title: Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences.
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.
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors: Bin
authors: B
authors: Wu
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA;
authors: Veronika
authors: V
authors: Miskolci
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA;
authors: Hanae
authors: H
authors: Sato
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA;
authors: Evelina
authors: E
authors: Tutucci
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA;
authors: Charles A
authors: CA
authors: Kenworthy
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors: Sara K
authors: SK
authors: Donnelly
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA;
authors: Young J
authors: YJ
authors: Yoon
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors: Dianne
authors: D
authors: Cox
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA robert.singer@einstein.yu.edu louis.hodgson@einstein.yu.edu.
authors: Robert H
authors: RH
authors: Singer
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA robert.singer@einstein.yu.edu louis.hodgson@einstein.yu.edu.
authors: Louis
authors: L
authors: Hodgson
mesh_terms: D000818:Animals; D001483:Base Sequence / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D003062:Codon / Q000235:genetics; D015870:Gene Expression; D005821:Genetic Techniques; D006801:Humans; D017909:Levivirus / Q000235:genetics; D051379:Mice; D059372:Nucleotide Motifs; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D003062:Codon
keywords: MS2; biosensor; genome integrity; retroviral reporter; single molecule