Watson-Crick base pairs are the major component of double helical structures in RNA;their structures and stabilities are well understood.Non-Watson-Crick base pairs are less well characterized,despite the fact that they are critical in (de)stabilizing RNA secondary structures,shaping RNA into complex tertiary structures,and providing unique local and global structures for ligand binding.Here,we used optical tweezers and to study the contribution of U·U and U·C pairs in an internal loop to the mechanical folding energy landscape of a hairpin in human telomerase RNA.Our single-molecule mechanical unfolding studies show that a U·U mismatch is more stable than a U·C mismatch,consistent with our ensemble thermal melting studies.The U·U to U·C mutation,which disrupts one hydrogen bond and is located 6 Watson-Crick base pairs away from the terminal end of the hairpin,lowers the mechanical unfolding energy barrier allosterically (by lowering the unfolding force by ~1 picoNewton (pN) per mutation) without affecting the unfolding transition state position.The mechanical folding transition state position from the moves towards the fully folded hairpin structure upon U·U to U·C mutation,suggests a 13-nucleotide misfolded state exists for the mechanical folding of the hairpins.Thus,a conservative single mutation within a non-Watson-Crick region may affect RNA stability and biological functions involving RNA (un)folding.