For over 50 years the G-quartet has been a defining self-assembled structure in biology and non-covalent synthesis. It is shown here for the first time that the G-quartet is isolatable in water in the absence of stabilizing G-quartet stacking or cations through the construction of a phosphate-linked template-assembled synthetic G-quartet. Synthetic design has facilitated preservation of the guanine base, ribose sugar, and phosphate components with correct linkage chemistry relative to G-quadruplex DNA. Thus, a minimal synthetic model of G-quadruplex DNA, as in that associated with human gene promoter or telomere regions, is represented by this system. An application as a probe for interactions between G-quadruplex DNA and potential anticancer therapeutical binding ligands is demonstrated. Binding constants of 10(5)-10(7) M(-1) magnitude and 1:1 stoichiometries for TMPyP4, piper, and azatrux ligands were determined, whereas perturbations in BSU1051 and BRACO19 ligand signal were not observed. These data suggest a unique test for critical end-stacking interactions at the exclusion of intercalative or looping interactions for G-quadruplex binding ligands.