The binding interaction of a prospective anti-cancer photosensitizer, norharmane (NHM, 9H-pyrido[3,4-b]indole) with double stranded RNA reveals a primarily inter-calative mode of binding. Steady-state and time-resolved fluorescence spectroscopic results demonstrate the occurrence of drug-RNA binding interaction as manifested through environment-sensitive prototropic equilibrium of NHM. However, the key finding of the present study lies in unraveling the complexities in the NHM-RNA binding thermodynamics. Isothermal Titration Calorimetry (ITC) results reveal the presence of two thermodynamically different binding modes for NHM. An extensive temperature-dependence investigation shows that the formation of Complex I is enthalpically (Delta H-I < 0) as well as entropically (T Delta S-I > 0) favored with the enthalpic (entropic) contribution being increasingly predominant in the higher (lower) temperature regime. On the contrary, the formation of Complex II reveals a predominantly enthalpy-driven signature (Delta H-I < 0) along with unfavorable entropy change (T Delta S-I <0) with gradually decreasing enthalpic contribution with temperature. Such differential dependences of Delta H-I and Delta H-II on temperature subsequently lead to opposing heat capacity changes underlying the formation of Complex I and II (Delta C-p(I) < 0 and Delta C-p(II)). A negative Delta C-p underpins the pivotal role of 'hydrophobic effect' (release of ordered water molecules) for the formation of Complex I, while a positive Delta C-p marks the thermodynamic hallmark for 'hydrophobic hydration' (solvation of hydrophobic (or nonpolar) molecular surfaces in aqueous medium) for formation of Complex II. A detailed investigation of the effect of ionic strength enables a component analysis of the total free energy change (Delta G). (C) 2018 Elsevier Inc. All rights reserved.