Intramolecular G-quadruplexes (G4s) are G-rich nucleic acid structures that fold back on themselves via interrupting loops to create stacked planar G-tetrads, in which four guanine bases associate via Hoogsteen hydrogen bonding. The G4 structure is further stabilized by monovalent cations centered between the stacked tetrads. The G-tetrad face on the top and bottom planes of G4s are often the site of interaction with proteins and small molecules. To investigate the potential impact of interrupting loops on both G4 structure and interaction with proteins/small molecules, we characterized a specific G4 from the 3'-UTR of PITX1 mRNA that contains loops of 6 nucleotides using biophysical approaches. We then introduced mutations to specific loops to determine the impact on G4 structure and the ability to interact with both proteins and a G4-specific ligand. Our results suggest that mutation of a specific loop both affects the global G4 structure and impacts the ability to interact with a G4 binding protein and small molecule ligand. (C) 2017 Elsevier Inc. All rights reserved.