SHAPE (Selective 2’ Hydroxyl Acylation Analyzed by Primer Extension) is a method used by scientists to study the shape and structure of RNA molecules. There are many different variations and versions of of SHAPE that can be done by a lab, this article is designed to give you an overview of the of the type of SHAPE chemical probing that is most commonly used by researchers who work with Eterna. This technique involves adding a chemical to the RNA molecule, which reacts with different parts of the RNA depending on their flexibility and accessibility. There are several types of chemicals used in SHAPE, but the chemical that is most frequently used by labs who work with Eterna, is 1-methyl-7-nitroisatoic anhydride (1M7).
1M7 specifically reacts with the 2’-OH groups of unpaired nucleotides. The modified RNA, called a SHAPE adduct, is then reverse transcribed, producing complimentary DNA (cDNA).The more reactive a nucleotide is the more likely that that nucleotide is not participating in base pairing. By analyzing the patterns of this chemical reactivity, researchers can determine the three-dimensional shape of the RNA molecule.
The data obtained from SHAPE chemical mapping experiments can be presented as a graph or chart showing the reactivity values of different parts of the RNA molecule. The higher the reactivity value, the less likely a nucleotide is to be involved in a stable base pair. However, it is worth noting that nucleotides with high levels of reactivity may still participate in non-stable base pairing, or non-canonical base pairing.
SHAPE chemical probing is one of the tools used by scientists, in addition to other structural and bioinformatic analysis methods. In SHAPE, "total counts" refer to the number of sequencing reads or data points obtained for each nucleotide position in an RNA sequence. When performing a SHAPE experiment, a large number of RNA molecules are typically analyzed and sequenced to generate the necessary data for accurate structure prediction.
A "total count > 10,000" indicates that a particular nucleotide position in the RNA sequence has been sampled and sequenced at least 10,000 times. This is a measure of data quality and reliability, and a high total count is generally desirable to ensure statistical significance and minimize the impact of experimental noise or artifacts. Different experiments and molecules may have different thresholds, but a minimum standard threshold of "total count > 10,000" is typically used for SHAPE data analysis experiments.
- http://www.chem.unc.edu/rna/pdf-files/2005_em_jacs.pdf the original 2005 scientific paper
- The figure 1 of 'Probing Retroviral and Retrotransposon Genome Structures: The "SHAPE" of Things to Come.' presents a good overview of this method.
- The Wikipedia article about the reverse transcriptase: http://en.wikipedia.org/wiki/Reverse_transcriptase
- Correlating SHAPE signatures with three-dimensional RNA structures
- Massively Parallel RNA Chemical Mapping with a Reduced Bias MAP-seq Protocol
Matthew Seetin, Wipapat Kladwang, J. P. Bida, and Rhiju Das
- McGinnis, J. L.; Dunkle, J. A.; Cate, J. H. D.; Weeks, K. M. The Mechanisms of RNA SHAPE Chemistry. Journal of the American Chemical Society 2012, 134 (15), 6617–6624. https://doi.org/10.1021/ja2104075.
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- Tian, S.; Cordero, P.; Kladwang, W.; Das, R. High-Throughput Mutate-Map-Rescue Evaluates SHAPE-Directed RNA Structure and Uncovers Excited States. RNA 2014, 20 (11), 1815–1826. https://doi.org/10.1261/rna.044321.114.
- Kladwang, W.; VanLang, C. C.; Cordero, P.; Das, R. Understanding the Errors of SHAPE-Directed RNA Structure Modeling. Biochemistry 2011, 50 (37), 8049–8056. https://doi.org/10.1021/bi200524n.
- Aviran, S.; Pachter, L. Rational Experiment Design for Sequencing-Based RNA Structure Mapping. RNA 2014, 20 (12), 1864–1877. https://doi.org/10.1261/rna.043844.113.
- Spitale, R. C.; Flynn, R. A.; Torre, E. A.; Kool, E. T.; Chang, H. Y. RNA Structural Analysis by Evolving SHAPE Chemistry. Wiley Interdisciplinary Reviews: RNA 2014, 5 (6), 867–881. https://doi.org/10.1002/wrna.1253.