In this mini review, we discuss how our understanding of ribonucleic acid (RNA) properties becomes significantly deepened when a broad range of modern chemical and biophysical methods is applied. We span our perspective from RNA solid-phase synthesis and site-specific labeling to single-molecule fluorescence-resonance-energy-transfer imaging and NMR spectroscopy approaches to explore the dynamics of RNA over a broad timescale. We then move on to Fourier-transform-ion-cyclotron-resonance mass spectrometry (FT-ICR-MS) as a powerful technique for RNA sequencing and modification analysis. The novel methodological developments are discussed for selected biological systems that include the thiamine-pyrophosphate riboswitch, HIV and ribosomal A-site RNA, and transfer RNA. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 1114–1123, 2013.