Amarachi Ahiarah's SOD1 Aptamer Project (2017)
Anti-Superoxide Dismutase Aptamer for the Inhibition of Amyotrophic Lateral Sclerosis
Introduction
Millions of Americans are impacted by neurologic disorders caused by the death of nerve cells. 1 The deterioration of these cells cause diseases such as Amyotrophic Lateral Sclerosis (ALS). In this disease, the degradation of nerve cells causes loss of muscle function, gradually inhibiting necessary daily functions such as walking, eating and even breathing. 2 Studies have shown that ALS is related to the mutation of the protein called Superoxide Dismutase. Previous research suggest the mutation of this protein causes it to have toxic activity that promotes aggregation, and that its mutant form may be related to the nerve cell death that causes these diseases. 3 Amyotrophic Lateral Sclerosis; overexpression and aggregation of the mutated Superoxide Dismutase was found to be linked to motor neuron diminishment. 5
As seen in figure 1, Superoxide Dismutase is an enzyme used to catalyze the redox reaction that forms oxygen (O2 ) or hydrogen peroxide (H2O2 ) from harmful superoxide radicals (O2 - ). The protein is present in most all oxygen containing organisms, is mainly located amongst the cytoplasm and mitochondrial intermembrane space, and is in the form of 16.8 kDa homodimer in vascular tissue. 9
An aptamer is a sequence of oligonucleotides that binds specifically to a target. Aptamers are often compared to antibodies because of their shared high specificity and affinity to target molecules. A benefit of aptamers versus their counterpart antibodies is the fact that aptamers are acquired through chemical synthesis, meaning that they can be mass produced, and their production cost is less. 8 Aptamers are beneficial because of their high affinity towards a specific protein of choice. In this experiment, an aptamer that binds to Superoxide Dismutase will be sought in order to inhibit its function in the case of mutation, making the aptamer a therapeutic.
To find an aptamer against Superoxide Dismutase, Selection, or the Systematic Evolution of Ligand by Exponential enrichment (SELEX) method will be performed. Bead based selection is used because it is the process by which target bound RNA is partitioned from the unbound RNA pool. The target, after being immobilized with beads that act as a scaffold to hold the protein, is subjected to incubation with an oligonucleotide pool. Then through successive washes with a buffer solution, the unbound or weakly bound pool is washed away. Once the strongest binding pool is obtained, it is amplified and converted to double stranded DNA through the Polymerase Chain Reaction, then converted back into single stranded DNA through translation. Once the RNA polymerase recognizes the promoter sequence during translation, it is able to begin building the unincorporated nucleotide triphosphates into the growing RNA chain. The subsequent RNA is then purified and visualized through Polyacrylamide Gel Electrophoresis and then quantified using spectrophotometry.
A round of selection is currently underway, and the steps through RNA quantification has been completed. However due to the low concentration of RNA acquired, the steps through transcription will be redone.
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References
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