User:Stormyruiz/sandbox: Difference between revisions
Content deleted Content added
Stormyruiz (talk | contribs) m wording |
Gtsourdinis (talk | contribs) →Chaperone-Assisted RNA Crystallography (CARC): I added comments next to the sentences, sections that I thought could use some improvement. Overall, very interesting and novel contribution! Cheers! |
||
| (16 intermediate revisions by one other user not shown) | |||
| Line 1: | Line 1: | ||
{{dashboard.wikiedu.org sandbox}} |
{{dashboard.wikiedu.org sandbox}} |
||
= Chaperone-Assisted RNA Crystallography (CARC) = |
|||
''This is meant to be a subsection within the Biography section of the main article entitled [[Antoine Lavoisier]]'' |
|||
[[File:RNA-Fab crystal.jpg|thumb|Example of a crystal formed by CARC using a Fab]] |
|||
Chaperone-Assisted RNA Crystallography (CARC) is a method of [[RNA]] [[Crystallography]] that utilizes a binding protein ([[Chaperone (protein)|chaperone]]) to facilitate crystal formation. Chaperones may include [[Antibody|antibodies]]<ref name="Fab CARC">{{cite journal|last1=Sherman|first1=Eileen|last2=Archer|first2=Jennifer|last3=Ye|first3=Jing-Dong|title=Fab Chaperone-Assisted RNA Crystallography (Fab CARC)|journal=Methods Molecular Biology|date=2016|volume=1320|pages=77-109|doi=10.1007/978-1-4939-2763-0_7|pmid=26227039}}</ref><ref name="Abs as RNA Crystal Chaperones">{{cite book|last1=Huang|first1=Hao|title=Antibodies as RNA crystallization chaperones|date=2015|publisher=DAI-B 76/08(E), Dissertation Abstracts International|location=Ann Arbor|isbn=9781321645439|pages=171|url=https://search.proquest.com/docview/1667702187?accountid=14657}}</ref>, [[DARPin|DARPins]]<ref name="DARPins">{{cite journal|last1=Sennhauser|first1=Gaby|last2=Grutter|first2=Markus|title=Chaperone-Assisted Crystallography with DARPins|journal=Cell Structure|date=October 8, 2008|volume=16|issue=10|pages=1443–1453|doi=10.1016/j.str.2008.08.010}}</ref>, and other types of proteins.<!-- I'm not an expert in this, but it might be helpful to provide one last concrete example in this trio of protein chaperone examples, in order to guide readers genuinely interested in researching this more --> |
|||
== Purpose of a chaperone == |
|||
While crystallography is a popular method for determining the structures of biological molecules <ref name="PDB Statistics"> [http://www.rcsb.org/pdb/static.do?p=general_information/pdb_statistics/index.html RCSB Protein Data Bank Statistics] </ref> (see the PDB's<!-- perhaps spell out what PDB stands for for clarity --> website on number of structures obtained by experimental method [http://www.rcsb.org/pdb/static.do?p=general_information/pdb_statistics/index.html here]), less than 1,500 of the approximately 130,600 structures in the [[Protein data bank]] are of RNA. RNA is negatively charged and has extensive secondary but not tertiary structure, thus RNA does not form appropriate crystal contacts to make a lattice structure. While adding ions help to stabilize the negatively charged backbone, the issue still remains of locking RNA into one rigid conformation so that crystals form and phasing is good. |
|||
To combat this problem, chaperones are used to help stabilize RNA and form a regular lattice structure. Common chaperones used in research are [[Fragment antigen-binding|Fabs]]<ref name="Fab CARC" /><ref name="Abs as RNA Crystal Chaperones" />, the region of an antibody that binds the [[Antigen|antigen]] (in this case, RNA), including the [[Paratope|paratope]]. |
|||
==== Research benefitting the public good ==== |
|||
== Fab CARC Process == |
|||
While Lavoisier is commonly known for his contributions to the sciences, he also dedicated a significant portion of his fortune and work toward benefitting the public<ref name="Supplement">{{cite book|last1=Duveen|first1=Dennis I.|title=Supplement to a bibliography of the works of Antoine Laurent Lavoisier, 1743-1794|date=1965|publisher=Dawsons|location=London}}</ref><ref name="Bibliographic">{{cite book|last1=McKie|first1=Douglas|title=Bibliographic Details Antoine Lavoisier, the father of modern chemistry, by Douglas McKie ... With an introduction by F. G. Donnan|date=1935|publisher=V. Gollancz ltd.|location=London}}</ref><ref name="Bibliographic P">{{cite book|title=Bibliographic Details Lavoisier in perspective / edited by Marco Beretta.|date=2005|publisher=Deutsches Museum|location=Munich}}</ref><ref name="Lavoisier in the year one">{{cite book|last1=Bell|first1=Madison Smart|title=Lavoisier in the year one|date=2005|publisher=W.W. Norton|location=New York}}</ref>. Lavoisier was a humanitarian – he cared deeply about the people in his country and often concerned himself with improving the livelihood of the population by agriculture, industry, and the sciences<ref name="Bibliographic">{{cite book|last1=McKie|first1=Douglas|title=Bibliographic Details Antoine Lavoisier, the father of modern chemistry, by Douglas McKie ... With an introduction by F. G. Donnan|date=1935|publisher=V. Gollancz ltd.