Guatam Rangappa's CIAP Aptamer Project (2019)

Identification of an Anti-CIAP RNA Aptamer for Detection of Prostate Cancer Protein (KLK-15) Utilizing an ELONA

Over the years, the rate of prostate cancer cases has risen drastically and leads as one of the most prominent causes of deaths among men. In 2014 alone, 20.1 per 100,000 men died due to prostate cancer (Alam et al., 2014). Prostate cancer is caused by mutations in DNA lead to uncontrollable growth of cells in the prostate of men (Figure 2). To treat prostate cancer many antibodies, such as monoclonal antibodies, are currently being administered and the cost of treatment is very high. Examples of monoclonal antibodies include anti-human epidermal growth factor receptor-2 (HER2) MAb trastuzumab, anti-epidermal growth factor receptor (EGFR) MAbs cetuximab and panitumumab, and the antivascular endothelial growth factor (VEGF) MAb bevacizumab (Benham and Harris, 1979). Monoclonal antibodies work by identifying tumors and working as a therapeutic to suppress pain and inflammation. Monoclonal antibodies bind to the same epitope/antigen, detect tumors, and secrete molecules that purify tumors, thus reducing pain. Compared to antibiotics, aptamers are easily selected against small molecules, are ten times smaller than antibiotics, and use chemical synthesis, which reduces lot-to-lot variability. More research into aptamers can promote a cost-effective way to replace antibodies with more long-lasting effects.

CIAP, or calf intestinal alkaline phosphatase, is a reporter molecule used to remove phosphate groups from the 5’ end of the DNA strands to prevent self-ligation (Figure 3) of a linearized plasmid (Bilski et al., 2017). It is found in the mucus of calf intestines. It weighs 69 kDa and functions as a dimer (Chen et al., 2010). It has been used to try and reduce inflammation and detect the presence of other cancer proteins, such as prostinogen (KLK15). Prostinogen is a genetic mutation that is a leading cause of prostate cancer (Estaki et al., 2014). In my previous work with the Aptamers Stream, research was done to further explore and identify an inhibition aptamer that would be used for detection of prostinogen. In 2012, Vincent Huynh discovered an aptamer against CIAP. Further research is being done in the Ellington lab at the University of Texas at Austin to learn more about the aptamer that was selected for and CIAP itself. The application that will be worked on this semester is a diagnostic to test for the prostinogen(KLK15), a prostate cancer protein. Prostinogen is activated by a molecule binding to its active site. An ELONA, or enzyme-linked oligonucleotide assay, will be used to aid as a test to help with detection. In the presence of prostinogen along with an inhibiton aptamer, CIAP will send a response to fluorescents to illuminate. Thus, a color change will occur and be visible to the human eye. Selection against CIAP would use CIAP’s capability as a receptor molecule and enhance it to respond in the presence of prostate cancer proteins.

Aptamers, or oligonucleotide molecules that bind to a specific target molecule, are created by selecting them from a large random sequence pool. By selecting for aptamers, one can develop applications for their use. Previous research involving CIAP has led to the creation of therapeutics to reduce inflammation and detection of other cancer proteins (Fawley and Gourlay, 2016). A diagnostic to detect prostate cancer proteins will be developed, because not that much attention is being directed toward early detection of the condition. The diagnostic would be helpful since CIAP will bind to streptavidin beads and detect the presence of prostinogen with an inhibition aptamer, thus causing fluorescents to stimulate a response that changes color. The purpose of the diagnostic is to allow for better detection of prostate cancer proteins and would reduce the burden many men facing prostate cancer encounter.

The basic steps of streptavidin bead-based aptamer selection against CIAP using the N71 RNA pool were as follows. To begin bead based selection, 100 uL of magnetic streptavidin beads were used to bind to 10 uL of CIAP target. 10 uL of N71 pool was then immobilized to the beads and successive washes were performed. Ethanol precipitation of the selection washes were performed and reverse transcription was then executed to make ssDNA to run cycle course PCR. The ideal cycle of E1 was then determined and used to run large scale PCR to verify the ideal cycle was correctly identified. During PCR, ssDNA is converted into dsDNA. After a band appeared in lsPCR, ethanol precipitation is again performed along with transcription to convert dsDNA back into RNA. PAGE is then run to extract purified RNA. When visualizing the PAGE gel, a shadow appeared, thus verifying purified N71 RNA was present. Ethanol precipitation was once again performed, and the RNA was quantified by using the nanodrop to measure the concentration. Calculations were then executed to ensure enough volume of the RNA was collected for the RNA concentration. Round 1 is almost completed for aptamer selection, but a positive primer check will need to be run on a PAGE gel to identify whether the issue is with the reagents.

CIAP has an isoelectric point of 5.7. Instead of binding nucleic acids, CIAP prevents self-ligation. It has an activator of Mg-2, Zn, and CA-2 and its high bonding affinity arises from uneven positive and negative charges (Ren et al., 2012). CIAP is stable and functional in any buffer used around body temperature. It is stored in 10 mM Tris-HCl and Tris-HCl buffer can be used as its selection buffer (Riggle et al., 2013). For my application, since it is a bodily application, I will use 1X PBS buffer as the selection buffer because it most ideally mimics body conditions. Both PBS buffer and the human body have a pH around 7.4.

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