Ted Rodriguez's CIAP Aptamer Project (2016)

RNA Aptamers Against Calf Intestinal Alkaline Phosphatase for Detection of Parkinson’s Disease

Introduction/Background

Currently, there is no standard diagnostic test for Parkinson’s (4). Current diagnosis of this disease is typically left to a family physician and is dependent on family history and a physical screening (4). While mostly accurate these screenings can have their drawbacks, mainly in that the diagnosis is currently reliant upon subjective examination by a doctor with no biochemical proof of the disease. A study done on the accuracy of the diagnosis of Parkinson’s disease showed that 15% of those diagnosed do not fulfill all clinical criteria for Parkinson’s (5). The same study further showed that 20% of patients with Parkinson’s who have been brought to medical attention were not diagnosed (5).

The exact way in which Parkinson’s occurs is still unknown. Current Research has shown it to occur from inflammation of the Substantia Nigra, a part of the brain known to control a person’s movement (2)(6). This inflammation is thought to be a reaction to Tumor Necrosis Factor Alpha (TNF-α), a messaging chemical released by the Liposaccharide Mediated Pro-Inflammatory Response System (LPS) (2). The inflammation caused by the LPS is thought to create pressure buildup on the NA resulting in the death of present cortical neurons (2). These neurons cannot be grown back and so any damage done is permanent. This is where an aptamer for CIAP is needed (6).

As stated before, an Aptamer is a sequence of oligonucleotides with a high bonding affinity for a specific enzyme (Figure 2). This sequence can be made from RNA or DNA sequences. This high bonding affinity arises from the unique positive and negative charges specific to each enzyme and RNA or DNA sequence (7). No uniform sequence of nucleotides is evenly charged throughout its entire structure (7). By finding a complementary sequence (aptamer) due to its own uneven charges we can effectively bond the enzyme and aptamer together. Aptamers are a unique possibility for medical treatment in that they can do many of the functions that anti-bodies do (7). Advantages that aptamers contain over anti-bodies is that they are more easily edited to a unique target (7). They are cheaper and easier to produce than anti-bodies and work in-vitro and in-vivo environments (7).

CIAP is a naturally occurring enzyme found in the mucus membrane of calf intestines. It has a molecular weight of 69,000 Da. Its primary function is to dephosphorylate the 5’ phosphorylated of vector DNA (Figure 3). Doing this prevents self-ligation of DNA strands. It has an Isoelectric point of 5.7 and is dimeric (8). It has an optimum pH range of 9.8 and has an inhibition level below a pH of 4.5 (8). CIAP has an activator of Mg+2, Zn, and Ca+2 and is a dimeric enzyme (8).

The ability of CIAP to detoxify LPS has already been shown in previous experiments in which CIAP was injected into rats that had received lethal doses of an Escherichia Coli infection and produced a survival rate of 80% (9). In another later experiment on piglets CIAP injections were shown to reduce the effects of TNF-α with up to 98% (9). Additionally, in the piglets these CIAP treatments were raised up to 400x the dosage that showed 98% effectiveness in reducing TNF- α for the period of 28 days, at the end of which, no malicious effects could be observed from either the TNF-α or the large doses of CIAP themselves given to the piglets (9). While not positive proof, this gives promise that CIAP can be easily regulated in the body. Finding an Aptamer that attached to CIAP could, in concept, be used as a diagnostic tool if it is found to conglomerate in certain areas of the brain, a sign that there is inflammation present at said locations.

While meant to be purely a diagnostic tool for Parkinson’s an aptamer could theoretically be altered to carry vital compounds that could reduce or effectively cure Parkinson’s (10). Further research would need to be done, however, in concept, once an aptamer is found for CIAP, it could be altered for much more other than that. Many diseases exhibit inflammation as a symptom and CIAP with an aptamer and detection molecule could in effect be used to treat any of these with a little bit of optimization.

Current research into this aptamer is still in round 1 of selection. It remains eluted in a pool of other nucleic acid sequences. It must go further rounds of selection to amplify the desired sequence and remove all lower-affinity binders.

Click Here for the Final Report

References