Prakrut Mehta's DNA Polymerase (A12) Aptamer Project (2016)

Nickel-Bead Based Aptamer Selection Inhibition Against A12 DNA Polymerase

Introduction/Background

To run any sort of Polymerase Chain Reactions (PCR), DNA polymerase is the most prevalent enzyme required to make the reaction work. Researchers most commonly make use of the "Taq DNA polymerase," (Figure 1a) which is obtained from a species of bacteria - Thermus Aquaticus, that can tolerate high temperatures. This highly important enzyme, only when added to the master mix of a PCR reaction, can enable the reaction to proceed properly (Figure 1b). Due to its highly active state at room temperatures, it is most commonly preserved on ice before added to a master-mix. A master mix is a mixture of all the reagents in bulk necessary to perform a PCR reaction, which can then be separated into individual aliquot quantities as required for different washes. Although the PCR reaction is prepared with utmost care, the results sometimes do show signs of failure or a high margin of error. This mainly occurs due to the binding of different reagents to one another, leading to formation of heteroduplexes or arbitrary products, that do not bind to and react with the genetic material to amplify it. In most PCR reactions, binding of non-specific primers leads to the formation of some non-specific products, which reduce the overall yield of results. Primer can also form primer-dimers, leading them to attach to each other and serve as template for each other and amplify themselves in a higher quantity. These errors can decrease the efficiency of a PCR reaction by as much as 50 - 60 times, which is a major problem in the research field. Hence, this is a common problem faced by most researchers while trying to correctly perform a PCR reaction. This is where the use of an aptamer comes into play. With the finding of an aptamer against the A12 variant of DNA polymerase (target), this problem can easily be resolved. DNA polymerase is an enzyme that is most prevalent in DNA replication procedures. It makes DNA molecules by synthesizing DNA nucleotides, and works in a pair to make two identical DNA strands from one single parent template strand. Although "Taq DNA Polymerase" is most commonly used, other heat-loving bacterial sources have also shown increased levels of fidelity within the PCR, some of which are Thermococcus kodakaraensis (KOD) and Pyrococcus furiosus (Pfu). These different bacterial polymerases synthesized in the Ellington lab - KOD DNA polymerase and Pfu DNA polymerase, can finish a replication process at a much faster rate than 'Taq' and also have high elongation rate. Jimmy Gollihar of The Ellington Lab at UT - Austin created this “A12” DNA polymerase through the process of recombination of the products of DNAP shuffling of KOD and Pfu DNA polymerases. This target is deemed to be a multimer because it forms a dimer when it attaches to the DNA template strand to synthesize it. The molecular weight of this target is also assessed to be at 92 kDa. Throughout research procedures conducted by scientists, this target is generally used for detection purposes comparing against Taq and other forms of DNA Polymerase, to detect different synthesizing speed, functioning of the polymerases and margin of errors, using the A12 variant as a standard.

The aptamer, usually found through the method of SELEX, would ideally bind to the polymerase's active site as a substrate (Figure 1b), and act as an inhibitor to prevent the polymerase from functioning and starting the reaction until a high enough temperature has been reached. SELEX is basically exposing a wide range of an oligonucleotide pool to a protein target (DNA Polymerase A12 in this case) and later eluting and amplifying the bound sequences by PCR to prepare for the next successive rounds of selection, where the elution stringency is increased to find the tightest-binding oligonucleotide sequence[1]. The A-12 DNA Polymerase storage buffer is used to store this target protein, and because the target is found to be most stable with this buffer solution, this storage buffer can be used as the selection buffer itself. Reactivity of this protein target is different because it was manually synthesized, and due to its variant properties and isoelectric point (pI), different types of salts would have to be added to other generally used selection buffers like HEPES or TRIS to make the protein target stable, which is why its storage buffer is highly preferred. At higher temperatures, the aptamer would simply degrade and drift away from the active site of the DNA Polymerase and allow it to function without any setbacks or binding to non-specific reagents. An aptamer is generally a nucleic acid specie that binds to any protein (target molecule) and allows for various benefits like Therapeutics, Inhibition, Diagnostics and Drug delivery. Aptamers are highly beneficial compared to antibodies since aptamers can self-refold and also do not have a hydrophobic core of protein. They do not convulse inwards also and have a higher affinity and specificity for their targets. To date, an aptamer has not been found for this variant of DNA Polymerase. Finding an aptamer will be immensely beneficial to any scientist around the globe who used a PCR reaction with the A12 variant of DNA Polymerase as it will help reduce the yield of errors in results while quickly completing the replication process and even reducing formation of unwanted products by binding tightly to the polymerase enzyme. Shown below are two pictures which briefly describe how the aptamer will bind to the protein target, and later how that aptamer will function as an inhibitor during a PCR reaction, serving its ultimate purpose.

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References