In this research, we employed tetra(ethylene glycol) (EG(4)) to substitute bases at the loop region of the intramolecular DNA i-motif formed by (CCCTAA)(3)CCC, and systematically studied the influence of such nonbase components on the stability and conformation of the formed structures by circular dichroism (CD), UV-vis spectroscopy and gel electrophoresis. We found that with all loop bases substituted, the i-motif structure can still form. The stability of the i-motif generally got weaker with the increase of the substitution number. Substitution at different positions might lead to different topologies. The findings above demonstrate that bases at the loop region play an important role on the stability and topology of the intramolecular DNA i-motif.