The interaction of two natural protoberberine alkaloids berberine and palmatine and the synthetic derivative coralyne with the RNA triplex poly(U).poly(A)(*)poly(U) was Studied using various biophysical and calorimetric techniques. All the three alkaloids bind noncooperatively to the triplex. The affinity of berberine and palmatine was in the order of 10(5) M-1, while that of coralyne was one order higher as inferred from spectroscopic Studies. The alkaloids stabilized the Hoogsteen base-paired third strand of the triplex without affecting the stability of the duplex. Fluorescence quenching and viscosity studies gave convincing evidence for the partial intercalation of berberine and palmatine and a true intercalative binding of coralyne to the triplex. This was further Supported from the significant polarization of the emission spectra of the complex and the energy transfer from the base triplets to the alkaloids. Circular dichroic studies suggested that the conformation of the triplex was perturbed significantly by the binding of the alkaloids, being more by coralyne, compared to berberine and palmatine and also evidenced by the generation of strong induced optical activity in the bound coralyne Molecules. Isothermal titration calorimetric studies revealed that the binding to the triplex was favored by a predominantly large negative enthalpy change (Delta H degrees = -5.42 kcal/mol) with small favorable entropy contribution (T Delta S degrees = 2.02 kcal/mol) in berberine, favored by almost equal negative enthalpy (Delta H degrees = -3.93 kcal/mol) and entropy changes (T Delta S degrees = 3.89 kcal/mol) in palmatine and driven by predominant entropy contributions (Delta H degrees = -1.84 and T Delta S degrees = 7.44 kcal/mol) in coralyne. These results advance Our knowledge oil the binding of small molecule isoquinoline alkaloids that are specific binders of RNA structures, particularly triplexes.