Samantha Finkenstaedt's PK6 aptamer project (2014)
Nucleic Acid Aptamer Selection against PK6 DNA Polymerase for Development of a Hot Start DNA Polymerase
Thermus aquaticus (Taq) DNA polymerase, used in artificial amplification of DNA through PCR and ligase chain reaction (LCR), frequently causes off target amplification of target sequences, small product yield, and substantial quantities of primer dimers. The poor fidelity and low thermostability of Taq DNA polymerase make it necessary to find an alternative DNA polymerase for use in artificial amplification1.
In order to inhibit the DNA polymerase, an aptamer, or specific nucleic acid sequence that binds with a high affinity to a protein target, will be used to inhibit the DNA polymerase. The aptamer-based inhibitor will bind reversibly to the enzyme and inhibit its capabilities at temperatures below 45oC, but release the enzyme during normal PCR conditions2. This will permit the assembly of reactions at room temperature and will not require a separate high temperature incubation step to activate the enzyme.
The aim of my project is, first, to discover an aptamer that has a high binding affinity for PK6 DNA Polymerase. This will be achieved using the in vitro SELEX3 method of aptamer selection. After an aptamer is found, my second aim is to modify the aptamer in order to create a Hot Start DNA polymerase mechanism. The aptamer produced through multiple rounds of sequencing, binding assays, and sequencing, will need to be modified and then tested to see if it is capable of inhibiting PK6 DNA Polymerase activity at ambient temperatures.
The two completed rounds of selection have brought the project closer to finding a feasible aptamer against PK6. In conjunction with ongoing research concerning Hot Start DNA polymerases, the project shows promise in creating a new Hot Start DNA Polymerase.
The target is not one that can be purchased from a vendor; Jimmy Gollihar synthesized the target through the cloning, expression, and purification of Pfu DNA polymerase and KOD DNA polymerases in the Ellington lab. PK6 DNA polymerase is a 91.8 kDa protein.
Figure 1. By inhibiting the DNA polymerase at room temperature, there will be fewer primer dimers, reduced off target priming, and a higher product yield.
Link to Full Proposal: Proposal
References:
1. Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B., Oka, M. and Imanaka, T. “Applied and Environmental Microbiology” 1997.
2. New England Biolabs, Inc. “EpiMark Hot Start Taq DNA Polymerase.” Ipswich, MA
3. Stoltenburg, Regina, Christine Reinemann and Beate Strehiltz et al. “SELEX – A revolutionary method to generate high-affinity nucleic acid ligands” 2007
4. Gold, Larry et al. “Nucleic acid ligands.” Patent 5,475,096. December 1991.
5. Lebedev, A, Paul, N., Yee, J., Timoshchuk, V., Shum, J., Miyagi, K., Kellum, J., Hogrefe, R., and Zon, G. (2008) “Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance” 2008.
6. Russel, Connolly et al. “The 3’ – 5’ proofreading exonuclease of archaeal family-B DNA polymerase hinders the copying the template strand deaminated bases” 2009.
7. Carr, Steven et al. “The Polymerase Chain Reaction”
8. Gold, Larry et al. “Nucleic acid ligands.” Patent 5,670,637. March 27, 1995.