SHAPE-Seq

SHAPE-Seq

Selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)  provides structural information about RNA. In this method, a unique barcode is first added to the 3’ end of RNA, and the RNA is then allowed to fold under pre-established in vitro conditions. The barcoded and folded RNA is treated with a SHAPE reagent, 1M7, that blocks reverse transcription. The RNA is then reverse-transcribed to cDNA. Deep sequencing of the cDNA provides single-nucleotide sequence information for the positions occupied by 1M7. The structural information of the RNA can then be deduced.

Pros:

• Provides RNA structural information
• Multiplexed analysis of barcoded RNAs provides information for multiple RNAs
• Effect of point mutations on RNA structure can be assessed
• Alternative to mass spectrometry, NMR, and crystallography

Cons:

• Need positive and negative controls to account for transcriptase drop-off
• Need pre-established conditions for RNA folding
• The folding in vitro may not reflect actual folding in vivo
  

• SHAPE-Seq: Lucks J. B., Mortimer S. A., Trapnell C., Luo S., Aviran S., et al. (2011) Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proc Natl Acad Sci U S A 108: 11063-11068

1. Spitale R. C., Flynn R. A., Zhang Q. C., Crisalli P., Lee B., et al. (2015) Structural imprints in vivo decode RNA regulatory mechanisms. Nature 519: 486-490
2. Loughrey D., Watters K. E., Settle A. H. and Lucks J. B. (2014) SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing. Nucleic Acids Res 42:
3. Lee J., Kladwang W., Lee M., Cantu D., Azizyan M., et al. (2014) RNA design rules from a massive open laboratory. Proc Natl Acad Sci U S A 111: 2122-2127