Adrian Mihalcea's DNA Polymerase (A12) Aptamer Project (2016)

Cancer cells are somatic cells, or any cell in the body that does not function in reproduction, that do not follow the normal control system of the cell cycle3. The human body is a robust piece of machinery, with individual parts that work together to keep it functioning. However, cancer cells are faults in this interconnected web of working pieces. As more faults (cancer cells) develop there is a greater chance for the whole system to go critical. These cells divide quickly and limitlessly1, because they have no need for growth factors, since they make their own factors. Normally, there are cues in the body that cause cells to stop dividing or when to divide under certain conditions. Two examples of this are density-dependent inhibition and anchorage dependence. Density-Dependent inhibition is when too many cells begin to crowd around each other, they stop dividing. Anchorage dependence is when a cell to wants to divide it has to be attached to a substratum. Cancer cells ignore all these cues, including the ones for apoptosis as shown in Figure 3. Cancer that develops at the heart is usually a malignant tumor, which is when abnormal cells from one location in body who’s genetic and cellular changes enable them to survive and spread towards new tissues9. One way the body attempts to halt indefinite cell division is through the use of p53 tumor suppressor genes. The p53 gene produces proteins that help in halting the cell cycle, which allows p53 to produce specific proteins that assist in DNA repair. If all else fails, p53 will produce proteins that trigger apoptosis in malfunctioning cells. These genes are replicated with the help of A12 DNA polymerase.

A12 DNA polymerase is a protein that assists other DNA polymerases in the replication of DNA through the synthesis of new nucleotide sequences. The DNA polymerase recognizes and binds to a RNA primer sequence placed on top of the DNA sequence, because DNA polymerases can only add nucleotides to an existing foundation. When individual nucleotides are incorporated onto an existing strand, each is connected by a phosphodiester bond, which is a covalent bond between the 5’ phosphate molecule of one nucleotide and the 3’ hydroxide group of another nucleotide. A12 was developed in Elington lab with the use of DNAP shuffling of the KOD and Pfu DNA polymerase through recombination. Related genes with identical sequences were digested with DNase I. As shown in figure 4, the resulting fragments were then thermocycled in a PCR without primers, in which fragments anneal to one another and mutually serve as primer and template. The products were then amplified in a standard PCR, yielding a shuffled pool8. This protein functions as a monomer because it does not combine with other proteins to form complex structures. Medical applications for A12 DNA polymerase, such as the detection of a varieties of cancers, are being developed by major laboratories like the Laboratory of Chemical Biology, NY USA and the Institute of Molecular Genetics, Moscow Russia.

An aptamer is a modified oligonucleotide sequence, which has a high affinity for attaching to a specific molecular target; a cell, a protein, another nucleotide sequence, etc. Aptamers are designed specifically for their target, and only have a high binding affinity for that target. Targets can be distinguished by a single, small difference, which means aptamers do not need a lot of identification to gain a fix on their target. For example, a stem cell through cell differentiation can turn into a multitude of cell types (e.g. RBCs). A key benefit in the development of aptamers is that they are cheaper to produce in comparison to antibodies, which means less of a cost constraint towards all applications. Since aptamers can be designed for a diverse range of targets, their real world applications are without limits. Most RNA aptamers are designed for therapeutic and diagnostic applications6. If the necessary controls are maintained and the appropriate reagents are used, the original pool of RNA (N71) is expected to transcribe into ssDNA, that DNA to amplify, and for the reformation of RNA.

One possible method for detecting the presence of cancer cells earlier in production would be the use of a fluorescent tagged aptamer, developed in pH found in the human body, which would only produce a yellow fluorescence under UV light when attached to A12 DNA polymerase during replication of the p53 tumor suppressor gene. A12 DNA polymerase has been proven to be functional in 10X buffer with the following components: (500 mM tris-HCl pH 8.8, 100mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1.0% Triton-x, and 0.01% BSA). However, for application purposes this buffer would not be a desired selection buffer for Aptamer selection. If a diagnosis for heart cancer, through the use of an aptamer, cannot be developed, then an aptamer can be designed to fit in the active site of A12 DNA polymerase to competitively inhibit the enzyme. RNA aptamers have been created for the direct inhibition of A12 DNA polymerase, which would help to halt the replication of DNA in order to fix errors in the coding. In comparison to the normal amounts of p53 in the body on average, brightly fluorescent samples would indicate that there is an abnormal amount of cell division occurring and that the presence of cancer cells will soon follow4.

Identification of an A12 aptamer is in progress. So far the first gel ran for lsPCR revealed good technique maintenances, but not promising results. The ladder came out properly and there were no bands under the NTC, but there was not a band under the W335 column. After having reviewed the results, a positive control would be performed to see if RNA was collected from bead-based selection.

Second gel ran for lsPCR revealed good technique practices, and promising results. The ladder came out clearly, there were no bands under the NTC column, and there was a band of correct length under the W030 column. For the next round of selection, more washes will be performed during bead-based selection to increase the stringency of the pool. A larger batch of pool candidates will increase the chances of one of those candidates being developed into an aptamer. An aptamer that will serve as a biomarker test for proper diagnostic of heart cancer.

(11)Wang, F., & Yang, W. (2009, December 24). Structural Insight into Translesion Synthesis by DNA Pol II. Cell, 139(7), 1279-1289. doi:10.1016/j.cell.2009.11.043