<p>Through interactions with small molecules and proteins, nucleic acids store and retrieve genetic information. In the current investigation, the energetics of binding of three natural plant based cytotoxic beta carboline alkaloids, harmaline, harmalol and harmine, with, single stranded poly(rA), ds A- form of poly(rC)· poly(rG), tRNA^phe of clover leaf and double stranded Calf thymus DNA (CT DNA) of varied motifs and conformation have been investigated using isothermal titration calorimetry (ITC). Harmaline exhibited the highest affinity for poly(rA), followed by poly(rC)· poly(rG), CT DNA, and tRNA^phe. Harmalol on the other hand showed maximum interaction with CT DNA followed by poly(rC)· poly(rG), tRNA^phe and least with poly(rA). Both harmaline and harmalol provided entropy-driven binding with poly(rA) and tRNA^phe, but enthalpy-driven binding with harmaline and harmalol was observed with double-stranded CT DNA and poly(rC)· poly(rG). While harmine reported maximum affinity towards poly(rA) followed by CT DNA, tRNA^phe and least with poly(rC)· poly(rG) and it was enthalpy driven binding with CT DNA while with the other three polynucleotides harmine showed entropy driven binding. Heat capacity (Δ<i>C</i><Stack> _p ^o </Stack>) analysis because of binding of harmaline, harmalol, and harmine with CT DNA were observed to be −&#xa0;0.148, −&#xa0;0.137, and −&#xa0;0.150&#xa0;kcal mol⁻¹&#xa0;K⁻¹, respectively, using the temperature dependency of the binding enthalpies calculated from ITC experiments. The values were further extended to calculate the corresponding Δ<i>G</i><Stack> _hyd ^o </Stack> as −&#xa0;11.84, −&#xa0;10.96 and −&#xa0;12.00 /kcal mol⁻¹. The findings indicated that the interaction of harmine, a planar molecule, with the above nucleic acid motifs was stronger and thermodynamically more favored followed by harmaline and harmalol, which are partially puckered planar molecules.</p>