Lulia Abraha's CIAP Aptamer Project (2019)

RNA Aptamer Against Calf Intestinal Alkaline Phosphatase for an

Alternative ELIZA Test to Diagnose Systemic Lupus Erythematosus

Systemic lupus erythematosus is an autoimmune disease that causes systematic inflammation. In a healthy body, the immune system has antibodies designed to attack any foreign invaders and cancers. In lupus, the immune system starts to attack the body’s own healthy tissues. Lupus is often misdiagnosed because of the wide range of symptoms, including fatigue, weight loss, hair loss, and fever, that are not unique to the disease, making it difficult to diagnose (Paz 2017). The most accurate test for lupus currently is the Antinuclear Antibodies Test (ANA). This test is used to test for an overabundance of antibodies in a patient’s blood sample as shown in Figure 1.1 (ANA 20118). The sample from the patient is placed in a plate containing specific type of cells, usually extracted from sections of the liver, kidney, or human tissue culture cell lines (Petri 2019). If an antibody is present (shown in blue in figure 1.1), it attaches to the cells. Any unbound antibody is washed away. An antibody selected against human antibody (yellow) binds to the human antibody. The sample is incubated with a secondary antibody selected against human antibody and tagged with a florescent dye. Incubation allows the human antibody with the developed antibody to attach indicated by the florescent dye. In a positive ANA test, the sample will fluoresce (shown in green in figure 1.1) indicating that the person has lupus.

Although the ANA test is very effective when diagnosing lupus as well as other autoimmune diseases, the use of antibodies makes it more expensive. Replacing the antibody with an aptamer, a small oligonucleotide that has high affinity to bind to other molecules, is a more cost-effective option. Because antibodies are developed in vivo, they are more expensive to develop making aptamers more ideal. Access to healthcare is not available to everyone due to difference in socioeconomic status. As doctors order more tests to rule out certain diseases, the medical bill just increases. Utilizing the cheaper, yet effective, materials available can significantly decrease the bill.

Aptamers are oligonucleotides that bind to a specific target. Aptamers function similar to antibodies. Over the past years, aptamers have been used in variety of applications. An aptamer can be used for diagnostic purposes as it can be modified to signal the presence of another molecule. They can be used for therapeutic purposes as they can be modified to inhibit a protein function or a pathway in the body. Lastly, they can also be used to improve targeted drug delivery of functionalized payloads. An aptamer selected against CIAP can be used in many different applications, mostly serving as diagnostic tools. Because CIAP shows a colorimetric response upon phosphorylation, it can detect the presence of another molecule through a colorimetric response. In previous years, an aptamer against CIAP was found at the University of Texas at Austin. This aptamer was developed as an alternative reagent that is readily amendable to conjugation for ELISA. (Huynh, Wei, and Stovall 2015).

Although very similar in function, there are many reasons that makes aptamers ideal over antibodies. Aptamers are developed at the bench, using vitro selection. They are less costly, can be produced in large amounts, and are stable in extreme temperature and pH (Park 2014). A more effective diagnostic tool for lupus can be developed using aptamers. An aptamer against CIAP, a widely used reporter molecule, can be used to replace the secondary antibody in this ANA assay. The patients’ blood will be placed on a plate containing cells. Antibodies found in an affected person’s blood will attach to the cells in the plate as shown in figure 1.2. An aptamer developed against CIAP will then be conjugated to the antibody using streptavidin protein and biotin. Because streptavidin has high binding affinity to biotin, the antibody can be tagged with biotin which will then be expected to bind to an aptamer with streptavidin protein. In the presence of the antibody, CIAP is expected to show a colored signal due to dephosphorylation of p-Nitrophenyl phosphate (pNPP).

The first steps of developing the diagnostic tool is to select an aptamer that best binds toCIAP. The selection process, selecting a high binding RNA/DNA from a large RNA pool, isperformed using the SELEX (Systematic evolution of ligands by exponential enrichment)method (Prediger 2016). It begins with a large pool of nucleic acids. The nucleic acids are thenincubated with the target which allows some of the nucleic acids to bind. Those that are unbound are washed away and collected. The ones that do bind are eluted from the target and collected. This process of removing the bound nucleic acids from unbound is done using streptavidin bead-based selection. Beads are used to bind target protein. CIAP has a biotin affinity tag which has a high affinity to bind to streptavidin. All the bound and unbound RNA is reverse transcribed to DNA and then amplified using PCR. Once PCR is successful, the dsDNA can now be used for transcription. Transcription is done to transcribe RNA from the DNA template. After multiple rounds of selection, a potential aptamer is developed. Binding assays are used confirm that an aptamer with a high binding affinity to CIAP is developed. Upon completion of the following procedure, an aptamer against CIAP, a reporter molecule, to replace the antibody in the existing antinuclear antibody test should be developed for a more cost-effective diagnostic tool for lupus. This is done by performing multiple rounds of selection using the SELEX method. Currently, two rounds of selection against CIAP is complete. The target to pool ratio on the first round was 1:1. A total of 3 washes were performed. The resulting bound RNA from the first round of selection is 67 uM. The results showed that the majority of the RNA is binding to the target and very little is washed away. The next step is to perform another round of selection. To increase stringency, a total of 5 washes is done. The target to pool ratio will also be changed to 3:4. I will use 300 pmols of RNA with 400 pmols of target on the next round of selection. Currently, close to 1.5 million people are affected by lupus. Upon completion, this diagnostic tool will provide a cheaper, more accessible method to accurately diagnose lupus.

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References

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https://www.hopkinslupus.org/lupus-tests/lupus-blood-tests

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www.fasebj.org/doi/abs/10.1096/fasebj.29.1_supplement.562.6.

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WebMD, WebMD, 7 May 2019, www.webmd.com/lupus/arthritis-lupus#1.