Live Cell Imaging using RNA-Stabilized Fluorogenic Proteins

Background & Unmet Need

• RNA imaging with fluorescent microscopy enables the study of RNA localization and spatiotemporal dynamics
• One technique involves the use of fluorogenic RNA aptamers, which induce fluorescence by binding not otherwise fluorescent molecules
• However, there are currently few fluorogenic dyes compatible with live cell imaging, as they must be added exogenously and often display nonspecific fluorescence activation by cellular lipids or DNA
• Unmet Need: Inducible RNA imaging system compatible with live cells

Technology Overview

• The Technology: A method for imaging mRNA using genetically encoded fluorogenic proteins that are specifically activated by an RNA aptamer named “Pepper”
• The inventors converted constitutively active fluorescent proteins, such EYFP and mNeonGreen, into conditionally fluorescent proteins through incorporation of a degradation tag (tDEG) that contains an RNA-binding site
• In the absence of Pepper RNA, tDeg-tagged proteins are rapidly degraded, enabling RNA-dependent imaging with fluorescent proteins
• PoC Data: EYFP-tDEG construct exhibits 38-fold increase in fluorescence in presence of Pepper RNA
• Doesn’t require addition of nuclear localization elements to the fluorescent protein
• Ideal tool for tracking and quantifying RNA expression in live cells

Technology Applications

• Visualization and imaging of cellular mRNA using standard microscopy techniques
• Temporal measurement of RNA expression
• Screening for novel RNA-binding proteins in live cells
• Tunable regulation of protein expression for synthetic biology applications

Technology Advantages

• Minimal impact to mRNA turnover rates and translation efficiencies
• Compatible with numerous fluorogenic proteins, providing a wide range of spectral properties
• Regulated reporter degradation delivers low background fluorescence