My name is Auden Wilson and I am a rising sophomore. I first started working in the Pollock lab last spring semester, but this is my first summer term. We are researching novel anticancer drugs that potentially interact with DNA. DNA interactions are the mechanism behind many effective chemotherapies, but compounds that interact with DNA can be very dangerous, so these interactions must be carefully researched.
DNA intercalators, or compounds that go in between the nucleotide steps of the DNA ladder (see figure one) make effective chemotherapies because they can inhibit DNA replication by stopping an enzyme that straightens DNA (Braña, M. F., 2001).
To detect DNA interactions, there is an agarose gel electrophoresis experiment that separates segments of DNA according to size. If the DNA treated with a compound runs through the gel differently than the DNA running through the gel alone, the compound is likely interacting with the DNA. DNA is negatively charged, so it is attracted to positive charges. In an electrophoresis experiment, a current is sent through the agarose gel; DNA samples (treated and untreated with the compounds) are loaded into wells on one side of the gel. The current runs from negative on the side with the wells to positive on the opposite side, which pulls the DNA through the gel. It is important to use an agarose gel because agarose forms a complex 3D structure when it sets at room temperature that limits the movement of molecules. Shorter fragments of DNA can travel faster through the gel, so they can get farther than the longer strands. Running gel electrophoresis experiments is a good first step to understand if certain compounds are interacting with DNA because they will often change the way it moves. If a compound is interacting with DNA, we would expect the treated DNA to not show up on the gel, or to appear somewhere else compared to the DNA running alone. Once a compound has shown that it interacts with DNA in gel experiments, it is important to experiment further to determine the mechanism of action.
To get a better idea of what is going on, a DNA melting temperature experiment can detect DNA intercalation. The melting temperature of DNA is representative of the temperature where half of the double stranded DNA in a sample has become single-stranded. This melting temperature changes when a DNA intercalator interacts with the DNA. DNA intercalators, the compounds that go in between the nucleotide steps of the DNA ladder, typically stabilize DNA and raise the melting temperature. This adjustment in melting temperature is apparent when comparing the UV spectra at 260 nm (the primary wavelength of light DNA absorbs) over a temperature range from treated and untreated DNA samples. If there is a change in melting temperature after treatment with a compound, most likely, it is an intercalator. However, there are no absolute negatives in science, so if the melting temperature has not changed, there could still be a different type of interaction with the DNA that would require additional experimentation to evaluate.
DNA strands lengthen as base pairs separate to accommodate DNA intercalators. This change causes an increase in DNA viscosity. This change can be measured to determine the binding mechanism of a compound that interacts with DNA. Other mechanisms of binding can affect DNA viscosity, but DNA intercalaltors have the clearest, most significant change. If this test was also inconclusive, intercalation is still not off the board, but the compound should also be tested for other DNA interactions.
DNA intercalators can also be effectively optimized as anticancer therapies if they can poison or inhibit an enzyme called Topoisomerase II (Nitiss JL., 2009). Finding the exact mechanism of action is important to create safe drugs, but also to optimize drugs for anticancer properties while minimizing risk of secondary malignancies or toxicities.
Braña, M. F., Cacho, M., Gradillas, A., de Pascual-Teresa, B., & Ramos, A. (2001). Intercalators
as anticancer drugs. Current pharmaceutical design, 7(17), 1745–1780. https://doi.org/10.2174/1381612013397113
Nitiss JL. Targeting DNA topoisomerase II in cancer chemotherapy. Nat Rev Cancer. 2009
May;9(5):338-50. doi: 10.1038/nrc2607. Epub 2009 Apr 20. PMID: 19377506; PMCID: PMC2748742.