Unexpected homoadenine self-duplexes are formed when pyrene units are bound covalently to the deoxyadenosine bases at specific distances (1,4 relationships). This discovery illustrates how small-molecule pyrene intercalators can be used to drive unknown nucleic acid assembly with a concomitant change in fluorescence. When a pair of pyrene fluorophore units is located within an oligodeoxyadenylate chain, the system can display three different colors (reddish-orange, green, or blue) depending on the relative location of the fluorophores. A unique fluorescence signal, a reddish band peaking at 580 nm, appears when the oligomers possess more than two spacers between the pyrene fluorophores(1,4 relationships). Several spectroscopic experiments, for example, recording variable-concentration spectra, CD, UV, melting temperature, and gel electropherogram, indicate that this new reddish band came from an intermolecular homoadenine self-duplex. Time-resolved fluorescence measurements using both TCSPC and upconversion methods indicate that this unique fluorescence has a long lifetime.