POT1 and RPA compete for the 3′ overhang of ssTEL4 in 150 mM K+. (A) The RPA-mediated GQ unfolding was significantly reduced using the ssTEL construct terminating with a short TT-3′ overhang (ssTEL4TT), rather than the TTAG-3′ overhang of ssTEL4. Even at 1,000 nM RPA, only ∼50% of the GQ molecules remain folded. (B) The RPA-mediated GQ unfolding at 1 µM POT1. (C and D) The Langmuir isotherm fits to GQ unfolding in the absence and presence of POT1, respectively. POT1 addition did not significantly affect Keq for GQ unfolding, compared with the 12-fold increase observed in ssTEL4 terminating with a TTAG overhang.