Determination of association constants and FRET in hydrazide-based molecular duplex strands

Abstract

FRET pair pyrene and perylene fluorophores were incorporated into hydrazide-based molecular duplex strands. The dimerization constants were determined via monitoring the pyrene excimer emission band. When non-fluorescent hydrazide-based oligomers with complementary hydrogen-bonding sites were added, pyrene excimer–monomer transition was observed. The supramolecular substitution reactions from less tightly hydrogen-bonded homoduplexes to more tightly hydrogen-bonded heteroduplexes were further used to determine the association constants. By mixing oligomers with pyrene and perylene fluorophores, respectively, supramolecular substitution reactions induced efficient FRET from the donor (pyrene) and the acceptor (perylene). The FRET process could be further modulated via varying mixing concentrations, temperature, and solvent polarity, and addition of non-fluorescent oligomers.