Skip to main content
. Author manuscript; available in PMC: 2020 Aug 17.
Published in final edited form as: Nat Chem Biol. 2020 Feb 17;16(7):801–809. doi: 10.1038/s41589-020-0478-0

Figure 1. A single-molecule telomerase extension assay using high-resolution dual-trap optical tweezers.

Figure 1.

a, Experimental design to measure processive telomerase catalysis using dual-optical tweezers. b, Schematic showing the molecular details of tether formation to monitor DNA elongation by telomerase. c, Western blot of purified 3xFLAG- and 3xFLAG-HaloTag-TERT containing telomerase samples (representative example of 3 independent purifications), probed with anti-FLAG-M2-HRP (top) and poly-HRP-streptavidin (bottom). d, Northern blot of RNA extracted from purified 3xFLAG- and 3xFLAG-HaloTag-TERT containing telomerase samples, probed with three phosphorylated DNA oligonucleotides complementary to TR (representative example of 3 independent purifications). Standards are in vitro transcribed full-length TR. e, Western blot of purified 3xFLAG- and 3xFLAG-HaloTag-TERT containing telomerase samples, probed with anti-FLAG-M2-HRP (top), TCAB1 (middle), and dyskerin (bottom) antibodies (representative example of 3 independent purifications). f, Direct telomerase primer extension assay of 3xFLAG-HaloTag-TERT modified with biotin and 3xFLAG-TERT containing telomerase in reaction buffer containing 10 μM or 50 μM dNTPs and 50 mM KCl (LC = loading control). Relative activity is total lane intensity normalized to LC and the respective 3xFLAG-TERT telomerase activity (n = 3, mean ± s.d.). Specific activity is additionally normalized to the relative amount of TR (Fig. 1d). For uncropped gel images please see supplementary figure 5.