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

SHAPE RNA Structure Probing Applications

As our understanding of RNA biology increases, so do the number of functions and interactions RNA is shown to have. Coding and non-coding RNAs play a variety of roles in cells, and the structure of the RNA can be pivotal in defining and enabling these functions. Thus, the ability to probe RNA structure is critical in understanding these myriad cellular interactions. SHAPE is a method of accurately and reproducibly measuring RNA secondary structure, information that can enable drug discovery, synthetic RNA engineering, identification of novel RNA misfolding events, and more.

SHAPE measures RNA flexibility at single nucleotide resolution in both, cellular conditions (in cellulo) and purified, deproteinized RNA (in vitro). In vitro SHAPE reveals flexibility across an RNA strand. In cellulo shows flexibility in regions not bound by proteins, which can help predict RNA’s secondary structure. Combining in cellulo and in vitro mutation rates, another method called ΔSHAPE (delta SHAPE), can identify which RNA positions interact with RNA Binding Proteins.

Advancing therapeutic research by SHAPE RNA Structure Probing

Example 1: Cancer Research – HOTAIR LncRNA Interaction with RBPs

Long non-coding RNAs, or lncRNAs, play a variety of roles in cancer, where they can function as enhancer RNAs, decoys, and scaffolds, in addition to other roles in cross-talk and transcriptional regulation. One gene that has been shown to repress metastasis and tumor repressor genes is called HOX transcript antisense intergenic RNA, commonly known as HOTAIR. This is a widely studied lncRNA known to interact with LSD1 and PRC2. These interactions are involved with cancer metastasis and thus represent a potential therapeutic target. By utilizing SHAPE RNA Structure Probing, research has shown that the secondary structure of HOTAIR is a key factor in its interactions with LSD1 and PRC2 which aids drug development tremendously.

Example 2: Alzheimer Disease

Alzheimer’s Disease (AD) research is another area in which SHAPE is advancing therapeutic opportunity. APP, or Alzheimer precursor protein, is one of the progressors of AD. The translation of this protein is accelerated by the influx of Iron, and as a result, metal chelation is one of the therapeutic strategies for AD treatment. Research has utilized SHAPE to elucidate the structure of an RNA element that binds to an iron-responsive element (IRE), enabling the translation of APP, and thus the progression of the disease. This structural element, now mapped because of SHAPE, presents a potential therapeutic target for AD treatment.

eSHAPE: Efficient, fast, and cost effective SHAPE RNA Structure Probing

The information provided by SHAPE is clearly instrumental in progressing our understanding of RNA biology and opening doors to drug development for widespread diseases. One remaining barrier for the researcher, however, has been the burden of difficulty, time, and cost associated with SHAPE so far. While labs can perform SHAPE assays using published methods in literature, there are challenges that this approach presents. The uncertain consistency and reliability of DIY reagent preparation can introduce variability in the assay’s performance. Furthermore, there can be high barriers to entry in terms of sequencing cost. In the world of SHAPE, more is better when it comes to read depth, and as a result, sequencing costs can become astronomical when performing SHAPE experiments. And while individual SHAPE experiments themselves can be costly, to obtain ΔSHAPE data, one must run both the in cellulo and in vitro SHAPE experiments, followed by extensive bioinformatic analysis.

To help researchers break through these barriers, Eclipsebio now offers its eSHAPE technology as the solution – application of NAI for structure probing and opimization of subsequent cDNA synthesis and NGS library preparation enable you to perform SHAPE RNA Structure probing at a fraction of time and cost.

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