A two-dimensional mutate-and-map strategy for non-coding RNA structure

• Published in: Nature chemistry Vol. 3; no. 12; pp. 954 - 962
• Main Authors: Kladwang, Wipapat, VanLang, Christopher C., Cordero, Pablo, Das, Rhiju
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.12.2011; Nature Publishing Group
• Subjects: 631/337/384; 639/638/45/535; 639/638/92; Accuracy; Algorithms; Analytical Chemistry; Biochemistry; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Crystallography; Glycine - chemistry; Hypotheses; Inorganic Chemistry; Models, Molecular; Mutation; NMR; Nuclear magnetic resonance; Nucleic Acid Conformation; Organic Chemistry; Physical Chemistry; Protein synthesis; Riboswitch; RNA, Untranslated - chemistry; United States > US; California
• ISSN: 1755-4330; 1755-4349; 1755-4349
• DOI: 10.1038/nchem.1176

Non-coding RNAs fold into precise base-pairing patterns to carry out critical roles in genetic regulation and protein synthesis, but determining RNA structure remains difficult. Here, we show that coupling systematic mutagenesis with high-throughput chemical mapping enables accurate base-pair inference of domains from ribosomal RNA, ribozymes and riboswitches. For a six-RNA benchmark that has challenged previous chemical/computational methods, this ‘mutate-and-map’ strategy gives secondary structures that are in agreement with crystallography (helix error rates, 2%), including a blind test on a double-glycine riboswitch. Through modelling of partially ordered states, the method enables the first test of an interdomain helix-swap hypothesis for ligand-binding cooperativity in a glycine riboswitch. Finally, the data report on tertiary contacts within non-coding RNAs, and coupling to the Rosetta/FARFAR algorithm gives nucleotide-resolution three-dimensional models (helix root-mean-squared deviation, 5.7 Å) of an adenine riboswitch. These results establish a promising two-dimensional chemical strategy for inferring the secondary and tertiary structures that underlie non-coding RNA behaviour. Non-coding RNAs are ubiquitous biomolecules with intricate three-dimensional folds that are difficult to characterize. This Article presents an information-rich strategy for inferring RNA structure by combining nucleotide-by-nucleotide mutagenesis with single-nucleotide-resolution chemical mapping.