Chris Kujalowicz's HRP Aptamer Project (2017)

Aptamer​ ​Complex​ ​Against​ ​Horseradish Peroxidase​ ​and​ ​C4​ ​Gene​ ​as​ ​a​ ​Diagnostic Marker​ ​for​ ​Schizophrenia

Introduction and Background

Presently, there is a dearth in the field of schizophrenia diagnostics. Patients often experience a better prognosis if the disease is diagnosed early and treatment begins immediately. Oftentimes, however, the disease cannot be diagnosed without the presence of symptoms, making early intervention difficult and prognosis poor. I aim to create an effective and accessible diagnostic tool for the disease, based on a patient’s genetic predisposition to the disease. This tool will be cost-effective and ethically sourced since aptamers and HRP will be used to engineer this diagnostic tool. Individuals with a family history of schizophrenia would be able to determine their susceptibility to the disorder quickly, which would enable them to take measures to impede the onset of the disease. Additionally, early treatment would help hinder the disease’s progress, which would alleviate the financial weight of the patient care and treatment required in the late stages of the disease. Overall, I hope to make a lasting impact on the state of schizophrenia research and better the lives of the millions of people affected by the disease worldwide.

Horseradish Peroxidase (HRP) is a naturally-occurring enzyme found in the roots of the horseradish plant. The enzyme itself is often used by joining it chemically to other compounds, making molecules called conjugates. HRP produces a colored, luminescent derivative when applied in the proper conjugate, allowing for the quantification of the said derivative, making HRP an effective reporter molecule. In contrast to more widely-used reporter molecules like CIAP, HRP is smaller, less expensive, and typically more stable, making it an more ideal choice for aptamer selection. The enzyme’s molecular weight is 44,173.9 Daltons (Sigma-Aldrich, 2017). HRP is a monomer although it is generally only applied when working in a conjugate with another protein.

The nature of a reporting molecule is to indicate or amplify the presence of a specific target, which can be a helpful attribute in the field of diagnostics. Past research on HRP in diagnostic applications includes the tracking of invasive breast cancer through the use of a chemiluminescent inhibitory aptamer, allowing for an accurate and reliable way to diagnose and observe the metastasis of the disease. One study published in the Hindawi Scientific World Journal explored the use of an HRP Aptamer to detect tetracycline in milk. Furthermore, recent developments in neurobiology have discovered that HRP is small and stable enough to be inducted into neural pathways, and using its chemiluminescent derivatives, can track and mark neural pathways. Most labs working with HRP use its chemiluminescent property as a way to amplify the presence of certain molecules, but the primary lab finding aptamers against HRP is UT’s FRI Aptamer Stream.

An aptamer is a short oligonucleotide sequence that, due to its specific structure, has a high binding affinity to a target of interest. Using the SELEX method, the target of interest is bound to magnetic beads. When exposed to a random RNA pool, sequences with a high binding affinity for the target will adhere to the beads and remain in the selection, while the unbound species will be removed. After a round of selection is complete, only the most specifically bound oligonucleotides will remain. Rounds of selection continue until an aptamer against the target is discovered. To assess the specificity of the potential aptamer, the pool can be submitted to Sanger sequencing to give a full assessment of the pool’s binding affinity. Due to the specific nature of aptamers, the molecules can have a wide range of applications, including therapeutics, diagnostics, drug delivery, industry, among others. Aptamers being used in therapeutics could be engineered to inhibit a protein involved with an affected process, and aptamers against reporter proteins would be effective in diagnostics.

In order for a diagnostic tool to be designed, the disease of interest must first be understood. Diseases like schizophrenia can be influenced by genetics, in conjunction with environment and brain chemistry fluctuations. Therefore, it should be noted that individuals with certain genetic composition can be predisposed to developing certain mental disorders. A recent Harvard Medical School study performed a revolutionary set of research using “36,989 schizophrenia cases and 113,075 controls, and identified 108 regions of our DNA where genetic variants increase a person's risk of schizophrenia” (Crew, 2017), which also reinforces the notion that genetics play a pivotal role in the development of diseases like schizophrenia. The study also revealed that complement component 4 (C4) is a gene involved with brain development and immune responses, which varies greatly between schizophrenia patients and healthy individuals. Those who experience an overexpression of the gene can manifest increased synaptic pruning, which when occurring outside of normal developmental stages can sever crucial neural pathways. Since HRP can be inducted into neural pathways, an aptamer against it could have a practical downstream application within neurons, which could help track the integrity of neuronal connections. Furthermore, as a reporter molecule modified to signal the presence of its target, HRP could be an optimal way to quantify the overexpression of an “HRP-ylated” C4 gene, which could potentially help diagnose schizophrenia before its initial symptoms appear. According to a recent comparison study, the enzyme also “has a high turnover rate that allows generation of strong signals in a relatively short time span” (Beyzavi, 1987). According to Aptagen aptamer index, an HRP aptamer for PC-3 Human Prostate Carcinoma has been discovered, which suggests that finding a gene-binding HRP aptamer is possible. For my application, I will use a primary aptamer against the overexpressed C4 gene, which I will then bind to a second aptamer against HRP through the use of a complimentary base pair linkage sequence, extending each aptamer by a few single-stranded base pairs that are complimentary to each other. Once the two aptamers join, a chemiluminescent enzyme can adhere to the HRP molecule bound to the second inhibitory aptamer, which makes visualization of the overexpressed gene possible.

Finding this aptamer will be done using the SELEX process, shown in the figure below. SELEX begins with a random or modified RNA pool being exposed to target-bound beads, resulting in the subsequent binding of RNA species that have a high binding affinity to the target. Since the beads are magnetic, they are separated and washed, collecting the unbound pool and three pool washes. The tightly-binding species are eluted from the beads and collected. These species are then amplified and can either be sequenced or exposed to another round of selection to further increase selectivity and specificity of the bound species. Rounds of selection continue until an aptamer is found.

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References

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