Discussion & Future Experiments. Using a dual labeled aptamer containing the quencher Iowa black we expected a decrease in fluorescence as the aptamer folded. Instead we saw an increase in fluorescence. In addition, the absolute intensity of the fluorescence fluctuated between experiments. A number of possible experimental factors including instrumental settings (excitation and detection wavelength and monochromator slit width) were examined to explain these results, nothing could explain for the increase in fluorescence as the distance decreased between the donor and quencher In future studies we plan to use a modified TBA (CCaaCggTTggTgTggTTgg) dual labeled with FAM and Dabcyl. The delay between scans will be performed submerged in heated water to ensure for complete unfolding. STUDIES IN FLUORESCENCE RESONONANCE ENERGY TRANSFER IN A FAM/IOWA BLACK LABELED THROMBIN-BINDING APTAMER Background Aptamers are single strands of DNA or RNA 15 to 50 bases long that bind strongly and specifically to a molecular target. These molecules are increasingly popular in diagnostics, research and therapeutics. Thrombin-binding aptamer (TBA) is one aptamer getting a lot of attention. Thrombin is a protein important in blood coagulation. TBA folds into a unique “chair” structure when it binds to thrombin or to certain cations. TBA’s interaction with the cations may allow for a better understanding of the its folding characteristics. In the chair structure TBA has two guanine- quadruplexes that are arrangements of 4 guanines stabilized by hydrogen bonds. G-quadruplexes have received much attention because guanine rich sequences are commonly found in the human genome and have mechanisms linked to cancer, HIV and other diseases. Fluorescence resonance energy transfer (FRET) is the transfer of energy from a donor to an acceptor and is distance dependant. Hypotheses Using a dual labeled TBA, as ions are added to the solution total fluorescence will decrease when using FAM and Iowa Black-FQ as a result of FRET. Acknowledgements: UW-Whitewater Undergraduate Research Program Dr. Paul House Ms. Marsha Goodell Summary A dual labeled thrombin binding aptamer produced an increase in fluorescence for ions that other researchers have shown should bring the donor/acceptor pair FAM/Iowa black closer together. This is not expected – Iowa black should decrease the FAM fluorescence. Careful study of how the experiment was done did not reveal the cause of this inconsistency. Jay Blatnik UW-Whitewater Mentor: Dr. Paul House UW-Whitewater Study Design We monitored the folding of TBA with a variety of cations including previously well studied (Na +, K + ) and two seldom looked at (Rb +, Cs + ). All experiments were conducted at room temperature. Data was corrected for the increase in volume as the salt solution was added; the percent difference in fluorescence was calculated and graphed. Our previous work with TBA dual labeled with FAM and TAMRA on the 3’ and 5’ ends, is graphed below. It shows an increase in fluorescence with the addition of monovalent cations but a decrease in fluorescence with the addition of divalent cations. These results are inconsistent with work from other researchers who have shown both K + and Sr 2+ cause TBA to fold. These inconsistent results led us to use a differently labeled aptamer. Iowa black is an acceptor that leads to decreased fluorescence when FRET occurs. Results for FAM/TAMRA labeled probe with a 10 minute wait between scans (λ EX : nm, monitored at nm and 5.0 nm slit widths) [Salt] (mM) Results of FAM/Iowa Black-labeled probe with a 10 minute wait between scans (λ EX : nm, monitored from to nm and 5.0 nm slit widths) Jay Blatnik and Paul G. House University of Wisconsin–Whitewater, Department of Chemistry Whitewater, Wisconsin