Aspirin is an old drug, which belongs to the nonsteroidal anti-inflammatory drugs. It is used extensively as a painkiller, and recently, it has been suggested to be effective against colorectal cancer. The aim of this study is to investigate the interaction of yeast RNA with aspirin in aqueous solution at physiological pH with drug/RNA(P) (P = phosphate) molar ratios of r = 1/80, 1/40, 1/20, 1/10, 1/4, 1/2, and 1. Fourier transform infrared (FTIR) difference spectroscopy is used to determine drug binding mode, sequence selectivity, and RNA secondary structure, as well as the structural variations of the aspirin-RNA complexes in aqueous solution. Spectroscopic evidence showed that at low drug concentration (r = 1/80), aspirin-RNA interaction is through the G-C and A-U base pairs and the backbone PO2 group. Such interaction largely perturbs the G-C vibrations at 1698 and 1488 cm(-1) and the A-U bands at 1654 and 1608 cm(-1) as well as the phosphate antisymmetric stretch at 1244 cm(-1). At r = 1/40, minor structural changes occur for the ribose-phosphate backbone geometry with RNA remaining in the A-conformation. At r > 1/20, a partial helix destabilization occurs. The drug distributions around the double helix are about 50% G-C, 30% A-U, and 20% PO2 groups. A comparison between aspirin-RNA and asprin-DNA complexes shows minor differences. The aspirin anion binding is via drug OCO and COOCH3 groups.