The purpose of this research project is to focus on producing signals for the specific detection of clinically relevant biomarkers. Sensors in general consist of two components that include target detection and signaling. Once the probe detects a target molecule a signal is produced. We are working towards building an accurate and efficient biosensor for important small molecules such as let-7i that is an important miRNA. For the current detection strategy, aptamers are used for detecting specific targets. The target recognition is in combination with signaling via reactivation of Glucose oxidase (GOx) with its co-factor flavin adenine dinucleotide (FAD). The aptamer probe is designed to bind FAD (anti-FAD) and was modified to also bind let-7i in a different binding pocket. The FAD is released from the aptamer upon let-7i binding. The signaling mechanism of this experiment is dependent on the response received upon FAD based reactivation of apo-GOx (enzyme without co-factor). Enzyme kinetics are used to measure the reactivation rate of the inactive enzyme once FAD is reintroduced. Preliminary data were used to design the modified aptamer to bind FAD and let-7i in different binding regions. Additionally, binding experiments were performed for FAD to the aptamer. Our current work consists of elucidating the affinities of the modified aptamer to FAD and the specificity of the aptamer for let-7i. Aptamer and enzyme based sensing mechanisms are targeting towards enhancing selectivity and sensitivity of the detection of clinical biomarkers.

Let-7i binding signaled by enzyme reactivation

251st National Meeting (2016)

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