|location=London}}</ref>. The first instance of this occurred in 1765, when the [[French Academy of Sciences]] when he submitted an essay on improving urban street lighting<ref name="Bibliographic">{{cite book|last1=McKie|first1=Douglas|title=Bibliographic Details Antoine Lavoisier, the father of modern chemistry, by Douglas McKie ... With an introduction by F. G. Donnan|date=1935|publisher=V. Gollancz ltd.|location=London}}</ref><ref name="Bibliographic P">{{cite book|title=Bibliographic Details Lavoisier in perspective / edited by Marco Beretta.|date=2005|publisher=Deutsches Museum|location=Munich}}</ref><ref name="Lavoisier in the year one">{{cite book|last1=Bell|first1=Madison Smart|title=Lavoisier in the year one|date=2005|publisher=W.W. Norton|location=New York}}. |
|||
[[File:Phage display cycle.png|thumb|Phage display cycle]] |
|||
A common method to select appropriate chaperones is by [[Phage display]].<ref name="Phage Display net">{{cite web|title=Phage Display Technology|url=http://phagedisplay.net/|website=Phage Display}}</ref> This method utilizes Fab-bound phage in order to allow for easy selection and subsequent amplification. Tight-binding Fabs are selected for against the desired RNA, and the unbound Fab-phage complexes are washed away. Fab-phage complex is then eluted and cell culture is infected with the phage in order to amplify the Fabs that passed that round of selection.<ref name="Fab CARC" /><ref name="Phage Display Technique">{{cite journal|last1=Hammers|first1=Christoph|last2=Stanley|first2=John|title=Antibody Phage Display: Technique and Applications|journal=Journal of Investigative Dermatology|date=2013|volume=134|pages=1-5|doi=10.1038/jid.2013.521}}</ref> Then, another round of selection and amplification can be performed and after sufficient rounds, the Fab can be sequenced to determine the primary structure sequence of Fabs that bind most efficiently. |
|||
However, Phage display alone is insufficient to find a chaperone for crystallography. Simply because the Fab may have high affinity for the RNA, this does not necessarily mean that the Fab-RNA complex will crystallize.<!-- I would recommend rephrasing in order to sound more encyclopedic, like: "The Fab's high affinity to the RNA does not guarantee that the Fab-RNA complex will crystallize." --> To test if crystals are capable of forming, small-scale crystal trials are performed under various conditions. If crystals form, large scale trials can be set up and structure can be determined by [[X-ray crystallography|X-ray diffraction]]. |
|||
Three years later in 1768, he focused on a new project to design an aqueduct. The goal was to bring in water from the river Yvette into Paris so that the citizens could have clean drinking water. But, since the construction never commenced, he instead turned his focus to purifying the water from the Seine. This was the project that interested Lavoisier in the chemistry of water and public sanitation duties<ref name="Lavoisier in the year one">{{cite book|last1=Bell|first1=Madison Smart|title=Lavoisier in the year one|date=2005|publisher=W.W. Norton|location=New York}}</ref>. |
|||
Another method to be used that selects instead for an RNA against a specific chaperone is called [[Systematic evolution of ligands by exponential enrichment|SELEX]]. This works in the opposite way of phage display, in that a large pool of RNA is repeatedly selected for against a binding molecule and then amplified between each round by reverse transcription and PCR.<!-- I would spell PCR out, too and link it to the main wiki article, since it's one of the most popular biology assays --> |
|||
He additionally was interested in air quality, and spent some time studying the health risks associated with gunpowder’s effect on the air<ref name="Bibliographic P">{{cite book|title=Bibliographic Details Lavoisier in perspective / edited by Marco Beretta.|date=2005|publisher=Deutsches Museum|location=Munich}}</ref>. In 1772, he performed a study on how to reconstruct the Hôtel-Dieu hospital, after it had been damaged by fire, in a way that would allow proper ventilation and clean air throughout<ref name="Lavoisier in the year one">{{cite book|last1=Bell|first1=Madison Smart|title=Lavoisier in the year one|date=2005|publisher=W.W. Norton|location=New York}}</ref>. |
|||
== Applications == |
|||
