Although synthesized as linear polymers, RNA transcripts fold into intricate structures crucial for cellular physiology. It is now evident that RNA functions extend far beyond the established roles of messenger, transfer and ribosomal RNAs, with examples in RNA splicing and editing, telomere maintenance, protein secretion, small-molecule sensing and reaction catalysis, to name just a few. How RNA achieves its arsenal of functions with a limited assortment of building blocks is a question of great interest, and the answer often lies in deciphering RNA structure at its different levels of complexity.

RNA-structure determination, however, is far from trivial. Many RNAs are poorly conserved, and their function cannot be inferred by simple homology searches based solely on primary structure. Covariation analyses of secondary structure conservation are therefore often preferred, and they can even guide the computational prediction of functional three-dimensional RNA modules.

To decipher RNA secondary structure in the first place, several high-throughput experimental approaches have been developed. Recent advances in chemical and enzymatic RNA footprinting have demonstrated the possibility for high-throughput secondary-structure mapping with single-nucleotide resolution. With careful design, such techniques can even provide information on the native three-dimensional fold of RNA transcripts.

Going from sequence to tertiary RNA structure (structural information is from Structure 19, 1413–1423; 2011).

An even higher-resolution picture of RNA tertiary structure can be obtained by classical structural biology methods. These, however, face limitations as throughput is usually low, nuclear magnetic resonance remains limited to fairly small molecules, and crystallization of large, negatively charged RNAs is far from trivial. Computational prediction methods have provided a valuable alternative, but they are often limited to conserved RNA folds, require substantial computer resources and cannot account for the full complexity of environment-dependent and intramolecular interactions influencing RNA structure.

With single-molecule diffraction deemed theoretically possible and the ongoing development of X-ray free electron laser technology, a high-resolution picture of the 'RNA structurome' seems plausible. Concerted efforts can increase data collection and analysis throughput, and results can greatly facilitate prediction and high-fidelity modeling of additional transcripts. Although it could take some time for this to happen, we will certainly be watching for reliable new methods for RNA-structure determination.