstat should prevent their formation. (B) Still images from three-color live-cell single-molecule imaging experiments. Telomeres were marked by mEOS3.2-TRF2, telomerase was detected using a 3xFLAG-HaloTag-TERT conjugated to JF646, and Cajal bodies were visualized using BFP-coilin (scale bar = 5 µm). (C) Kymographs of TERT and TRF2 trajectories from HeLa cells stably expressing 3xFLAG-HaloTag-TERT and mEOS3.2-TRF2 from their respective endogenous loci. Cells were treated with 2 µM imetelstat, 2 µM mismatched control oligonucleotide, or no drug. (D) Diffusion coefficient distributions of TERT trajectories from HeLa cells stably expressing 3xFLAG-HaloTag-TERT treated with 2 µM imetelstat, 2 µM mismatched control oligonucleotide, or no drug (n = 4000–5000 trajectories per condition). (E) Quantification of the fraction of cells with at least one long-static telomerase–telomere interaction after treatment with 2 µM imetelstat, 2 µM mismatched control oligonucleotide, or no drug (NNo Drug = 3, NMM Control = 3, Nimetelstat = 4, where N = number of independent experiments, n = 13–36 cells per condition, mean ± SD, t test). (F) Quantification of the fraction of cells with at least one long-static telomerase–telomere interaction as a function of imetelstat concentration (n = 28–44 cells per condition). (G) Model for telomerase recruitment to chromosome ends derived from the current study. Short probing interactions require TERT–TPP1 protein–protein binding, while the productive interaction of telomerase with the DNA 3′-end (far right) also requires base pairing with the template region of TR. Whether telomerase can hop or slide to the 3′ end from internal binding sites or, alternatively, is released back into the nucleus and must collide directly with the 3-terminus is an open question (?).