Using Spectroscopy to Guide the Adaptation of Aptamers into Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors utilize the binding-induced conformational change of an electrode-attached, redox-reporter-modified aptamer to transduce target recognition into an easily measurable electrochemical output. Because this signal transduction mechanism is single-step and rapidly reversible, EAB sensors support high-frequency, real-time molecular measurements, and because it recapitulates the reagentless, conformation-linked signaling seen in vivo among naturally occurring receptors, EAB sensors are selective enough to work in the complex, time-varying environments found in the living body. The fabrication of EAB sensors, however, requires that their target-recognizing aptamer be modified such that (1) it undergoes the necessary binding-induced conformational change and (2) that the thermodynamics of this "conformational switch" are tuned to ensure that they reflect an acceptable trade-off between affinity and signal gain. That is, even if an "as-selected" aptamer achieves useful affinity and specificity, it may fail when adapted to the EAB platform because it lacks the binding-induced conformational change required to support EAB signaling. In this paper we reveal the spectroscopy-guided approaches we use to modify aptamers such that they support the necessary binding-induced conformational change. Specifically, using newly reported aptamers, we demonstrate the systematic design of EAB sensors achieving clinically and physiologically relevant specificity, limits of detection, and dynamic range against the targets methotrexate and tryptophan.

Real-Time, Seconds-Resolved Measurements of Plasma Methotrexate In Situ in the Living Body.

Dose-limiting toxicity and significant patient-to-patient pharmacokinetic variability often render it difficult to achieve the safe and effective dosing of drugs. This is further compounded by the slow, cumbersome nature of the analytical methods used to monitor patient-specific pharmacokinetics, which inevitably rely on blood draws followed by post-facto laboratory analysis. Motivated by the pressing need for improved "therapeutic drug monitoring", we are developing electrochemical aptamer-based (EAB) sensors, a minimally invasive biosensor architecture that can provide real-time, seconds-resolved measurements of drug levels in situ in the living body. A key advantage of EAB sensors is that they are generalizable to the detection of a wide range of therapeutic agents because they are independent of the chemical or enzymatic reactivity of their targets. Three of the four therapeutic drug classes that have, to date, been shown measurable using in vivo EAB sensors, however, bind to nucleic acids as part of their mode of action, leaving open questions regarding the extent to which the approach can be generalized to therapeutics that do not. Here, we demonstrate real-time, in vivo measurements of plasma methotrexate, an antimetabolite (a mode of action not reliant on DNA binding) chemotherapeutic, following human-relevant dosing in a live rat animal model. By providing hundreds of drug concentration values, the resulting seconds-resolved measurements succeed in defining key pharmacokinetic parameters, including the drug's elimination rate, peak plasma concentration, and exposure (area under the curve), with unprecedented 5 to 10% precision. With this level of precision, we easily identify significant (>2-fold) differences in drug exposure occurring between even healthy rats given the same mass-adjusted methotrexate dose. By providing a real-time, seconds-resolved window into methotrexate pharmacokinetics, such measurements can be used to precisely "individualize" the dosing of this significantly toxic yet vitally important chemotherapeutic.

Light-Directed Migration of D. discoideum Slugs in Microfabricated Confinements.

This paper investigates the light-driven migration of the multi-cellular microorganism Dictyostelium discoideum as a potential bio-actuation mechanism in microsystems. As a platform for slug migration we use microscale confinements, which consist of intersecting microchannels fabricated from solidified agar-water solution. The agar surface provides necessary moisture to the slugs during the experiment while remaining sufficiently stiff to allow effective slug migration. The movements of the slugs in the microchannels are driven and guided by phototaxis via controlling light transmitted through optical fibers. The microchannels impose geometrical confinements on the migrating slugs, improving the spatial precision of the migration. We demonstrate that slugs that form in a microchamber can be driven to migrate through the microchannels, as well as steered to a particular direction at microchannel intersections. Our experimental results indicate that slug movements can be more effectively controlled in microchannels, and potentially useful for bio-actuation applications.

Mutation of the Dictyostelium fbxA gene affects cell-fate decisions and spatial patterning.

Cell-fate decisions and spatial patterning in Dictyostelium are regulated by a number of genes. Our studies have implicated a gene called fbxA, which codes for an F-box protein, in these pathways. The FbxA protein is one of the controls on a cAMP phosphodiesterase called RegA, mediating its degradation via ubiquitin-linked proteolysis. Using marked strains, we showed that the fbxA- mutant has defective cell-type proportioning, with a dearth of prestalk cells compared to prespore cells. In this work, we show that this effect occurs earlier during the 24 hour developmental cycle than previously thought. The normal sorting of the prestalk and prespore cells in aggregates and mounds is not affected by the mutation. The mutant cells sort abnormally at the tipped mound stage, when prespore and prestalk cells normally distribute into their proper compartments. The fbxA- mutant forms pre-stalk cells in low numbers when not in chimeras, but in the presence of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form non-viable stalk cells. In an attempt to identify the signal transduction pathway that mediates proportionality in prestalk and prespore cells, we asked whether certain signal transduction mutants were immune to the effects of the fbxA- cells and formed spores in chimeras.

Genetic interactions of the E3 ubiquitin ligase component FbxA with cyclic AMP metabolism and a histidine kinase signaling pathway during Dictyostelium discoideum development.

Dictyostelium discoideum amoebae with an altered fbxA gene, which is thought to encode a component of an SCF E3 ubiquitin ligase, have defective regulation of cell type proportionality. In chimeras with wild-type cells, the mutant amoebae form mainly spores, leaving the construction of stalks to wild-type cells. To examine the role of fbxA and regulated proteolysis, we have recovered the promoter of fbxA and shown that it is expressed in a pattern resembling that of a prestalk-specific gene until late in development, when it is also expressed in developing spore cells. Because fbxA cells are developmentally deficient in pure culture, we were able to select suppressor mutations that promote sporulation of the original mutant. One suppressor mutation resides within the gene regA, which encodes a cyclic AMP (cAMP) phosphodiesterase linked to an activating response regulator domain. In another suppressor, there has been a disruption of dhkA, a gene encoding a two-component histidine kinase known to influence Dictyostelium development. RegA appears precociously and in greater amounts in the fbxA mutant than in the wild type, but in an fbxA/dhkA double mutant, RegA is restored to wild-type levels. Because the basis of regA suppression might involve alterations in cAMP levels during development, the concentrations of cAMP in all strains were determined. The levels of cAMP are relatively constant during multicellular development in all strains except the dhkA mutant, in which it is reduced at least sixfold. The level of cAMP in the double mutant dhkA/fbxA is relatively normal. The levels of cAMP in the various mutants do not correlate with spore formation, as would be expected on the basis of our present understanding of the signaling pathway leading to the induction of spores. Altered amounts of RegA and cAMP early in the development of the mutants suggest that both fbxA and dhkA genes act earlier than previously thought.