UVM Theses and Dissertations
In this project, we selected three groups of aptamers binding 2-AA from a singlestranded DNA (ssDNA) library that contained a large amount of 91-base ssDNA (1.1 x 10¹⁸) using in vitro selection called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The selected aptamers were modified to the structure switching fluorescence signaling aptamers (SSFSAs). All ofthe SSFSAs were able to signal the presence of 2-AA, and Aptamer III generated the most significant fluorescence enhancement and showed the highest affinity to 2-AA. Five small molecules that were similar to 2-AA in chemical structure were used to test the specificity ofthe SSFSAs, and Aptamer II showed the highest specificity to not only 2-AA but also acetophenone (A). Additionally, the affinity ofthe aptamers to acetophenone (A) were higher than 2-AA. The result might be resulted from two factors, which included that 1) the 2-AA solution (98%) used in SELEX contained acetophenone, and 2) the intrinsic quenching effect of 2AA on fluorophore molecules caused the decrease in fluorescence. Fortunately, acetophenone (A) was also validated as another unique volatile compound generated by Pseudomonas aeruginosa. Therefore, acetophenone (A) can be used as a candidate-biomarker for the identification of Pseudomonas aeruginosa as well.
We have successfully selected the aptamers that can bind two very small molecules and designed the signaling aptamers with fluorescence signal generation ability. However, in contrast with those known fluorescence signaling aptamers, the fluorescence enhancements generated by our SSFSAs are still limited. The improvements can be done by optimizing the design ofthe fluorophore-DNA (FDNA) and the quencher-DNA (QDNA) molecules that play the most important roles in generating fluorescence signal.