SHAPE: Difference between revisions

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<p>The <strong>SHAPE</strong> acronym&nbsp;stands for "<strong>S</strong>elective 2&prime; <strong>h</strong>ydroxyl <strong>a</strong>cylation analyzed by <strong>p</strong>rimer <strong>e</strong>xtension". It is the primary method used to evaluate designs in [[EteRNA]] [[lab]] experiments.</p>
__TOC__<p>SHAPE (Selective 2’ Hydroxyl Acylation Analyzed by Primer Extension)  is a method used by scientists to study the shape and structure of RNA molecules. <ref>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. <nowiki>https://doi.org/10.1021/ja2104075</nowiki>.</ref>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).<ref>Mortimer, S.; Weeks, K. M. A Fast-Acting Reagent for Accurate Analysis of RNA Secondary and Tertiary Structure by SHAPE Chemistry. ''Journal of the American Chemical Society'' '''2007''', ''129'' (14), 4144–4145. <nowiki>https://doi.org/10.1021/ja0704028</nowiki>.</ref></p>
<p>&nbsp;</p>
[[File:Fig A- SHAPE with 1M7.jpg|alt=1M7 interacts with the 2' hydroxyl (OH)  groups on the unpaired bases of the RNA creating a modified nucleotide called a SHAPE adduct|center|thumb|784x784px|''Fig A:'' Schematic of 1M7 interacting with the 2' hydroxyl (OH)  groups on the unpaired bases of the RNA creating a modified nucleotide called a SHAPE adduct. Image created with Biorender.com]]
<p style="padding-left: 30px;"><em>Warning: this page is under heavy construction. The material is being accumulated, before it is refined. You are (very) welcome to participate.</em></p>
 
<p>&nbsp;</p>
1M7 specifically reacts with the 2’-OH groups of unpaired nucleotides<ref>Busan, S.; Weidmann, C. A.; Sengupta, A.; Weeks, K. M. Guidelines for SHAPE Reagent Choice and Detection Strategy for RNA Structure Probing Studies. ''Biochemistry'' '''2019''', ''58'' (23), 2655–2664. <nowiki>https://doi.org/10.1021/acs.biochem.8b01218</nowiki>.</ref>. 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.<ref name=":0">Watters, K. E.; Lucks, J. B. Mapping RNA Structure in Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq). ''Methods in molecular biology'' '''2016''', 135–162. <nowiki>https://doi.org/10.1007/978-1-4939-6433-8_9</nowiki>.</ref>   By analyzing the patterns of this chemical reactivity, researchers can determine the three-dimensional shape of the RNA molecule.<ref>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. <nowiki>https://doi.org/10.1261/rna.044321.114</nowiki>.</ref>
<p>__TOC__</p>
[[File:Fig B.jpg|alt=Fig B: During Mutational profiling(MaP), the SHAPE adducts in the RNA create mutations in the complimentary DNA (cDNA).  The cDNA then makes thousands of copies called double stranded DNA (dsDNA). Image created with Biorender.com|center|thumb|764x764px|''Fig B'': During Mutational profiling(MaP), the SHAPE adducts in the RNA create mutations in the complimentary DNA (cDNA). Image created with Biorender.com]]
<p>&nbsp;</p>
 
