Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences

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 in...

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Veröffentlicht in:Genes & development 2015-04, Vol.29 (8), p.876-886
Hauptverfasser: Wu, Bin, Miskolci, Veronika, Sato, Hanae, Tutucci, Evelina, Kenworthy, Charles A, Donnelly, Sara K, Yoon, Young J, Cox, Dianne, Singer, Robert H, Hodgson, Louis
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container_end_page 886
container_issue 8
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container_title Genes & development
container_volume 29
creator Wu, Bin
Miskolci, Veronika
Sato, Hanae
Tutucci, Evelina
Kenworthy, Charles A
Donnelly, Sara K
Yoon, Young J
Cox, Dianne
Singer, Robert H
Hodgson, Louis
description 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.
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subjects Animals
Base Sequence - genetics
Capsid Proteins - genetics
Cell Line
Cells, Cultured
Codon - genetics
Gene Expression
Genetic Techniques
Humans
Levivirus - genetics
Mice
Nucleotide Motifs
Repetitive Sequences, Nucleic Acid - genetics
Resource/Methodology
Saccharomyces cerevisiae - genetics
title Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences
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