Harnessing cooperativity to achieve high-precision in vivo measurements

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Summary. The ability to measure molecules and monatomic ions in the body in real-time and with high-precision would revolutionize many aspects of both biomedical research and clinical practice. It would, for example, provide clinicians with immediately actionable information monitoring regarding electrolyte imbalances, and the plasma levels of drugs of dangerous narrow therapeutic windows. To this end, we are developing Electrochemical Aptamer-Based (EAB) sensors, a demonstrably generalizable platform technology for measuring analyte concentrations in situ in the body. Using this technique, we have already demonstrated the real-time, seconds-resolved measurement of more than a dozen drugs, metabolites and protein biomarkers in the veins, brains, and peripheral tissues of live rats and the subcutaneous space of human subjects for periods of up to 24 h. Building on this, we propose here aptamer selection and aptamer-engineering approaches aimed at improving the sensitivity of these receptors to small changes in the concentration of their target ligands. Our first approach to this end is overcome the often-poor affinity of small-molecule-binding aptamers, thus “tuning” of their affinities to optimally match the concentration range of clinical interest. To achieve this, we are developing unprecedented new selection schemes, including analog-selection, an approach for obtaining initial, if sometimes low-performance, aptamers against difficult targets, and insertion-reselection, which recursively (and dramatically) increases the structural complexity, and thus the performance, of these initial aptamers. Our second aim uses the excess binding energy (i.e., dissociation constants several-fold below the necessary measurement range) afforded by these advanced selection schemes as a basis for introducing allosteric cooperativity, a mechanism that greatly steepens binding curves. In the near term, the expected outcome of the proposed research will be a suite of high-precision, in-vivo EAB sensors against a set of clinically important, narrow-clinical-window drugs, metabolites, and electrolytes. The expected long-term impact of our work however, is much broader, as our success will establish approaches by which the responsiveness of biomolecular receptors to changing ligand concentrations can be rationally improved, a development that will positively impact many receptor-based biotechnologies.