Julliet Ogu's SOD1 Aptamer Project (2017)
Superoxide Dismutase 1 Aptamer Selection: A Potential Tool for the Development of Novel Anti-Cancer Therapeutics
Introduction and Background
Cells are the basic building blocks of the human body. These microscopic subunits differentiate and form the tissues and organs that facilitate the body’s vital processes. Each cell is controlled by thousands of genes which determine the cell’s function. As cells divide, this genetic information is passed onto newer cells. This process of cell division is usually stringently controlled by numerous mechanisms within the cells’ genetic makeup which prevent the proliferation of damaged cells. Cancer occurs when damaged cells begin to grow and divide uncontrollably, destroying healthy cells and invading other tissues. As cancer cells continue to multiply, they can form solid masses of living and necrotic tissue called tumors. These masses interfere with bodily systems and release hormones that alter bodily functions, leading to disability and death. As the world’s population continues to live longer, cancer rates are expected to soar in the next two decades (1). This has precipitated a greater need for the development of new anti-cancer therapeutics.
Recent research has revealed the potential of SOD1 as a novel target for cancer therapy (3). SOD1 stands for superoxide dismutase 1. This enzyme breaks down superoxide radicals, which are charged, toxic molecules that are damaging to cells. These radicals are also referred to as reactive oxygen species (ROS), and are produced as a by-product of a cell’s metabolic processes (4). To support their rapid division and growth, cancer cells are more metabolically active than healthy cells and thus are more susceptible to producing lethal amounts of reactive oxygen species (2). To combat this intrinsic state of oxidative stress, malignant cells rely heavily on antioxidant enzymes such as SOD1 to counteract ROS production (2). This leads to the overexpression of this enzyme in these cells (4).
SOD1 is a soluble cytoplasmic protein found in most eukaryotic organisms. The molecular weight of human SOD1 is 15,936 Da. This protein binds copper and zinc in a homodimer complex, and is one of two isozymes that are responsible for breaking down free superoxide molecules into hydrogen peroxide and molecular oxygen (6). This process aids in preserving the delicate balance between antioxidants and superoxidases that is vital for cells to survive. SOD1 is a somatic enzyme that functions optimally at a pH similar to that found in blood (7.35- 7.45) and has an overall negative charge and isoelectric point of 7.4. The active site of this enzyme has a positive charge due the zinc and copper ions embedded in this area. The copper binding site is the catalytic site, and is where free radical dismutation occurs. The binding of copper ions at this site is vital for the catalytic function of the enzyme.
This positively charged active site is favorable for nucleic acid binding (6). PBS (phosphate-buffered saline) has been shown to be a successful buffer for SOD1, as it has the desired pH and the enzyme is stable in this solution (5). No aptamer has yet been developed against SOD1. SOD1 has also been implicated in the pathophysiology of amyotrophic lateral sclerosis, a neuromuscular degenerative disease where certain mutations in the gene lead progressive muscle weakness, inability to control movement, and loss of muscle mass (6). Currently, arimoclomol is in clinical trial for the treatment of SOD1 mutant ALS patients. Labs owned by Orphazyme, a biopharmaceutical company, are spearheading the research and development of this drug. Thus far, this drug has been proven to delay disease progression in mice models (5).
Aptamers are oligonucleotide sequences that bind to a target molecule with high specificity. An aptamer selected against a desired target can be used in therapeutics, diagnostics, and the development of biosensors. These nucleic acids can be considered a valid alternative to antibodies, as they are more durable and cheaper to produce. An aptamer selected against SOD1 can be used as an anti-cancer therapeutic by allosterically inhibiting SOD1 in malignant cells. As stated previously, cancer cells express SOD1 at high levels due to their heightened susceptibility to ROS toxicity. An SOD1 aptamer can be functionalized to detect rapidly dividing cells that overexpress this protein and inhibit its expression and/or function. This would prevent these cells from efficiently eliminating toxic reactive oxygen species, thus inducing apoptosis. This experiment will attempt to find an SOD-1 aptamer using the Systematic Evolution of Ligands by Exponential Enrichment, or SELEX method (6). The process begins with the immobilization of the protein target onto magnetic nickel beads. A pool of undifferentiated RNA sequences is introduced to the immobilized target, and the bound RNA species eluted. The RNA species is reverse transcribed into ssDNA via reverse transcription and cycle course PCR is used to determine the optimal number of cycles for large scale PCR amplification. The amplified dsDNA is transcribed back into RNA and is then purified and concentrated using PAGE and ethanol precipitation. A binding assay will then be performed to confirm the presence of an aptamer. The nucleotide makeup of the aptamer will then be sequenced. The SELEX process will be repeated for several more rounds so as to increase the binding affinity of the binding species and enrich an aptamer.
Mock round of selection is currently underway. As of November, 1st 2017, the SELEX method has been performed up to ccPCR. In the first attempt, the results of ccPCR were inconclusive due to suspected discrepancies in PCR. Bead based selection and ccPCR were repeated, and contamination was found in the NTC after gel electrophoresis was conducted. Several contamination tests were performed until the sources of the contamination were identified and eradicated, and ccPCR may now progress. The objective of successfully completing ccPCR is to determine the most effective PCR cycle by which to amplify the nucleic acid sample. Large scale PCR can then be performed to obtain larger samples of the nucleic acids and the rest of 1st round selection can then be completed. After 1st round, more rounds of selection will be performed to increase the odds of finding an aptamer against SOD1. This aptamer can be functionalized for use in anti-cancer therapeutics.
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References
1)Information on increasing cancer rates: http://www.medicaldaily.com/cancer-trends-2017-why-are-cancer-rates-increasing-407270
2) E.O., Liu, J., Albitar, M. et al. “Intrinsic oxidative stress in cancer cells: a biochemical basis for therapeutic selectivity Hileman,” Cancer Chemother Pharmacol (2004) 53: 209. doi:10.1007/s00280-003-0726-5
3) Papa L, Manfredi G, Germain D ; “SOD1, an unexpected novel target for cancer therapy”. From the Department of Medicine, Division of Hematology/Oncology, Tisch Cancer Institute Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY (2008).
4) Bostwick, D. G., Alexander, E. E., Singh, R., Shan, A., Qian, J., Santella, R. M., Oberley, L. W., Yan, T., Zhong, W., Jiang, X. and Oberley, T. D. (2000), Antioxidant enzyme expression and reactive oxygen species damage in prostatic intraepithelial neoplasia and cancer. Cancer, 89:-
5) Son, Marjatta et al. “Biochemical Properties and in Vivo Effects of the SOD1 Zinc Binding Site Mutant (H80G).” Journal of neurochemistry 118.5 (2011): 891–901. PMC. Web. 1 May 2017.
6) Domenica Musumeci, Daniela Montesarchio, Polyvalent nucleic acid aptamers and modulation of their activity: a focus on the thrombin binding aptamer, Pharmacology & Therapeutics, Volume 136, Issue 2, November 2012, Pages 202-215, ISSN 0163-7258,
7) Sullivan, Lucas & S Chandel, Navdeep. (2014). Mitochondrial reactive oxygen species and cancer. Cancer & metabolism. 2. 17. 10.1186/2049-3002-2-17.
8) Domenica Musumeci, Daniela Montesarchio, Polyvalent nucleic acid aptamers and modulation of their activity: a focus on the thrombin binding aptamer, Pharmacology & Therapeutics, Volume 136, Issue 2, November 2012, Pages 202-215, ISSN 0163-7258,