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Team:UCLA/Project/mrnaDisplay
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<h4>How does it work?</h4> | <h4>How does it work?</h4> | ||
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Revision as of 23:19, 25 September 2013
Contents |
Historical Background
Richard Roberts and Jack Szostak from Harvard developed this technique in 1997. They were able to generate and identify a myc epitope starting from a random mrna library using this technique.
What is it?
mRNA display is a technique used to select for proteins that bind to specific targets. It allows for the identification of these selected proteins because they are covalently attached to the DNA that codes for them.
How does it work?
- First generate a library of DNA using pcr or some other method.
- Next DNA library is transcribed in vitro to an RNA library.
- With the help of a DNA splint, this RNA strand is ligated to a single strained DNA that contains a puromycin protein at the 3 ' end
- This RNA-puromycin fusion is then translated in vitro.
- The Puromycin mimics a tRNA and is incorporated into the A site of the ribosome. However the puromycin has a non hydrolizable amide bond, therefore terminating translation and releasing the polypeptide, which is still bound to the RNA template through the puromycin.
- You now have a library of proteins covalently attached to the RNA that coded for them.
- In order to create a more stable structure, the RNA is converted to a DNA-RNA duplex through in vitro reverse transcription
- Now out you can screen for the protein out of your library that best binds your target molecule.
- Once you have your protein, it can be easily identified because it is still bound to the DNA that coded for it.
- Simple pcr of the DNA followed by sequencing will give you the identity of your protein, (ie the sequence that codes for it.)
What does the MTD have to do with mRNA display?
The MTD protein will be our scaffold for our MRNA display experiment. Most proteins are not capable of undergoing the mutation necessary to create a large library while still maintaining their structure and function. Therefore we needed a protein whose structure allows it to be mutagenized in certain sites and still remain stable. The MTD has evolved in the natural system of bordatella bacteriophage to have precisely this property. Therefore it acts as an effective scaffold because the majority of the protein is able to remain constant and stable while a small localized region of each protein gets mutagenized.
Other approaches for directed protein targeting?
-Phage display: An in vivo technique similar in principle to mrna display in which bacteriophage are allowed replicate to create a diverse library with different binding affinities. The DNA from the bacteriophage that bind the target can be isolated and then sequenced.
-Ribosome display: Another in vitro technique that fuses the rna transcript to the protein it codes for. Instead of a puromycin linker between the transcript and the protein, it is the ribosome that links the protein and the template that codes for it.
What are the advantages of this route / why did we choose this route for our project?
One of the major advantages of the mRNA display approach is that it takes place completely in vitro. This eliminates the limitations and complications that come with working in vivo and gives us more control over the entire process. Also, using an in vitro method allows us to create a more diverse library. Generating the library in vitro can potentially yield several orders of magnitude more protein variants than in vivo systems such as phage display.
With respect to ribosome display, which is also an in vitro method, mRNA has an advantage when it comes to screening for protein binding. The disadvantage of ribosome display is that the linker between the protein and the template DNA is the actual ribosome. This is an issue because the ribosome is a very large protein and it has the potential to interact with the target protein, thereby throwing off the selection process. In contrast, the puromycin link in mRNA display is much smaller and is not likely to interfere in any way with the target protein.
Issues With using this route?
What we are essentially doing with this project is taking the MTD protein which comes from an in vivo system (bordatella phage) and using it as the scaffold in our in vitro protein selection system. We know that in that native system of bordatella phage, a large library of phage are generated each with unique variants of the mtd protein on its tail fibers. Out of this large library, some phage are able to effectively bind to a certain target using the mtd protein.
A major concern is that the mtd protein may not behave the same way in vitro as it does in the natural bordatella system. Although we are theoretically able to generate more variants of mtd than in the natural phage system, it is possible that by isolating it from the phage could seriously affect its binding affinity. ‘For example, the native phage has several tail fibers, each with a copy of the mtd that could potentially bind a target. It may be that one isolated mtd has a very low binding affinity and that it is necessary to have several in order to create a strong enough binding interaction with the target.
