SHAPE RNA Structure Probing

eSHAPE: Enhanced SHAPE RNA Structure Probing with NAI Reagent


For NGS-based accurate prediction of RNA secondary structures on a high-throughput scale

eSHAPE from Eclipse BioInnovations is a method to obtain RNA structure probing data for in vitro transcribed RNA formulations or from Total RNA. In vitro transcribed/synthetic RNAs or Total RNA are used as input for RNA structure probing with the NAI reagent. eSHAPE is available as kits for end user completion, and as a full service offering with results delivered as a data package containing RNA structure information as reactivity values.

Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is a chemical probing method that measures RNA flexibility at single nucleotide resolution. As licensee of the patented technology, Eclipse Bio apply NAI to the RNA probing reaction, which forms adducts with the free 2′-OH on the RNA backbone in single stranded regions.  In contrast to other probing agents, NAI is unbiased and modifies all 4 RNA bases in single stranded RNA regions.

During the reverse transcription reaction these adducts induce mutations in the newly formed cDNA at a higher rate than the DMSO control sample. Mutation rates are then calculated for each position along the RNA, and later computed into SHAPE reactivities that guide the folding of the RNA by indicating the pairing status for each position. The SHAPE technology measures local RNA flexibility in purified, deproteinized RNA in vitro or in cellulo enabling prediction of RNA secondary structures.

Deep coverage from eSHAPE

  • Extremely deep RNA coverage of > 10,000x with minimal sequencing depth
  • Elevated mutation rates in NAI probed samples compared to DMSO control sensitively identify RNA positions with high reactivity/unpaired bases

Reactivities of high sensitivity & specificity
  • Reactivity values are predictive of paired and unpaired base status in an RNA secondary structure fold
  • Computed reactivities from eSHAPE single RNA experiments are consistent with whole transcriptome approaches

Detect RNA structural changes due to sequence variants
  • Compare reactivities of similar RNAs to identify regions of RNA folding variability
  • Reveal differences in RNA folding across conditions or due to an outside binding factor