<p>== Overview ==</p>
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. <ref name=":0" />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.<ref>Kladwang, W.; VanLang, C. C.; Cordero, P.; Das, R. Understanding the Errors of SHAPE-Directed RNA Structure Modeling. ''Biochemistry'' '''2011''', ''50'' (37), 8049–8056. <nowiki>https://doi.org/10.1021/bi200524n</nowiki>.</ref>
<p>Experimental methods exist for determining, at the level of individual atoms, the various inter-atomic bindings that determine how an [[RNA|RNA molecule]] folds.&nbsp; However, this type of experiment can take months to perform, for a single RNA [[sequence]].&nbsp; The EteRNA cloud lab process is now synthesizing more than 1000 novel RNA molecules per month, and as players/scientists, we expect the results for all 1000 sequences to be available in weeks, if not days.&nbsp; To meet this need, the EteRNA biochemistry team is pioneering the development of high-throughput RNA analysis, and well as high-throughput RNA synthesis.&nbsp; This high-throuhput analysis is under continual development and evolution, and improvements are continally being introduced into the EteRNA cloud labs. &nbsp;EteRNA players who participate in the cloud labs and question the results that get back are actually part of the scientific review process.</p>
[[File:Fig c.jpg|alt=Fig C: A SHAPE profile is created of the reactive bases. From that profile, proposed structures are able to be modeled.|center|thumb|809x809px|''Fig C: A SHAPE profile is created of the reactive bases. From that profile, proposed structures are able to be modeled. More reactive bases are shown in yellow or red.'' ]]
<p>At a very high level of abstraction, SHAPE analysis works by mixing a chemical reagent (e.g. [[1M7]]), called the [[SHAPE probe]], into a solution containing many thousands of copies of each distinct RNA molecule being analyzed. The [[SHAPE probe]] has the potential for chemically attacking and modifying every base of every RNA in the solution. However, the speed with which the probe modifies the individual [[base]]s varies over several orders of magnitude.&nbsp; By carefully controlling the time of the exposure, the SHAPE procedure can limit the reaction so that only a small percentage of all the bases are modified.&nbsp; Using modern technology for processing nucleic acid chains, the number of times any analogous base has been modified can be counted, and the base-probe reaction rate for each base position of each distinct RNA moleculte can be estimated.</p>
 
<p>This base-probe reaction rate is <span style="text-decoration: underline;">not</span> a direct measurement of whether the base is or is not bound to a [[Complementarity|complementary base]] via one of the three [[Base Pair|pair bond]]s recognized in EteRNA [[puzzle]]s -- [[GC Pair|CG]], [[AU Pair|AU]] and [[GU Pair|GU]]. It's not even known exactly what details of a base's configuration goes into determning the reaction rate.&nbsp; But the general consensus is that the reaction rate is an indication of how flexible the RNA's [[backbone]] is at each base position.&nbsp; This flexibility is highly correlated with (but not wholly determined by) whether or not the base is paired to one of its complements (C with G, G with either C or U, etc).&nbsp; This high correlation is the justification for using the SHAPE data as a measure of whether or not the base is bound, for the purpose of assigning the "score" for the molecule.&nbsp; But the fact that it is not a perfect correlation ensures that there will be an ongoing discussion among the most engaged players and the EteRNA scientists about how the SHAPE data should be presented and interpreted.</p>
 
<p>== Background: The high-level structure of RNA ==</p>
 
<p>&nbsp;</p>
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.<ref>Aviran, S.; Pachter, L. Rational Experiment Design for Sequencing-Based RNA Structure Mapping. ''RNA'' '''2014''', ''20'' (12), 1864–1877. <nowiki>https://doi.org/10.1261/rna.043844.113</nowiki>.</ref>
<p>== Background: Laboratory procedures ==</p>
 
<p style="padding-left: 30px;"><em>It would seem that nearly everything we want to know about Cloud Lab procedures is described in http://arxiv.org/pdf/1304.1072.pdf</em></p>
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.<ref>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. <nowiki>https://doi.org/10.1002/wrna.1253</nowiki>.</ref><p style="padding-left: 30px;"></p>
<p>=== PCR ===</p>
 
