The four-stranded i-motif (iM) conformation of DNA has importance to a variety of biochemical systems that include nanomaterials and oncogene regulation. Fundamental studies to understand iM formation and its structure in solution still remain to be done. In this dissertation, we will discuss the use of the fluorescent base analog tC° to determine the structural state of DNA in solution. We will also demonstrate the use of tC° to determine both the hydrodynamic properties and the folding mechanism of an iM. We also determined the loop length dependence on iM formation and stability. Our hydrodynamic studies show that the iM structure has rotational correlation times similar to unfolded single-strand DNA, but displays the same structural rigidity as duplex DNA. This combination of hydrodynamic properties is unique to the iM structure. Using kinetic rates, a mechanism is proposed for the folding of a random coil oligo into the iM. The fluorescence changes in tC° describes the hydrogen bonding of the cytosines that occur during the folding of the iM. This folding mechanism shows that all hydrogen bonds form on the same time scale as the iM forms, meaning that no one base is more important than the others in forming the iM structure. The loop length dependence studies show a distinct difference in stability. The differences in thermal stability and pKa observed when lengthening loops suggests a reasonable method for gaining fine control over the thermal stability and pKa of the iM that can be readily adapted to nanomaterial usage. Our research also shows that the optimal search algorithms for finding iMs in genomic databases should be different from the algorithms currently used.