Super-Resolution-Chip: an in-vitro platform that enables super-resolution microscopy of co-cultures and 3D systems.

The development of organs-on-a-chip platforms has revolutionized in-vitro cellular culture by allowing cells to be grown in an environment that better mimics human physiology. However, there is still a challenge in integrating those platforms with advanced imaging technology. This is extremely important when we want to study molecular changes and subcellular processes on the level of a single molecule using super-resolution microscopy (SRM), which has a resolution beyond the diffraction limit of light. Currently, existing platforms that include SRM have certain limitations, either as they only support 2D monocultures, without flow or as they demand a lot of production and handling. In this study, we developed a Super-Res-Chip platform, consisting of a 3D-printed chip and a porous membrane, that could be used to co-culture cells in close proximity either in 2D or in 3D while allowing SRM on both sides of the membrane. To demonstrate the functionality of the device, we co-cultured in endothelial and epithelial cells and used direct stochastic optical reconstruction microscopy (dSTORM) to investigate how glioblastoma cells affect the expression of the gap-junction protein Connexin43 in endothelial cells grown in 2D and in 3D. Cluster analysis of Connexin43 distribution revealed no difference in the number of clusters, their size, or radii, but did identify differences in their density. Furthermore, the spatial resolution was high also when the cells were imaged through the membrane (20-30 nm for x-y) and 10-20 nm when imaged directly both for 2D and 3D conditions. Overall, this chip allows to characterize of complex cellular processes on a molecular scale in an easy manner and improved the capacity for imaging in a single molecule resolution complex cellular organization.

Aptamer-Mediated Control of Polyadenylation for Gene Expression Regulation in Mammalian Cells.

Small aptamer-based regulatory devices can be designed to control a range of RNA-dependent cellular processes and emerged as promising tools for fine-tuning gene expression in synthetic biology. Here, we design a conceptually new riboswitch device that allows for the conditional regulation of polyadenylation. By making use of ligand-induced sequence occlusion, the system efficiently controls the accessibility of the eukaryotic polyadenylation signal. Undesirable 3'-extended read-through products are counteracted by the downstream insertion of a microRNA target site. We demonstrate the modularity of the system with regard to sensor aptamers and polyadenylation signals used and combine the newly designed riboswitch with well-known aptazymes to yield superior composite systems. In addition, we show that the switches can be used to control alternative polyadenylation. The presented genetic switches require very little coding space and can be easily optimized by rational adjustments of the thermodynamic stability. The polyadenylation riboswitch extends the repertoire of RNA-based regulators and opens new possibilities for the generation of complex synthetic circuits.