At the time, the prisons in Paris were known to be largely unlivable and the prisoners’ treatment inhumane<ref name="Supplement">{{cite book|last1=Duveen|first1=Dennis I.|title=Supplement to a bibliography of the works of Antoine Laurent Lavoisier, 1743-1794|date=1965|publisher=Dawsons|location=London}}</ref>. Lavoisier took part in investigations in 1780 (and again in 1791) on the hygiene in prisons and had made suggestions to improve living conditions, which were largely ignored<ref name="Supplement">{{cite book|last1=Duveen|first1=Dennis I.|title=Supplement to a bibliography of the works of Antoine Laurent Lavoisier, 1743-1794|date=1965|publisher=Dawsons|location=London}}</ref><ref name="Lavoisier in the year one">{{cite book|last1=Bell|first1=Madison Smart|title=Lavoisier in the year one|date=2005|publisher=W.W. Norton|location=New York}}</ref>. |
|||
CARC can be used in many different branches of research, including medicine, biochemistry, biology, and other related fields. CARC, along with other methods, allows researchers to determine the 3-D structure of RNA and see activity such as stalled RNA machinery (see [[Ribosome|Ribosomes]], [[Ribozyme|Ribozymes]], [[Riboswitch|Riboswitches]], [[Spliceosome]]) to determine the mechanisms by which they work and allow us to better understand their functions and how they work.<!-- I feel like this last sentence is saying the same thing thrice: "determine the mechanisms" = "their function" = "how they work," so I would eliminate 2/3 and perhaps highlight (with a few words and maybe a link to the page) one problem that CARC could alleviate in medicine [unless you already talk about that in your Scipedia digital museum] --> |
|||
Once a part of the Academy, Lavoisier also held his own competitions to push the direction of research towards bettering the public and his own work<ref name="Bibliographic P">{{cite book|title=Bibliographic Details Lavoisier in perspective / edited by Marco Beretta.|date=2005|publisher=Deutsches Museum|location=Munich}}</ref>. One such project he proposed in 1793 was to better public health on the “insalubrious arts.” |
|||
==== Sponsorship of the sciences ==== |
|||
Lavoisier had a vision of public education having roots in “scientific sociability” and philanthropy<ref name="Bibliographic P">{{cite book|title=Bibliographic Details Lavoisier in perspective / edited by Marco Beretta.|date=2005|publisher=Deutsches Museum|location=Munich}}</ref>. |
|||
Lavoisier gained a vast majority of his income through buying stock in the [[Ferme générale|General Farm]], which allowed him to work on science full time, live comfortably, and allowed him to contribute financially to better the community<ref name="Lavoisier in the year one">{{cite book|last1=Bell|first1=Madison Smart|title=Lavoisier in the year one|date=2005|publisher=W.W. Norton|location=New York}}</ref>. (It would also contribute to his demise during the [[Reign of Terror]] many years later<ref name="Antoine Lavoisier">{{cite book|last1=McKie|first1=Douglas|title=Antoine Lavoisier: scientist, economist, social reformer.|date=1952|publisher=Schuman|location=New York}}</ref>.) |
|||
It was very difficult to secure public funding for the sciences at the time, and additionally not very suitable financially for a living, so Lavoisier used his wealth to open a very expensive and sophisticated laboratory in France so that aspiring scientists could study without the barriers of securing funding for their research<ref name="Supplement">{{cite book|last1=Duveen|first1=Dennis I.|title=Supplement to a bibliography of the works of Antoine Laurent Lavoisier, 1743-1794|date=1965|publisher=Dawsons|location=London}}</ref><ref name="Lavoisier in the year one">{{cite book|last1=Bell|first1=Madison Smart|title=Lavoisier in the year one|date=2005|publisher=W.W. Norton|location=New York}}</ref>. |
|||
He also pushed for public education in the sciences. He founded two organizations, [https://fr.wikipedia.org/wiki/Lyc%C3%A9e_Lavoisier Lycée] and [[Musée des Arts et Métiers]] which were created to serve as educational tools for the public. Funded by the wealthy and noble, Lycée regularly taught courses to the public beginning in 1793<ref name="Bibliographic P">{{cite book|title=Bibliographic Details Lavoisier in perspective / edited by Marco Beretta.|date=2005|publisher=Deutsches Museum|location=Munich}}</ref>. |
|||
==Notes== |
==Notes== |
||
Latest revision as of 03:35, 2 June 2017
 | This is a user sandbox of Stormyruiz. You can use it for testing or practicing edits. This is not the sandbox where you should draft your assigned article for a dashboard.wikiedu.org course. To find the right sandbox for your assignment, visit your Dashboard course page and follow the Sandbox Draft link for your assigned article in the My Articles section. |
Chaperone-Assisted RNA Crystallography (CARC)
[edit]
Chaperone-Assisted RNA Crystallography (CARC) is a method of RNA Crystallography that utilizes a binding protein (chaperone) to facilitate crystal formation. Chaperones may include antibodies[1][2], DARPins[3], and other types of proteins.
