A unique sixth-order reversible assembly of hexameric mimetic nucleic acid

Complementary dipeptides bearing the non-canonical nucleobases melamine and cyanuric acid (alpha DPNA) assemble into a cylindrical hexameric structure containing a stacked heterocyclic core with its beta-sheet peptide backbones on the surface. Circular dichroism spectroscopy and singular-value decomposition show that the evolution of CD spectra after mixing equimolar strands is an apparent two-state process, accounted for by only the spectra of free strands and the assembled hexamer, with no detectable intermediates. Equilibrium measurements reveal a highly cooperative, enthalpically driven assembly with a molecularity of 6.2 +/- 0.2 and an association constant of (2.3 +/- 0.2)X10^22 " " M^(-5). Over total strand concentrations of 20 to 100 micromolar, the assembly progress curves are described by an apparent reversible sixth-order macroscopic rate law. This deceptively simple behavior cannot represent an elementary six-strand collision. The apparent forward rate constant has a negative activation energy, showing that the observed rate law masks additional steps in the assembly reaction. A minimal model in which strand pre-isomerization precedes hexamer formation restores a physically reasonable positive activation energy while retaining the observed two-state CD behavior. Constrained simulations further show that a sequential pathway with low-population, spectroscopically silent, intermediates can give rise to the same deceptively simple sixth-order progress curve. These results provide an unusual example of highly cooperative hexamer formation and show how a simple high-order macroscopic rate law can conceal a more complex microscopic assembly mechanism. The thermodynamics and kinetics provide insights into how and why the hexamer assembles, offering some design principles for supramolecular assemblies in water.