Engineering protein-RNA interfaces for monitoring and manipulating RNA in living cells
Embargo Date
2026-05-23
OA Version
Citation
Abstract
mRNA serves a critical role in eukaryotic biology as an intermediary between stably encoded genetic information and dynamic protein-based processes. As such, cells have developed complex sets of interacting mechanisms to regulate the localization, abundance and functionality of mRNA in both time and space. The development of tools that allow researchers to observe and manipulate these processes has led to better understanding of diverse biological phenomena ranging from subcellular regulation of synaptic plasticity to the multicellular complexities of development and embryogenesis. Improving and expanding the capabilities of these tools will be important for enabling new research in both fundamental and translational RNA biology. In this work, we describe the application of protein engineering to develop new and sensitive techniques to both target and manipulate mRNA in mammalian cells. In the first portion of this work, we describe the generation of conditionally stable RNA-binding proteins through a combination of circular permutation and degron masking. We found that the application of these destabilized coat proteins to live-cell mRNA imaging led to improvements in resolution by suppressing the background associated with unbound protein subunits. In the second portion of this work, we describe a new and modular framework for designing mRNA-based synthetic circuits. By engineering allosterically regulated and reversibly autoinhibited adenosine deaminase domains and their substrates, we demonstrate the ability to induce translation in response to small molecule drugs, intracellular antigens, proteolytic activity, and optical stimulation. We then combine these components to generate self-regulating mRNA molecules capable of inducing programmable gene expression within minutes of stimulus addition. Lastly, we present preliminary findings that highlight the potential of these tools to be integrated with additional layers of protein logic. This work simultaneously provides tangible improvements to existing and widely applied techniques in RNA biological studies, offers novel and modular synthetic means of controlling mRNA activity and translation, and demonstrates a set of protein design principles which may be of general utility and applicability to the field.