New regulatory roles continue to emerge for both natural and engineered 
noncoding RNAs, many of which have specific secondary and tertiary structures 
essential to their function. Thus there is a growing need to develop 
technologies that enable rapid characterization of structural features within 
complex RNA populations. We have developed a high-throughput technique, 
SHAPE-Seq, that can simultaneously measure quantitative, single 
nucleotide-resolution secondary and tertiary structural information for hundreds 
of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2'-hydroxyl 
acylation analyzed by primer extension (SHAPE) chemistry with multiplexed 
paired-end deep sequencing of primer extension products. This generates millions 
of sequencing reads, which are then analyzed using a fully automated data 
analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq 
experiment. We demonstrate the ability of SHAPE-Seq to accurately infer 
secondary and tertiary structural information, detect subtle conformational 
changes due to single nucleotide point mutations, and simultaneously measure the 
structures of a complex pool of different RNA molecules. SHAPE-Seq thus 
represents a powerful step toward making the study of RNA secondary and tertiary 
structures high throughput and accessible to a wide array of scientific 
pursuits, from fundamental biological investigations to engineering RNA for 
synthetic biological systems.