Intracellular sensor spatial multiplexing via RNA scaffolds

Abstract

To circumvent the limitations of spectrally multiplexing sensors, fluorescent sensors are clustered by type and spatially separated in the cytoplasm to avoid cross-talk. Each sensor is fused to an orthogonal viral capsid protein that binds to a long, repetitive strand of its corresponding RNA sequence. All sensors fluoresce green and are indistinguishable during recording but are identified with post-hoc antibody or FISH staining for each sensor-specific puncta. This spatial multiplexing strategy will allow for easier scaling of the number of fluorescent reporters of physiological activity.