Ryan Lannan, September 16th, Fall 2011, Pool N58, RNA, Target: KRAS

Ryan Lannan
September 16th, Fall 2011
Pool N58, RNA
Target: KRAS

Here's the link to my proposal and below is my abstract:
https://docs.google.com/document/d/1s5lTDrqHdf2tS0prrOIXlE25dJKhSJH7VBnjyni60Dk/edit?hl=en_US

Here's the link to my Final Report
http://dl.dropbox.com/u/51887677/Final%20Report%20-%20Ryan%20Lannan%20-%20KRAS%2C%20N58.doc

Creating a Model for Selection Against RHES: Creating an Aptamer Targeting KRAS

Huntington’s disease (HD) is an autosomal dominant disorder that leads to neurodegenerative disease (1,2). The genetic defect leading to Huntington’s occurs on chromosome 4, which when translated creates a mutant form of the huntington protein (mHtt) (1,2). Over time mHtt clumps, forming insoluble clusters of the protein in all cells of the body, but in the striatum of the brain it causes apoptosis and cell damage in the cerebrum (3). It was at first thought that these clumps were the cause of the damage seen in HD but the precise pattern of damage in HD has led scientists to find another culprit, RHES. RHES is a protein expressed heavily in the striatum that helps mHtt become soluble, and it has been found that the soluble form of mHtt is cytotoxic (3). This combination has been found to be extremely effective at killing cells when expressed together, 50% of cells die within 48 hrs when both proteins are present (3).
Since mHtt is a variable protein depending on the specific HD mutation, targeting RHES for inhibition may be more effective. Aptamers (RNA ligands displaying molecular recognition) could be a possible inhibitory agent. Aptamers are “functional nucleic acid sequences that have been enriched from a random sequence pool that display enhanced molecular recognition to a target” (Complete RNA Bead Selection Protocol, 2011). In other words, they are an RNA sequence selected for from a random RNA pool that binds to a protein with a very high affinity. They can have a countless number of possible unique 3-dimensional structures built from loops, bulges, stems, pseudoknots, hairpins, triplexes or quadruplexes (5).
In order to perform the SELEX process, significant quantities of protein are required, which is why it is not yet practical for our stream to perform a selection against RHES because of its high cost. But Jessica Hicks is in the process of applying for a grant from the Cure Huntington’s Disease Foundation (CHDI) and just needs preliminary results that show promise for a RHES selection. How we are going to do that without RHES protein is by selecting for a similar but affordable protein. Jessica Hicks has selected KRAS, a member of the same RAS family of proteins that RHES is a part of. KRAS has similar structure and binding capabilities as RHES (Jessica Hicks). Ordering information for KRAS is as follows (Abcam, 1-888-772-2226, ab96817, $260/100ug).

Specific Aim 1:Perform SELEX in order to select an aptamer against KRAS protein that will bind to a site similar to RHES’s sumoylation site, proving that a selection done by this lab against RHES is possible.
Specific Aim 2: Perform SELEX in order to select an aptamer against RHES protein that will deactivate its sumoylation site, hopefully deactivating it’s role in creating soluble mHtt (3).


Figure 1: The creation of a RNA ligand against RHES could interfere with its interactions with mHtt, potentially stopping nerve degeneration in cases of Hungtington’s.

References

1. Lang A. Other movement disorders. In: Goldman L, Ausiello D, eds. Cecil Medicine. 23rd ed. Philadelphia, Pa: Saunders Elsevier;2007:chap 434.\

2. Jankovic J, Shannon KM. Movement disorders. In: Bradley WG, Daroff RB, Fenichel GM, Jankovic J, eds. Bradley: Neurology in Clinical Practice. 5th ed. Philadelphia, Pa: Butterworth-Heinemann Elsevier; 2008:chap 75.

3. Subramaniam S, Sixt KM, Barrow R, Snyder SH. Rhes, a Striatal Specific Protein, Mediates Mutant-Huntington Cytotoxicity. Science. 2009;1327(324).

4. Ellington, Szostak, (1990) “In Vitro Selection of RNA molecules that bind specific ligands.” Nature. 818-822

5. Stoltenburg et al. (2007) “SELEX – A (r)evolutionary method to generate high-affinity nucleic acid ligands.” Biomolecular Engineering. 381-403