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. 2002 Jan 15;30(2):559–568. doi: 10.1093/nar/30.2.559

Figure 5.

Figure 5

Figure 5

Dissociation rate of primer from telomerase is increased in the presence of phosphoramidate oligonucleotide, GRN137159. Purified telomerase (G5000PW pool) was incubated with or without the phosphoramidate oligonucleotide for 15 min, followed by the addition of primer d(TTAGGG)3 (100 nM) at 30°C for 30 min. d(TTAGGG)4 was added (20 µM) and aliquots (18 µl) were removed at the times indicated above. A nucleotide mix (2 µl) containing 2 mM dTTP and 10 µCi [α-32P]dATP (3000 Ci/mmol) was added and the labeling was allowed to proceed for 5 min. Labeling will extend each primer by 3 nt. The samples were separated on an 18% poylacrylamide gel containing 7 M urea. (A) The gel was dried and analyzed by a PhosphoImager. (B) The bands in (A) were quantified and expressed as a percentage of the initial amount of ennzyme–primer complex. The amount of the primer labeled immediately following the challenge with the longer primer was assigned the value of 100%. The data were fit to the equation for exponential decay as described in Materials and Methods. The t1/2 for the primer’s dissociation from telomerase is ∼300 min without the presence of the NP oligonucleotide GRN137159, versus ∼35 min in the presence of GRN137159.