Developing a Quantitative Framework for Designing Responsive RNA Electrochemical Aptamer-Based Sensors and Applications

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Chemistry & Biochemistry



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Electrochemical aptamer-based sensors utilizing structure-switching aptamers are specific, selective, sensitive, and widely applicable to the detection of a variety of targets. The specificity is achieved by the binding properties of an electrode-bound RNA or DNA aptamer biorecognition element that is a single-strand of DNA or RNA selected for in vitro to bind to a specific target molecule. Signaling in this class of sensors arises from changes in electron transfer efficiency upon target-induced changes in the conformation/flexibility of the aptamer probe. The changes in aptamer flexibility can be readily monitored electrochemically. The signaling mechanism enables several approaches to maximize the analytical attributes (i.e., sensitivity, limit of detection, and observed binding affinity) of the aptamer sensor. The work in this dissertations describes the quantitative effects of two different approaches to control sensor signaling in order to rationally tune sensor performance. The first part of this dissertations describes the effects of nucleic acid sequence and structure on the signaling of a representative small molecule aptamer-based sensor for the detection of aminoglycoside antibiotics. Modifying aptamer sequences to undergo large conformation changes upon target addition improves and maximizes E-AB sensor signaling because the collisional frequency and electron transfer rate of the 3?-attached redox molecule exhibits strong distance dependence. This dissertations also discusses the effects of stabilizing a folded structure of the aptamer to conserve the signal change, but reduce the binding affinity in order to shift the functional region of the sensor towards the therapeutic window of aminoglycoside antibiotics. Finally, with a newly developed family of aptamer sequences, tunable and predictable sensor responses achieved by employing different ratios of two aptamers with different affinities for the same target molecule on one sensor surface. The studies here were performed on a test bed aminoglycoside E-AB sensor, however the design criteria and framework established to tune sensor responses are generally applicable to any aptamer-based sensor. The second part of this dissertations explains the use of hydrogels to protect RNA E-AB sensors to enable use in complex media, such as whole blood, serum, plasma, etc.. The motivation is to bring the promising attributes of E-AB sensors to the clinic or bedside for real-time therapeutic drug monitoring. However, RNA E-AB sensor application has been limited as a result of degradation of the RNA sensing element in biological samples. To improve E-AB sensor function in complex samples, this work describes the development of a biocompatible hydrogel material to protect the oligonucleotides from degradation and inhibit non-specific absorption of proteins to the sensor surface ? both of which impede sensor function. Specifically, RNA sensors for aminoglycoside antibiotics were coated with a polyacrylamide hydrogel and tested in serum. Coating the RNA sensors with the hydrogel enabled sensor stability for a period of 3h in serum, which is a significant improvement from the uncoated sensors. The hydrogel coating also did not significantly affect E-AB sensor function based on the comparable titration curves of the uncoated and coated E-AB sensors. While sensor function and stability were tested specifically with aminoglycoside targets the technique employed to coat sensors with a hydrogel should be generally applicable to any small molecule E-AB sensor.