Technology Overview
RNA structure plays a critical role in translation, stability, RNA processing, localization, and interaction with proteins and other molecules. Understanding the structure of RNA can provide key insights into RNA-based mechanisms of gene regulation and RNA-protein interactions, and can guide the design of therapeutic compounds such as mRNAs, small molecules, siRNAs, and ASOs.
eSHAPE (SHAPE-MaP, Selective 2′-Hydroxyl Acylation analyzed by Primer Extension and Mutational Profiling) is a chemical probing method that measures RNA flexibility at single nucleotide resolution. As the exclusive licensee of NAI, Eclipsebio utilizes the probing agent for eSHAPE services for basic researchers and therapeutic developers. NAI forms adducts with free 2’OH groups on the RNA sugar backbone, in single stranded regions. Adduct formation occurs at a rate of 1-3% and further induce mutations in the cDNA during reverse transcription. Mutation rates, above DMSO control, are calculated into reactivity scores that are used to guide folding.
Compared to alternative approaches such as NMR and crystallography, eSHAPE offers a robust, high-throughput workflow and provides greater accuracy than predictive algorithms. eSHAPE provides valuable insights into the folding patterns of RNA molecules in multiple biological contexts. Experimental designs for eSHAPE studies vary with options to probe endogenous transcripts, in vitro transcribed (IVT) RNAs before and after transfection, within liquid nanoparticles, or in the presence of RNA binding compounds either in vitro or in cellulo. Contact your Eclipsebio expert for guidance.
Technology Highlights
Deep coverage from eSHAPE Single RNA
- Extremely deep RNA coverage >10,000X with minimal sequencing depth
- Elevated mutation rates in NAI samples compared to the DMSO control 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
Deep coverage from eSHAPE Single RNA
- Extremely deep RNA coverage >10,000X with minimal sequencing depth
- Elevated mutation rates in NAI samples compared to the DMSO control 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
