Hailey Ferrell's CIAP Aptamer Project (2018)

Anti-Calf Intestinal Alkaline Phosphatase Aptamer Diagnostic Tool for Multiple Sclerosis

Multiple Sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS) leading to the deterioration of the myelin in nerve cells as well as axonal damage and scar tissue formation (Raphael et al., 2015). MS affects over 2.5 million people worldwide, and yet there is currently not a conclusive, permanent diagnostic test for this disease. Current studies have conducted tests to screen for mRNA (messenger RNA) produced by MS to determine a potential biomarker. A sequence of mRNA identified as IL7R has been categorized as a potential biomarker for the disease in three different studies (Raphael, et al., 2015). There are no current studies determining IL7R to be a product of any other disease, which means it could be specific to MS. This project will utilize an inhibitory anti-CIAP aptamer, the reporter enzyme CIAP, and a toehold displacement mechanism to develop an ELAA (Enzyme Linked Aptamer Assay) diagnostic tool for Multiple Sclerosis.

CIAP is a reporter molecule, meaning the product of its catalytic activity will fluoresce. An aptamer is a sequence of oligonucleotides with a high binding affinity to a target molecule, in this case CIAP. Previous work of Vincent Huynh from the University of Texas at Austin Aptamer Research Lab found an aptamer (H-50) against CIAP; however, this aptamer does not inhibit CIAP’s function (Huynh, 2012). An inhibitory aptamer found against CIAP would be modified to contain a strand of RNA complimentary to the mRNA potential MS biomarker, IL7R which will be referred to as the linker sequence. The linker sequence will be integrated into the stem loop of the inhibitory aptamer which is important to its three-dimensional structure and binding affinity to CIAP. The anti-CIAP aptamer modified with the linker sequence will be referred to as the anti- CIAP complex. The H-50 aptamer, CIAP, and the anti-CIAP complex will become the Enzyme Linked Aptamer Assay complex as shown in Figure 1.

The configuration of the ELAA is an aptamer/target/aptamer sandwich type (Park et al, 2014). Figure 1 demonstrates the setup of this diagnostic tool. The H-50 aptamer will act as the anchor in the ELAA because it does not inhibit the functionality of CIAP. The anchoring aptamer (H-50) is immobilized onto a glass plate, then CIAP and the modified anti-CIAP aptamer will be immobilized onto H-50 to create the ELAA complex. Once blood serum containing IL7R is introduced to the ELAA complex, IL7R will bind with a high affinity to the linker sequence. This will change the three-dimensional configuration of the anti-CIAP aptamer, thus displacing it from CIAP. The plate will be washed to remove the extraneous reagents leaving a certain number of functioning enzymes on the plate. CIAP’s substrate (pNPP and H2O) will be introduced to the plate and the functioning reporter molecules will be free to catalyze the substrate producing a colorimetric response. The amount of fluorescence will be measured quantitatively through spectroscopy first determining the normal amount of IL7R produced in the body then comparing this amount to the fluorescence produced by an MS patient. The results are expected to conclude a higher concentration is produced from the blood serum of an MS patient. This diagnostic tool is only lacking a modified inhibitory aptamer against CIAP which this research aims to find.

The SELEX method as shown in Figure 2, will be utilized to obtain the inhibitory aptamer employed in the ELAA. SELEX is a cyclical process to obtain aptamers and is comprised of multiple rounds of selection. For one round of selection, a pool of RNA, is introduced to the target protein CIAP which is biotinylated and immobilized onto streptavidin coated beads. The bound species is eluted then converted into DNA to enable amplification of the pool. The amplified pool is then converted back into RNA then purified and quantified to begin the next round of selection. Each round produces a less diverse and more specific pool. After multiple rounds of selection, the pool will be sent for sequencing to determine the presence of a repeated sequence of RNA. If one is found this means a selection has occurred, which could be an indication of an aptamer. Binding assays will then be performed with the aptamer and target molecule to see where and how well the aptamer has bound. One round of aptamer selection against CIAP has been completed. The second round of selection is ready to commence with a starting RNA pool concentration of 64.27 μM. The second round will utilize filter-based in vitro aptamer selection to obtain a pool more specific to the target protein. Five to six more rounds, including negative selections to eliminated unwanted binders, will need to be completed before the pool can be sent for sequencing to identify if a selection has occurred. An inhibitory aptamer, modified with the linker sequence, found against CIAP sequence would complete the ELAA diagnostic tool for Multiple Sclerosis.

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References

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https://drive.google.com/file/d/0Byh3yR_5- FWXSDlueS01bEVNNGtDcG1FM1IzSkVtS0dONEdR/view

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