Table 1.
Major categories of methods for the analysis of RNA structures and interactions
| Categories | Variety of methods | Features | General reference(s) |
|---|---|---|---|
| X-ray crystallography | In vitro, atomic resolution, smaller, and nearly static molecules | Shi 2014 | |
| NMR | In vitro, atomic resolution, smaller molecules, can be either static or flexible | Bothe et al. 2011 | |
| Cryo-EM | In vitro, near atomic resolution, large, and heterogeneous complexes | Bai et al. 2015 | |
| Thermodynamic methods | Nussinov algorithm, Zuker algorithm, etc. RNA structure, Sfold, Mfold, UNAfold, ViennaRNA Package, etc. | Guarantees theoretically optimal structures, slow when permitting pseudoknotted structures | Eddy 2004; Mathews et al. 2010 |
| Comparative sequence analysis | Fold and Align, Fold then Align, Align then Fold, etc. | Implies functional relevance, limited by alignments and efficiency (local structures) | Mathews et al. 2010 |
| Chemical probing | DMS-seq, Structure-seq, SHAPE, icSHAPE, Mutate and Map, etc. | In vitro and in vivo, one-dimension averaged reactivity/flexibility/accessibility profile, can be used to probe secondary, tertiary structures and RNA–protein interactions | Kladwang et al. 2011; Ding et al. 2014; Rouskin et al. 2014; Spitale et al. 2015 |
| Enzymatic probing | PARS, Frag-seq, etc. | In vitro, one-dimension averaged reactivity/flexibility/accessibility profile | Kertesz et al. 2010; Underwood et al. 2010 |
| Cross-linking (based on physical proximity) | PARIS, LIGR-seq, SPLASH, CLASH, hiCLIP, MARIO | In vitro and in vivo, direct physical base-pairing contacts (except MARIO), captures alternative conformations | Helwak et al. 2013; Sugimoto et al. 2015; Aw et al. 2016; Lu and Chang 2016; Nguyen et al. 2016; Sharma et al. 2016 |
NMR, Nuclear magnetic resonance, Cryo-EM, cryogenic electron microscopy; DMS, dimethyl sulfate; SHAPE, selective 2′-hydroxyl acylation by primer extension; icSHAPE, in vivo click SHAPE; PARS, parallel analysis of RNA structures; Frag-seq, fragmentation sequencing; PARIS, psoralen analysis of RNA interactions and structures; LIGR-seq, ligation of interacting RNA and high-throughput sequencing; SPLASH, sequencing of psoralen cross-linked, ligated, and selected hybrids; CLASH, cross-linking, ligation, and sequencing of hybrids; hiCLIP, RNA hybrid and individual nucleotide resolution cross-linking and immunoprecipitation; MARIO, mapping RNA interactome in vivo.