Sheetal Mohanty's CIAP Aptamer Project (2018)

Detecting Tuberculosis Using Calf Intestinal Alkaline Phosphatase (CIAP) and An Aptamer Against Mannose-capped lipoarabunomannan (ManLAM)

Tuberculosis (TB) is a communicable respiratory infection which results in chronic cough, and blood-containing sputum. It affects approximately one-third of the world’s population, although about 90% of them remain asymptomatic. That being said, over ten million people have active TB, and it is one of the top ten causes of death worldwide, remaining a severe global health problem. (“WHO”, 2017) Currently, there is a diagnostic for TB which consists of chest X-rays, and multiple sputum cultures. Further, there is one vaccination, Bacillus Calmette-Guerein, which reduces the risk of infection turning active by nearly 60% and decreases the risk of infection by 20%. Although, this vaccination provides some resistance to TB, diagnostics are ineffective and inefficient, especially given that the population most effected by the disease goes undiagnosed. As TB impacts the developing nations of the world, the WHO suggests an ASSURED approach - affordable, specific, user-friendly, rapid and robust, equipment free, and delivered. Developing nations most impacted by TB, have limited healthcare resources to expend on their large populations, thus finding cost-effective and efficient solutions is of utmost importance. Thus, the development of a rapid diagnostic for TB will prove especially useful, be affordable and will lead the way for therapeutic and drug delivery applications. A way that this diagnostic tool can be created is by utilizing in vitro aptamers in place of in vivo antibodies.

Aptamers are very unique oligonucleotide sequences that bind to a specific target sequence and have a variety of applications. They are cost effective, have thermal stability, are easily generated, reliable, and can be a replacement for antibodies—this includes for diagnostics, drug delivery, therapeutics, and research/engineering applications. Aptamers can bind to any target using a variety of chemical bonding such as hydrogen bonding, covalent bonding, van der waals forces or even pi bonding. The bonding process is controlled by adjusting the equilibrium in order to have higher interactions amongst the oligonucleotides and targets.

TB, when it has infected the body, releases an early antigen in the blood circulatory system known Mannose-capped lipoarabunomannan (ManLAM). ManLAM is a lipoglycan which serves as a major component of the cell wall. As levels of ManLAM change with the presence of TB in the body, this makes it a perfect disease marker. CIAP is particularly useful as a reporter molecule in terms of aptamer research as it records a color change when the presence of an antigen or disease marker it is able to bind to is detected. The purpose of this application is to create a diagnostic system using Calf Intestinal Alkaline Phosphatase (CIAP) as a reporter molecule, and a secondary complex to function with an aptamer that binds to Mannose-capped lipabunomannan which is found in the blood stream within patients who have active Tuberculosis. Many studies have been conducted and successful aptamers, such as ZLX-1 and T9 (Tang, 2016)(Pan, 2014) have been identified against ManLAM. Yet, these aptamers have so far been used to deliver drugs and not actually diagnose TB. This application focuses on diagnosis, and thus utilizes CIAxP as a reporter molecule. An aptamer which already binds to ManLAM, can be attached to CIAP, and create a one solution diagnostic tool, where a physical color change will occur if the CIAP and an aptamer against ManLAM complex binds to ManLAM which would be present in the bloodstream, and thus be indicative of TB infection within the body. This creates an indirect Enzyme-linked immunosorbent assay (ELONA). In this case the existing aptamers against ManLAM will form the secondary antibody conjugate and the primary antibody conjugate will be formed by the CIAP and the selected aptamer, which would work in conjunction to detect different levels of ManLAM present in the blood stream indicated by a color change and thus diagnosing TB. With success of this application, TB would be able to be detected with a simple serum utilizing a drop of blood. Further, given success of this complex, immunosuppressant (Tang, 2014) or other therapeutic drugs could also be added to the complex, and potentially also serve a drug delivery or therapeutic application.

In order to find an aptamer an iterative process, called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) will be performed. This process begins with binding and selection, which starts with oligonucleotides that are generated in pools of random sequences. This experiment will utilize N71 pool. During selection these pools are binded with the target of interest at specific conditions representative of the application. A selection buffer is chosen, and additional properties that facilitate binding such as equilibrium adjustment, incubation time, and incubation temperature are all controlled. Further the pool, wash volume and number of washes-which helps discern the binding affinity-and the ratio to pool to target are also regulated. The target in this lab is an aptamer against ManLAM which can be attached to CIAP. Biotin affinity streptavidin beads will be utilized, as CIAP contains a biotin tag. Once a round of binding and selection has occurred, the bound and unbound oligonucleotides are partitioned and then the bound RNA is then eluted. The entire purpose of the aptamer selection process is to end with a more stringent pool and repeat the process until one strongly binding aptamer can be identified.

The bound pool and elutions of the washes are kept as controls. Cycle Course PCR is run on these samples to determine the optimal amount of cycles required for proper amplification during pool selection. Lastly, a large-scale PCR is used to amplify the bound pool. Throughout the PCR process, agarose gel electrophoresis is used to check for size accuracy. The lsPCR product is used in a transcription reaction in order to mass generate RNA, which is then purified utilizing polyacrylamide gel electrophoresis (PAGE). With successful PAGE, one is able to visualize an RNA shadow, which is cut out of the gel. The gel is then placed in a solution and purified in order to elute the RNA. Lastly, spectroscopy is used to quantify the concentration of the samples. At the culmination of one round of the aptamer selection process, a new RNA pool is formed which is more enriched than the initial pool and can be used for the next round of selection.

Currently selection against CIAP, and this specific application is underway. The selection conditions have been identified and an application developed. Troubleshoots for cycle course PCR are being conducted. There are other students also working with CIAP in the FRI Aptamer Stream lab, and they have developed a variety of applications. The goal is to complete at least six or more rounds of selection in order to find a tightly binding aptamer which can be used in conjunction with CIAP to form a TB diagnostic.

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