DEVELOPMENT OF FLUORESCENT METHODS TO DETERMINE DISSOCIATION CONSTANT FOR SINGLE STRANDED DNA BIOSENSORS

Biosensors are tools designed to encounter a chemical and provide a measurable signal. Recently, there has been research interests in developing single stranded DNA (ssDNA) aptamers as biosensors. The process of identifying an aptamer, is called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Once several possible aptamers are identified their dissociation constants, ,will be evaluated through fluorescent assay analysis. One of the most accurate testing of aptamers is using the 6-carboxyfluorescein (6-FAM)/DABCYL system. However, this process is expensive and labor intensive. Thus, there is a need for a quick and robust screening fluorescent assay to determine best aptamer candidates. The purpose of our project is to develop a fluorescent assay method by using fluorescent dyes, Thiazole Orange (TO) and Thioflavin T (ThT), to determine the for ssDNA aptamers. We identified DNA-aptamers through the process of an in vitro selection and amplification- SELEX. Through multiple rounds of SELEX, ssDNA sequences which strongly interact with a target kavain will be favored for PCR amplification and would be more likely identified as binders. We completed 14 rounds of SELEX. To determine a method for fluorescent testing, we used aptamers previously tested with 6-FAM/DABCYL. We compared TO and ThT’s ability to bind to the ssDNA. From these experiments, we concluded that ThT is a better dye to use when testing preliminary ssDNA aptamer’s . We expect that our study will enable development of the more advanced biosensors for detection of challenging small molecules of interest.