Hairpin nucleic acids are frequently used in physical studies due to their greater thermal stability compared to their equivalent duplex structures. They are also good models for more complex loop-containing structures such as quadruplexes, i-motifs, cruciforms, and molecular beacons. Although a connecting loop can increase stability, there is little information on how the loop influences the interactions of small molecules with attached base-paired nucleic acid regions. In this study, the effects-of different hairpin loops on the interactions of A/T specific DNA minor groove binding agents with a common stem sequence have been investigated by spectroscopic and surface plasmon resonance (SPR) biosensor methods. The results indicate that the hairpin loop as little influence on the specific site interactions on the stem but significantly affects nonspecific binding. The use of a non-nucleotide loop (with a reduced negative charge) not only enhances the thermal stability of the hairpin but also reduces the nonspecific binding at the loop without compromising the primary binding affinity on the stem.