Rational Design of a Circularly Permuted Flavin-Based Fluorescent Protein.

Flavin-based fluorescent proteins are oxygen-independent reporters that hold great promise for imaging anaerobic and hypoxic biological systems. In this study, we explored the feasibility of applying circular permutation, a valuable method for the creation of fluorescent sensors, to flavin-based fluorescent proteins. We used rational design and structural data to identify a suitable location for circular permutation in iLOV, a flavin-based reporter derived from A. thaliana. However, relocating the N- and C-termini to this position resulted in a significant reduction in fluorescence. This loss of fluorescence was reversible, however, by fusing dimerizing coiled coils at the new N- and C-termini to compensate for the increase in local chain entropy. Additionally, by inserting protease cleavage sites in circularly permuted iLOV, we developed two protease sensors and demonstrated their application in mammalian cells. In summary, our work establishes the first approach to engineer circularly permuted FbFPs optimized for high fluorescence and further showcases the utility of circularly permuted FbFPs to serve as a scaffold for sensor engineering.

Exploring the potential of water channels for developing MRI reporters and sensors without the need for exogenous contrast agents.

Genetically encoded reporters for magnetic resonance imaging (MRI) offer a valuable technology for making molecular-scale measurements of biological processes within living organisms with high anatomical resolution and whole-organ coverage without relying on ionizing radiation. However, most MRI reporters rely on contrast agents, typically paramagnetic metals and metal complexes, which often need to be supplemented exogenously to create optimal contrast. To eliminate the need for contrast agents, we previously introduced aquaporin-1, a mammalian water channel, as a new reporter gene for the fully autonomous detection of genetically labeled cells using diffusion-weighted MRI. In this study, we aimed to expand the toolbox of diffusion-based genetic reporters by modulating aquaporin membrane trafficking and harnessing the evolutionary diversity of water channels across species. We identified a number of new water channels that functioned as diffusion-weighted reporter genes. In addition, we show that loss-of-function variants of yeast and human aquaporins can be leveraged to design first-in-class diffusion-based sensors for detecting the activity of a model protease within living cells.

Engineering ligand stabilized aquaporin reporters for magnetic resonance imaging.

Imaging transgene expression in live tissues requires reporters that are detectable with deeply penetrant modalities, such as magnetic resonance imaging (MRI). Here, we show that LSAqp1, a water channel engineered from aquaporin-1, can be used to create background-free, drug-gated, and multiplex images of gene expression using MRI. LSAqp1 is a fusion protein composed of aquaporin-1 and a degradation tag that is sensitive to a cell-permeable ligand, which allows for dynamic small molecule modulation of MRI signals. LSAqp1 improves specificity for imaging gene expression by allowing reporter signals to be conditionally activated and distinguished from the tissue background by difference imaging. In addition, by engineering destabilized aquaporin-1 variants with different ligand requirements, it is possible to image distinct cell types simultaneously. Finally, we expressed LSAqp1 in a tumor model and showed successful in vivo imaging of gene expression without background activity. LSAqp1 provides a conceptually unique approach to accurately measure gene expression in living organisms by combining the physics of water diffusion and biotechnology tools to control protein stability.