Optogenetically controlled RAF to characterize BRAF and CRAF protein kinase inhibitors.

Here, we applied optoRAF, an optogenetic tool for light-controlled clustering and activation of RAF proteins that mimics the natural occurring RAS-mediated dimerization. This versatile tool allows studying the effect on BRAF and CRAF homodimer- as well as heterodimer-induced RAF signaling. Vemurafenib and dabrafenib are two clinically approved inhibitors for BRAF that efficiently suppress the kinase activity of oncogenic BRAF (V600E). However in wild-type BRAF expressing cells, BRAF inhibitors can exert paradoxical activation of wild-type CRAF. Using optoRAF, vemurafenib was identified as paradoxical activator of BRAF and CRAF homo- and heterodimers. Dabrafenib enhanced activity of light-stimulated CRAF at low dose and inhibited CRAF signaling at high dose. Moreover, dabrafenib increased the protein level of CRAF proteins but not of BRAF proteins. Increased CRAF levels correlate with elevated RAF signaling in a dabrafenib-dependent manner, independent of light activation.

Transcription factor sensor system for parallel quantification of metabolites on-chip.

Steadily growing demands for identification and quantification of cellular metabolites in higher throughput have brought a need for new analytical technologies. Here, we developed a synthetic biological sensor system for quantifying metabolites from biological cell samples. For this, bacterial transcription factors were exploited, which bind to or dissociate from regulatory DNA elements in response to physiological changes in the cellular metabolite concentration range. Representatively, the bacterial pyruvate dehydrogenase (PdhR), trehalose (TreR), and l-arginine (ArgR) repressor proteins were functionalized to detect pyruvate, trehalose-6-phosphate (T6P), and arginine concentration in solution. For each transcription factor the mutual binding behavior between metabolite and DNA, their working range, and othogonality were determined. High-throughput, parallel processing, and automation were achieved through integration of the metabolic sensor system on a microfluidic large-scale integration (mLSI) chip platform. To demonstrate the functionality of the integrated metabolic sensor system, we measured diurnal concentration changes of pyruvate and the plant signaling molecule T6P within cell etxracts of Arabidopsis thaliana rosettes. The transcription factor sensor system is of generic nature and extendable on the microfluidic chip.

Microfluidic synthesis of pharmacologically responsive supramolecular biohybrid microgels.

Biohybrid hydrogels that change their mechanical properties in response to pharmacological cues hold high promises as externally controlled drug depots for biomedical applications. In this study, we devise a generically applicable method for the synthesis of micrometer-scale, injection-ready biohybrid materials. We use droplet-based microfluidics to generate monodisperse pre-microgel fluid droplets, wherein which we react fluorescein-modified 8-arm poly(ethylene glycol) with a thiol-functionalized humanized anti-fluorescein single chain antibody fragment and vinylsulfone-functionalized 8-arm poly(ethylene glycol), resulting in the formation of stable, narrowly dispersed supramolecular microgels (30 and 150 µm diameter). We demonstrate that the addition of free fluorescein to these microgels results in a weakening of their hydrogel structure, eventually leading to its disintegration. This method of formation of pharmacologically responsive biohybrid hydrogels in an injection-ready formulation is a pioneering example of a general approach for the synthesis of biohybrid hydrogel-based drug depots for biomedical applications.