View source for SHAPE

<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>
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<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>
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<p>== Overview ==</p>
<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>
<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>
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<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>
<p>=== PCR ===</p>
<p>Teaching about PCR</p>
<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>
<li>http://www.chem.unc.edu/rna/pdf-files/2005_em_jacs.pdf the original 2005 scientific paper</li>
<li>The [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362945/figure/fig1/ figure 1] of [http://www.ncbi.nlm.nih.gov/pubmed/22685659 'Probing Retroviral and Retrotransposon Genome Structures: The "SHAPE" of Things to Come.'] presents a good overview of this method.</li>
<li>The Wikipedia article about the reverse transcriptase:&nbsp;http://en.wikipedia.org/wiki/Reverse_transcriptase</li>
<li>[http://rnajournal.cshlp.org/content/17/9/1688.full.pdf Correlating SHAPE signatures with three-dimensional RNA structures]</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>
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