Figure 1: Specific Aim 1 proposes selection against the “Native monomer” of alpha-synuclein seen on the far left, Specific Aim 2 selects against the “Fibril” on the far left. The oligomer is a toxic intermediate conformation as the protein goes from monomer to fibril. (1, 14)Alpha-Synuclein (AS) is a protein that is expressed most abundantly in the brain and other nerve tissues. More is known about the dysfunction of AS than the normal function. (1) This protein is associated with up to 11 different gene mutations that can adversely affect the conformation of the protein and proteins it may interact with.(6) Genetic or environmental factors can cause AS to aggregate and form buildups of the protein in neuronal cell bodies, away from the cell membrane; the aggregations of AS are called Lewy bodies. These structures were originally thought to be the cause of the death of dopaminergic neurons characteristic of Alzheimer’s Disease. (6,11,13) More recent research suggests, however, that the pathogenic form of the protein is the five part oligomer that, when associated with cell membranes, forms a pore-like structure; the going theory is that the Lewy Bodies are the cell’s defense mechanism against the pore-like oligomer.(14)
Specific Aim 1: This research will seek to identify an RNA aptamer with an affinity for the functional monomer of the AS protein, Figure 1. Single stranded RNA aptamers will be selected from a pool of random RNA sequences through repeated rounds of selection in a "process of elimination" type assay. An aptamer could functionally inhibit the monomer, preventing it from forming the toxic oligomer. Adding a fluorescent tag to the aptamer would prove useful for helping to further research of how and why the protein misfolds to form the Lewy bodies by allowing for direct observation.(4)
Specific Aim 2: It would be doubly useful to perform simultaneous rounds of selection against samples of the aggregate forms of AS (see Figure 1) including negative selections in order to increase the in vivo affinity and specificity of the respective aptamers. An aptamer that could bind to the aggregated Lewy bodies would be integral in early diagnostics by adding visible tags to the aptamer.(8) Since Lewy body formation is also often associated with decreased proteosome effectiveness, an aptamer that was tagged with ubiquitin could be used in therapeutics to aid the cell in proper destruction of the aggregations. (5, 9,12)
This protein can be purchased through the GenWay website for $165 for every .1mg. The catalog number is 10-663-45667.
-Cori Booker
Here is the link to my proposal as of 09/16/2011:
This is the link to Progress Report #1:
Link to Final Manuscript via google.docs:
Link to Final Manuscript on Blogger:
6 comments:
This sounds really cool. I hope you'll get something that'll lead to greater understanding of the function of this protein in the neural system. Good luck!
How in the world did you make that figure? Is there a specific program that you need to make that?
Great job, Cori. Just add a little background about what aptamers are and you will be set.
I really like your protein, Cori! I hope you will be able to discover the effects of binding at different sites, and I am very much interested in the function of this protein. Good luck!
You have a very interesting protein that needs lots of research! If all goes well, your project could be very beneficial to the medical field. Good luck with your project!
Great work. This selection sounds very interesting. I know you guys are limited on price, but, if you are interested in an aptamer that only binds the functional form, it may make sense to acquire the mutated form for negative selection. This would be similar to to MBB protein selection open vs. close.
Post a Comment