Quang Nguyen's CIAP Aptamer Project (2017)

Aptamer​ ​against​ ​Calf​ ​Intestinal​ ​Alkaline​ ​Phosphatase to​ ​Diagnosis​ ​Acute​ ​Myeloid​ ​Leukemia​ ​(AML)

Introduction and Background

Leukemia is the group of different cancers that caused by abnormal growth and malfunction of white blood cells in bone marrow. Leukemia cells can accumulate and spill into the bloodstream to affect other organs’ normal function. According to Leukemia & Lymphoma society, leukemia is one of the highest factors that causes cancer deaths with an estimate of 345,422 people living with, or in remission in the U.S solely. Another thing to be noticed is that Acute Myeloid Leukemia (AML) is the more popular in adults who are over 45 years old (American Cancer Society) and it has lowest survival rate among all kinds of leukemia. In fact, the survival rate for over 65 years old patient is less than 5% (Thein, Mya S., et al 2013).

Treatments such as chemotherapy and stem-cell transplants can be applied for AML leukemia patient but drawbacks are still exist. The large variability in gene mutations and gene expression in AML patients (Kumar et al., 2011) leads to different responses and adverse cytogenetic abnormalities in chemotherapy. Stem cell transplant has a higher success rate, but the complication makes it has a higher risk (American Cancer Society). In relation to prognostic of the mutation, high expression of BAALC gene combines with genes mutations reduces the survival significantly (Yoon J.H et al., 2014). One approach for AML leukemia treatment is early diagnosis, however, the current diagnosis tool such as bone marrow biopsy, spinal fluid test can create discomfort and infection while couldn’t specify in diagnosis AML among many other kinds of leukemia. By using an application of an Aptamer and CIAP, this problem can be solved. An aptamer can inhibit Calf Intestinal Alkaline Phosphatase (CIAP) which functions as a report molecule and RNA strand displacement technologies can be used to develop the future technique for prognosis of AML leukemia by detecting the level of BAAC gene expression and evaluating the disease.

An Aptamer is the short, single stranded nucleic acid (RNA or DNA) that bind with high affinity to a variety of target of interest such as a molecule, a protein, a cell or even a organism. They are selected from the large, random pool through the process known as in vitro SELEX. During this process, bead-based selection, cycle course PCR, large scale PCR, PAGE gel and transcription are performed to increase the stringency to the CIAP target and purify the initial pool of RNA.

In Aptamer research, the RNA is commonly used as an aptamer rather than the DNA because RNA’s smaller size and the simple strand structure allow them to enters the cell easier than same-length of DNA. This phenomenon of aptamer leads to its wide-range applications including therapeutic, drug delivery, therapy, and diagnosis (Stoltenburg et al., 2007). In this application, Aptamer - CIAP complex is used to detect the presence of BAALC gene through color change in the similar manner to the ELISA immunosorbent assay that have been done in the antibodies. However, Aptamer have some advantages compared to the antibodies in research because of its ability to bind to the specific targets. It can distinguish between two enantiomer and can recognize the precise epitope of a target molecule (Michaud et al., 2003). Moreover, since aptamer is selected in the similar condition with final assay environment, it will maintain structure and not dissociate like antibodies. Another important advantage of the aptamer is that they are found in the in vitro selection which is both affordable and require less time to finish compared to antibodies which are usually found in the living cells using in vivo selection.

Calf Intestinal Alkaline Phosphatase (CIAP) is an enzyme that found in Cow's Intestine. Similar to ELISA technology which is commonly used in research, in this study, CIAP will be used as a report molecule to detect the level of BAALC gene expression in order to develop a prognosis tool for Acute Myeloid Leukemia. Particularly, the CIAP exists as a dimer with a molecular weight ~140 kDa ( 69 kDa for monomer) and isometric point at 5.7. CIAP’s pH is 9.8, so it will stable in any basic selection buffer. Because the CIAP has partially negative charge, divalent salt is added to the selection buffer in order to make the ionic bridge to connect CIAP molecule and negative charge nucleic acid.

In a recent research about AML leukemia, a study shows that higher level expression of BAALC gene would decrease the survival rate in Acute Myeloid Leukemia (AML) patients because it also incorporates with other mutation genes to worsen prognosis in all cytogenetic groups ( Damiani et al., 2013). Therefore develop an aptamer that can help to detect an overexpression of BAALC gene will help the early prognostic and development of an appropriate treatment plan for AML patients. Utilizing CIAP capacity to phosphorylate the substrate such as pNPP to produce detectable color change, an aptamer will be developed to inhibits CIAP’s function to phosphorylate the substrate by obstructing the substrate binding site (Ellington and Bradra et al., 2015). The aptamer that found in SELEX will be reengineered at 5’ and 3’ end by adding the BAALC gene complementary extension in order for the target mRNA to bind precisely (Zhang and Winfree et al., 2009). In the presence of BAALC gene, it will be hybridized to a complementary extension which will initiate toehold-mediated strand displacement (Figure 3). This formation will unfold the inhibitory aptamer and restores the substrate phosphorylation that will allow color change. The level of color change can be quantified to evaluate the expression level of BAALC gene. This detection system can be used as the supplementary tool to detect gene expression along with real-time PCR technology due to time efficiency, but still, provide a reliable result.

In other to find an aptamer, many rounds of SELEX are needed to increase the stringency and develop an aptamer with high binding affinity to a target from the random RNA pool (N50 pool). Foremost, The bead-based selection method is used to separate the RNA strands that bound precisely to the target (CIAP) from unspecific or weak RNA strands. The CIAP molecule has biotin tag on them which allow them to conjugates to beads. Then, other procedures which are cycle course PCR, large scale PCR and transcription are performed to produce the sufficient amount of RNA to analyzed and prepared for the next round. The important of the selection process is to increase the stringency of RNA to the protein, therefore, the ratio of the RNA pool vs target protein (increase pool: target ratio) as well as the number and volume of washes can be manipulated to achieve this desire. After several rounds of selection (4-5), Sanger sequencing and the radioactive binding assay can be done to examine the pool enrichment and affinity for the target.

The project is currently on round one without any significant result. The project was stucked on ccPCR and had been troubleshoot to move on. Around 70% of the round 1 selection has been done up to finishing the transcription which convert the selection products back to the RNA to be a pool for round 2 of selection. Before ending of this semester, round one will be tried to finish and other rounds of selection will be continued in the future. The goal for the project is to be accomplished to round 5 or round 7 of selection in order to perform the binding assay and examine the pool’s binding affinity to the target. If the Aptamer is found, it would be modified to work efficiently in AML diagnosis application in order to seek for FDA approval and incorporate into clinical uses.

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References

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