Purpose of a chaperone
[edit]While crystallography is a popular method for determining the structures of biological molecules [4] (see the PDB's website on number of structures obtained by experimental method here), less than 1,500 of the approximately 130,600 structures in the Protein data bank are of RNA. RNA is negatively charged and has extensive secondary but not tertiary structure, thus RNA does not form appropriate crystal contacts to make a lattice structure. While adding ions help to stabilize the negatively charged backbone, the issue still remains of locking RNA into one rigid conformation so that crystals form and phasing is good.
To combat this problem, chaperones are used to help stabilize RNA and form a regular lattice structure. Common chaperones used in research are Fabs[1][2], the region of an antibody that binds the antigen (in this case, RNA), including the paratope.
Fab CARC Process
[edit]
A common method to select appropriate chaperones is by Phage display.[5] This method utilizes Fab-bound phage in order to allow for easy selection and subsequent amplification. Tight-binding Fabs are selected for against the desired RNA, and the unbound Fab-phage complexes are washed away. Fab-phage complex is then eluted and cell culture is infected with the phage in order to amplify the Fabs that passed that round of selection.[1][6] Then, another round of selection and amplification can be performed and after sufficient rounds, the Fab can be sequenced to determine the primary structure sequence of Fabs that bind most efficiently.
However, Phage display alone is insufficient to find a chaperone for crystallography. Simply because the Fab may have high affinity for the RNA, this does not necessarily mean that the Fab-RNA complex will crystallize. To test if crystals are capable of forming, small-scale crystal trials are performed under various conditions. If crystals form, large scale trials can be set up and structure can be determined by X-ray diffraction.
Another method to be used that selects instead for an RNA against a specific chaperone is called SELEX. This works in the opposite way of phage display, in that a large pool of RNA is repeatedly selected for against a binding molecule and then amplified between each round by reverse transcription and PCR.
Applications
[edit]CARC can be used in many different branches of research, including medicine, biochemistry, biology, and other related fields. CARC, along with other methods, allows researchers to determine the 3-D structure of RNA and see activity such as stalled RNA machinery (see Ribosomes, Ribozymes, Riboswitches, Spliceosome) to determine the mechanisms by which they work and allow us to better understand their functions and how they work.
Notes
[edit]- ^ a b c Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong (2016). "Fab Chaperone-Assisted RNA Crystallography (Fab CARC)". Methods Molecular Biology. 1320: 77–109. doi:10.1007/978-1-4939-2763-0_7. PMID 26227039.
- ^ a b Huang, Hao (2015). Antibodies as RNA crystallization chaperones. Ann Arbor: DAI-B 76/08(E), Dissertation Abstracts International. p. 171. ISBN 9781321645439.
- ^ Sennhauser, Gaby; Grutter, Markus (October 8, 2008). "Chaperone-Assisted Crystallography with DARPins". Cell Structure. 16 (10): 1443–1453. doi:10.1016/j.str.2008.08.010.
- ^ RCSB Protein Data Bank Statistics
- ^ "Phage Display Technology". Phage Display.
- ^ Hammers, Christoph; Stanley, John (2013). "Antibody Phage Display: Technique and Applications". Journal of Investigative Dermatology. 134: 1–5. doi:10.1038/jid.2013.521.