Nucleic Acid Aptamer Selection for Myelin Basic Protein for the Prevention of Malignant Schwann Cells by Manjari Subramanian

Abstract:
Myelin basic protein (MBP) promotes growth of myelin sheath throughout the nervous system by binding to the lipids on the myelin membrane (Boggs, 2006). However when a malignant cell is formed, the abnormal cell inhibits the transmission of electric signals across synapses (Kaur et al., 2008). MBP has the ability to distinguish the difference between a normal and malignant Schwann cell due to deletions within the MBP gene sequence (Kaur et al., 2008).

Figure 1 Myelin Basic Protein: Computer model of the protein. Human myelin basic protein by binding it to a leukocyte antigen (Smith et al., 1998).
Aim 1: Select for the protein  
            A multi-layered process will be involved in the selection for MBP. Aptamers, used as RNA protein markers in this case, will be selected for myelin basic protein. Aptamers bind to proteins with high specificity and affinity to the specific protein target. This is because aptamers are about one-tenth the size of an antibody (Boggs, 2006). This provides a set of tools that aid in area of therapeutic diagnostics in medicine. MBP itself is not a large protein (only 18.5 kDa); thus using an aptamer, which is even smaller, will increase chances of binding to the protein (Saxe et al., 1985). By selecting for MBP, the gene sequence of the protein can be verified before further development of an aptamer to distinguish between a normal and malignant cell. If successful, further tests can be conducted to see if the aptamer can deliver a drug or select against a mutate MBP. The first step is to find an aptamer that binds to this specific protein.

The target can be ordered from US Biologicals at $327.00 per milligram and $2.40 per round (400pmol).
References:
Boggs JM (2006) Myelin basic protein: a multifunctional protein. Cell Mol Life Sci. 63: 1945–1961. 

Eylar EH, Brostoff S, Hashim G, Caccam J, Burnett P (1971). Basic A1 protein of the myelin membrane. The complete amino acid sequence. J. Biol. Chem. 246 (18): 5770–84. 

Gragoudas ES, Adamis AP, Cunningham ET Jr, Feinsod M, Guyer DR (2004). Pegaptanib for neovascular age-related macular degeneration. N Engl J Med. 351(27):2805-16.

Kaur GRoy I (2008). Therapeutic applications of aptamers. National Institute of Pharmaceutical Education and Research (NIPER). Expert Opin Investing Drugs.

Meinl, Weber, Drexler, Morelle, Ott, Saruhan-Direskeneli, Goebels, Ertl, Jechart, Giegerich (1993). Myelin basic protein-specific T lymphocyte repertoire in multiple sclerosis. Complexity of the response and dominance of nested epitopes due to recruitment of multiple T cell clones. J Clin Invest. 92(6):2633–2643
Saxe DF, Takahashi N, Hood L, Simon MI (1985). Localization of the human myelin basic protein gene (MBP) to region 18q22----qter by in situ hybridization. Cytogenet. Cell Genet. 39 (4): 246–9.
 Smith, K.J., Pyrdol, J., Gauthier, L., Wiley, D.C., Wucherpfennig, K (1998). Crystal structure of HLA-DR2 (DRA*0101, DRB1*1501) complexed with a peptide from human myelin basic protein. J.Exp.Med. 188: 1511-1520
Wang C, Neugebauer U, Bürck J, Myllykoski M, Baumgärtel P, et al. (2011). Charge Isomers of Myelin Basic Protein: Structure and Interactions with Membranes, Nucleotide Analogues, and Calmodulin. PLoS ONE 6(5): e19915.
Warren, K.J., Catz, I., Steinman, L. (1995). Fine specificity of the antibody response to myelin basic protein in the central nervous system in multiple sclerosis: The minimal B-cell epitope and a model of its features. Proc. Natl. Acad. Sci. USA. 92: 11061-11065.

Yin Zhang, Hao Hong, Weibo Cai (2011). Tumor-Targeted Drug Delivery with Aptamers. Current Medical Chemistry 1. 18(27): 4185–4194.

Research Proposal:

https://docs.google.com/document/d/1iiFltUcxxQ39oUpegZl-CNKEM1p1RdXQlP2_njopEnA/edit?usp=sharing

1st Progress Report:

https://docs.google.com/file/d/0By_LVGU6rig_aTc4b2I4aG5GWmc/edit?usp=sharing


The Brain's New Hero: Nucleic Acid Aptamer Selection Against Beta-Glucuronidase



Nucleic Acid Aptamer Selection Against Beta-Glucuronidase
Ellen Airhart
Nucleic Acid Pool: N71 RNA Pool
Target: Beta-Glucuronidase

Microglia are a type of glial cell found in the central nervous system. There, they act as the first line of defense of the nervous system.  Stimulation of microglia is a signal of infectious, inflammatory, and degenerative illnesses of the CNS. This inflammation could be indicative of many diseases, such as Alzheimer’s, dementia, and other neurodegenerative conditions. Studies have shown that there is a rise in the release of β- glucuronidase, or GUS, by stimulated microglia into the extracellular space at an area of neuroinflammation. GUS is involved in the hydrolysis of glycosaminoglycans on the cell surface and the degeneration of the extracellular matrix. Therefore, GUS might be a biomarker for continuing neurodegeneration induced by neuroinflammation (Antunes, Doorduin, Haisma, Elsinga, Waarde, Willemson, Dierckx & Vries, 2012).
An aptamer, or an olglionucleic acid or peptide molecule that have been carefully selected to bind to its target in a similar way to an antibody (Pai, Roberts & Ellington, 2008), has not been discovered for Beta-Glucuronidase. If an aptamer was developed to bind to GUS it could be used as a biomarker to assist in diagnosing neurodegenerative diseases.
Primary Specific Aim: Identifying an aptamer that binds to GUS-His


Figure 1 illustrates the active sites Glu451, Tyr504, Glu540, and Asn450. This high number of active sites increases the chance that there is an aptamer for this target.
Secondary Specific Aim: Use this aptamer as a biomarker for neurodegenerative inflammation

Target Order Information
Vendor: Ellington Lab
Vendor Website: https://www.ellingtonlab.org
Central Lab Telephone:  512-471-6445
Office Manager:  512-232-3426
Cost per Unit: $0
            Cost per Round: $0
Antunes, I., Doorduin, J., Haisma, H., Elsinga, P., Waarde, A., Willemson, A., Dierckx, R., & Vries, E. (2012). F-feanga for pet of β-glucuronidase activity in neuroinflammation. Journal of Nuclear Medicine, 53(3), 451-458.
Lawson L J, Perry V H, Gordon S (1992). "Turnover of resident microglia in the normal adult mouse brain". Neuroscience 48: 405–415.

Pai, S., Roberts , A., & Ellington , A. (2008). Aptamer amplification: divide and signal. Expert Opinion on Medical Diagnostics, 2(12), 1333-1346. doi: 10.1517/17530050802562016
Roffler, S., Wu, C., Schechinger, W., Chen, K., & Prijovic, Z. (2009). Human beta-glucuronidase mutants with elevated enzymatic activity under physiological conditions and methods for identifying as such. 
First Target Proposal
First Progress Report
Second Progress Report
Final Progress Report