In RNA site-directed spin labeling (SDSL) studies, structural and dynamic information at the individual RNA nucleotide level is derived from the observed electron paramagnetic resonance spectrum of a covalently attached nitroxide. A systematic approach for RNA SDSL is to establish a library that categorizes observed spectral lineshapes based on known RNA structures, thus enabling line-shape-based structure identification at any RNA site. To establish the first RNA SDSL library, selective secondary structure elements have been systematically engineered into a model RNA. Nitroxide lineshapes reporting features specific to each element were obtained utilizing a new avid in-tethering scheme for suppressing spectral effects due to uniform RNA tumbling. The data demonstrated two key features required for a SDSL library with a predicting power: (i) spectral divergence-distinctive lineshape for different elements; and (ii) spectral convergence-similar lineshape for the same element in different contexts. This sets the foundation for further RNA SDSL library development. (c) 2006 Elsevier Inc. All rights reserved.