Faculty: Dr. Mehnaaz Ali
Sensor Strategies: Aptamer-based electrochemical detection schemes
Readily available and adaptable diagnostic and detection methods are paramount to timely remediation or diagnosis.1, 2 Although existing technologies offer salient methods for detection,3, 4 the ease of adaptability to a variety of target types is limited.5 Thus the objective of this proposal is to develop electrochemical methods for the detection of clinical and environmental targets that are easily adaptable to the type of target. The central hypothesis of this application, which will be pursued via two separate but complementary strategies (see Figure 1), is that aptamer binding events to either small molecules (via Specific Aim 1) or oligonucleotides (via Specific Aim 2) will result in the direct release of a redox tag. Specifically, the cofactor flavin adenine dinucleotide (FAD) or its target analog conjugate will be released and in turn detected electrochemically. This hypothesis has been formed based on initial studies (published in Chemical Communications6) that show a target-aptamer binding event leading to the quantitative release of FAD and measured indirectly via the reactivation of an enzyme. The successful completion of the proposed work will provide a general strategy for detection that can be adapted to achieve our long-term goal of developing a robust end-user sensor for numerous target types.
Aim 1. To establish an aptamer-based small molecule detection strategy with the release of FAD upon target recognition. We will develop a detection strategy where aptamer recognition of the small molecule tryptophan (an essential amino acid and important biochemical precursor) will lead to displaced suboptimal tryptophan-FAD analogs. (Figure 1A). Initially we will generate tryptophan-FAD conjugates to serve as suboptimal targets for the anti-tryptophan aptamer. The binding affinity of tryptophan versus the tryptophan-FAD conjugate will be measured via isothermal titration calorimetry. Displacement of the suboptimal conjugate with addition of tryptophan (as a target) will be used to measure enzyme reactivation (which is indirectly indicative of target bound), and will be followed via a colorimetric coupled enzyme assay.7 The final studies will include measuring displaced tryptophan-FAD analogs directly via cyclic voltammetry. Finally, we will explore the selectivity of the system with the use of tryptophan derivatives as targets to induce displacement.
Aim 2. To establish an aptamer-based oligonucleotide detection strategy with the release of FAD upon target recognition. We will develop a detection strategy where aptamer hybridization to an oligonucleotide target will lead the release of FAD. Specifically, an aptamer for FAD will be re-designed with the complementary sequence for microRNA-21 (miR-21), a cancer associated microRNA,8 within the loop of the aptamer (Figure 1B). Selective binding of FAD to the re-designed aptamer will be followed via a colorimetric assay7. Hybridization of the miR-21 target to the aptamer loop will induce a conformational shift and release the FAD. Initial hybridization studies will be assessed via fluorescence spectroscopy by using a fluorophore quencher pair conjugated to the 5’ and 3’ ends of the aptamer respectively. Subsequently, we will explore the selectivity of the system by using mismatched sequences and single base pair mismatches. Finally, we will optimize the design of the recognition modules (complementary sequence within the loop of the aptamer) in order to increase selectivity as well as optimize signal to background measurements and determine the limits of detection of the system.
There have been numerous ‘reagentless’ electrochemical sensors based on the folding of aptamers. However, few have been able to accommodate the detection of environmentally relevant small-molecules as well as clinically relevant proteins and oligonucleotides. Preliminary work has shown free diffusing FAD to be easily detected via cyclic voltammetry with detectable concentrations as low as 10-11M. These results will provide a sensitive method to detect the target bound release of FAD and thus produce a measureable response from the proposed system. The major outcomes of this project are expected to be the generation of novel detection strategies, which will directly marry aptamer-based target recognition with the release of a measurable redox tag. These outcomes will have a positive impact by advancing the field of electrochemical sensor research and provide a unique perspective on biomolecule based sensor fabrication and signal output.
1. Bissonnette, L.; Bergeron, M. G., Diagnosing infections--current and anticipated technologies for point-of-care diagnostics and home-based testing. Clinical Microbiology and Infection 2010, 16, 1044-1053.
2. Huckle, D., Point-of-care diagnostics: an advancing sector with nontechnical issues. Expert Review of Molecular Diagnostics 2008, 8, 679-688.
3. Freeman, R.; Sharon, E.; Tel-Vered, R.; Willner, I., Supramolecular cocaine-aptamer complexes activate biocatalytic cascades. J. Am. Chem. Soc. 2009, 131, 5028-5029.
4. Bogomolova, A.; Aldissi, M., Real-time aptamer quantum dot fluorescent flow sensor. Biosens Bioelectron 2011, 26, 4099-4103.
5. Li, B.; Ellington, A. D.; Chen, X., Rational, modular adaptation of enzyme-free DNA circuits to multiple detection methods. Nucleic Acids Res. 2011, 39, e110.
6. Sitaula, S.; Branch, S. D.; Ali, M. F., GOx signaling triggered by aptamer-based ATP detection. Chem. Commun. 2012, 48, 9284-9286.
7. Blake, D. A.; McLean, N. V., A colorimetric assay for the measurement of D-Glucose consumption by cultured cells. Anal Biochem 1989, 177, 156-160.
8. Volinina, s.; Calin, G. A.; Liu, C.-G.; Ambs, S.; Cimmino, A.; Petrocca, F.; Visone, R.; Iorio, M.; Roldo, C.; Ferracin, M.; Prueitt, R. L.; Yanaihara, N.; Lanza, G.; Scarpa, A.; Vecchione, A.; Negrini, M.; Harris, C. C.; Croce, C. M., A microRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl. Acad. Sci. USA 2006, 103, 2257-2261.