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Selective Catalytic Chemistry Using DNA Aptamers V. Garbo Garborcauskas One of the greatest challenges to modern chemists is to chemically modify one target molecule in a mixture. A selective reaction of only the target molecule is very difficult because other background molecules in the mixture may have similar or identical functional groups and often react in the same way. Our goal is to only react the starting chemical with one of the molecules in the mixture, instead of all of them. (See Figure A above) Currently, although there are methods to selectively modify a target protein, this cannot be done for small molecules. 11 Our goal this summer was to use DNA-small molecule catalyst conjugates (DCats) to achieve selective chemistry. This will be something that no lab has ever been able to do with small molecules and could revolutionize biomedical sciences and biochemistry. A DCat is made using a DNA aptamer that is targeted to the molecule selected for reaction. A DNA aptamer is an oligonucleotide that has an affinity to the target molecule. If the target molecule is cholic acid, the DNA aptamer used will have an affinity to cholic acid. Attached to the DNA is a linker (DSG in figure B above). The linker enables attachment of a small molecule catalyst, which catalyzes a reaction of interest, to the DNA aptamer (See Figure A above). A small molecule catalyst can be many different molecules or functional groups, however, since we initially wish to perform a hydrolysis reaction, an imidazole group was chosen. This summer, we had three experimental goals: synthesis of the first DCat, the development and optimization of methods to monitor the desired reaction and testing the first DCat for selective catalysis. This summer, the first ever DCat was synthesized. The linker, DSG, was first reacted with the small molecule catalyst, histamine. DNA was then added to the mix, allowing the amine functional group on the DNA to react with the linker. After running the reaction for more than four hours, there was a combination of three different possible molecules in the reaction mix. It is possible to get the DNA aptamer back, the DNA aptamer with the linker, or the desired product: the DNA aptamer with the linker and the small molecule. These three different molecules separate out on a reverse phase high-pressure liquid chromatography C18 silica column (HPLC). The ratio and amount of time these solvents flow through the column can lead to complete separation, or only partial separation. This summer, time was spent optimizing this method to allow for complete separation of the three different molecules. Conditions were found to separate out the different molecules without any overlap from the previous molecule, giving a pure product. Methods were developed this summer to monitor the progress of the reaction while it is running. One of the instruments in the Center for Molecular Biology was particularly helpful: the nanodrop spectrophotometer. Most spectrophotometers use a large volume (>2mL) to measure the wavelength of light allowed to go through the liquid, however, the DCat reaction is run in very small volumes, only 20µL. The nanodrop spectrophotometer uses 2µL for each reading and gives a UV spectrum absorbance. Before the DCat reaction could be started, standard curves of the byproduct, nitrophenol, the starting material and the desired product needed to be created so that the formation of the byproduct and how much starting material was left could be monitored throughout the DCat reaction on the nanodrop spectrophotometer. Another possible way to monitor reactions when they are finished is to run them through the HPLC to determine the final concentration of byproducts, starting materials and desired products and to adequately separate them out. Since the DCat is a catalyst, recovering the DCat is possible, using an instrument that separates out the different molecules. In order to determine how much DCat, starting material, byproduct and desired product there is in a reaction, it was necessary to create standard curves on the HPLC. All of these experiments were preliminary tests before the DCat reaction could run in order to effectively monitor the progress and the end of the reaction. 2012 73
Once the DCat has been synthesized, reactions were started with them to see if selective chemistry occurred. The DCat reactions were monitored with the nanodrop spectrophotometer. The nanodrop spectrophotometer gives a reading of UV light absorbance at a certain wavelength. Since the desired product has a known reading at 410 nm, the formation of the product can be monitored, and using standard curves, the amount of turnover can be calculated. With the cholic acid DCat, a selective reaction was observed however, with the ibuprofen DCat, there was only a very slow hydrolysis reaction. In the near future, the goal of the Gorin lab is to create more and different types of DCats expanding the amount and type of selective chemistry that will be able to be reacted. (Supported by the Howard Hughes Medical Institute) Advisor: David Gorin References: 1 Gorin, David J. 2011. Selectivity in Complex Contexts: Directing Chemical Reactions with DNA-Small Molecule Conjugates. Single Investigator Cottrell College Science Award Application. 2012 74
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Brugia Malayi - Clark Science Center - Smith College

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