<p>Studies on RNA and RNA-targeting inhibitors that regulate its expression form a central foundation of contemporary medicinal chemistry research. A combination of experimental methods that is spectroscopy and calorimetry and computational studies was used to maneuver into the interactive phenomenon of two phenothiazinium dyes methylene blue and new methylene blue with four single stranded RNAs -poly(G), poly(U), poly(I) and poly(C). A thorough perusal of comparison between the dyes by McGhee-von Hippel equation revealed highest affinity to poly(G) and least to poly(C). Circular dichroism studies emphasized on alteration of native RNA structures due to binding with the optically inactive dyes. Major enthalpic contribution and favourable entropy changes in the complexation scenario of both the dyes to the RNAs were also demonstrated. Computation modeling study reveals the binding location and respective detailed investigation of binding forces such as hydrogen bonds, van der Waals forces etc. involved in the binding process. Thus, an improved understanding of RNA structures and functions, identification of RNA motifs as viable targets, and validation of their accessibilities for small molecules will significantly advance the discovery of RNA-targeted small-molecule drugs. The objective of the study lies in the integration of biophysical and computational studies which serves as a stepping stone for drug identification and screening, offering deep insights into RNA-ligand interactions and thereby ushering in a new era of therapeutic development.</p>