RNA targeting, an evolving new approach to anticancer therapeutics, requires identification of small molecules that can selectively target specific RNA structures. In this context, the capability of polyamines and analogs to target RNA is being viewed as an attractive anti cancer therapeutics. The interaction of biogenic polyamines spermine, and synthetic analog 1-naphthyl acetyl spermine with three double stranded RNA polynucleotides, poly(I).poly(C), poly(C).poly(G) and poly(A).poly(U) has been studied to understand the structural and thermodynamic basis of the binding and the comparative efficacy of the analog over the natural polyamine. Circular dichroism spectroscopy, thermal melting and ethidium bromide displacement assay were used to characterize the binding. Microcalorimetry studies were performed to deduce the energetics of the interaction and atomic force microscopy experiments lend further insights into the interaction at the molecular level. Structural perturbations were observed for the RNA polynucleotides on binding of the polyamines. Melting results showed enhanced stabilization of RNA-polyamine complexes with increase in the total standard molar enthalpy of transition. Atomic force microscopy imaging experiments revealed the ability of both polyamine and its analog to condense and circularize RNA molecules. The binding affinity was strongest for poly(I).poly(C) and varied as poly(I).poly(C) > poly(C).poly(G) > poly(A).poly(U). The interactions were characterized by total enthalpy–entropy compensation and high standard molar heat capacity values.