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Main Menu - Block
- Overview
- Anatomy and Histology
- Cryo-Electron Microscopy
- Electron Microscopy
- Flow Cytometry
- Gene Targeting and Transgenics
- Immortalized Cell Line Culture
- Integrative Imaging
- Invertebrate Shared Resource
- Janelia Experimental Technology
- Mass Spectrometry
- Media Prep
- Molecular Genomics
- Primary & iPS Cell Culture
- Project Pipeline Support
- Project Technical Resources
- Quantitative Genomics
- Scientific Computing Software
- Scientific Computing Systems
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- Vivarium
Note: Research in this publication was not performed at Janelia.
Abstract
Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts.