<p>Teaching about PCR</p>
== External links ==
<p>{{#widget:YouTube|id=2KoLnIwoZKU}}</p>
<p>=== Transcription (DNA to RNA) ===</p>
<p>Here's a short video on <a href="http://www.dnalc.org/resources/3d/12-transcription-basic.html">transcription</a> in the living cell, emphasizing how rapidly it occurs.</p>
<p>=== Folding ===</p>
<p>=== Chemical modification ===</p>
<p style="padding-left: 30px;"><em>http://www.hindawi.com/journals/mbi/2012/530754/fig6/</em><br /><em>Looks like a good figure, covering chemical mod and RT</em></p>
<p>=== Reverse transcription (RNA to DNA) ===</p>
<p>=== Data collection (Sequencing) ===</p>
<p>=== Data processing ===</p>
<p style="padding-left: 30px;"><em>possible reference for this paragraph:&nbsp;http://rnajournal.cshlp.org/content/17/9/1688.full.pdf section "Data processing"</em></p>
<p>== External links ==</p>
<ul>
<ul>
<li>http://www.chem.unc.edu/rna/pdf-files/2005_em_jacs.pdf the original 2005 scientific paper</li>
<li>http://www.chem.unc.edu/rna/pdf-files/2005_em_jacs.pdf the original 2005 scientific paper</li>
Line 33: Line 22:
<li>[http://arxiv.org/pdf/1304.1072.pdf Massively Parallel RNA Chemical Mapping with a Reduced&nbsp;Bias MAP-seq Protocol]<br />Matthew Seetin, Wipapat Kladwang, J. P. Bida, and Rhiju Das</li>
<li>[http://arxiv.org/pdf/1304.1072.pdf Massively Parallel RNA Chemical Mapping with a Reduced&nbsp;Bias MAP-seq Protocol]<br />Matthew Seetin, Wipapat Kladwang, J. P. Bida, and Rhiju Das</li>
</ul>
</ul>
[[Category:DNA]]
[[Category:Experimental Methods]]
[[Category:Labs]]
[[Category:RNA]]

Latest revision as of 01:32, 28 April 2023

SHAPE (Selective 2’ Hydroxyl Acylation Analyzed by Primer Extension)  is a method used by scientists to study the shape and structure of RNA molecules. [1]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).[2]

1M7 interacts with the 2' hydroxyl (OH) groups on the unpaired bases of the RNA creating a modified nucleotide called a SHAPE adduct
Fig A: Schematic of 1M7 interacting with the 2' hydroxyl (OH) groups on the unpaired bases of the RNA creating a modified nucleotide called a SHAPE adduct. Image created with Biorender.com

1M7 specifically reacts with the 2’-OH groups of unpaired nucleotides[3]. 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.[4]   By analyzing the patterns of this chemical reactivity, researchers can determine the three-dimensional shape of the RNA molecule.[5]

Fig B: During Mutational profiling(MaP), the SHAPE adducts in the RNA create mutations in the complimentary DNA (cDNA).  The cDNA then makes thousands of copies called double stranded DNA (dsDNA). Image created with Biorender.com
Fig B: During Mutational profiling(MaP), the SHAPE adducts in the RNA create mutations in the complimentary DNA (cDNA). Image created with Biorender.com

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. [4]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.[6]

Fig C: A SHAPE profile is created of the reactive bases. From that profile, proposed structures are able to be modeled.
Fig C: A SHAPE profile is created of the reactive bases. From that profile, proposed structures are able to be modeled. More reactive bases are shown in yellow or red.


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.[7]

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.[8]

External links

  1. ↑ 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.
  2. ↑ Mortimer, S.; Weeks, K. M. A Fast-Acting Reagent for Accurate Analysis of RNA Secondary and Tertiary Structure by SHAPE Chemistry. Journal of the American Chemical Society 2007, 129 (14), 4144–4145. https://doi.org/10.1021/ja0704028.
  3. ↑ Busan, S.; Weidmann, C. A.; Sengupta, A.; Weeks, K. M. Guidelines for SHAPE Reagent Choice and Detection Strategy for RNA Structure Probing Studies. Biochemistry 2019, 58 (23), 2655–2664. https://doi.org/10.1021/acs.biochem.8b01218.
  4. ↑ 4.0 4.1 Watters, K. E.; Lucks, J. B. Mapping RNA Structure in Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq). Methods in molecular biology 2016, 135–162. https://doi.org/10.1007/978-1-4939-6433-8_9.
  5. ↑ 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.
  6. ↑ 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.
  7. ↑ 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.
  8. ↑ 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.
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