Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences.
Genes Dev
; 29(8): 876-86, 2015 Apr 15.
Article
en En
| MEDLINE
| ID: mdl-25877922
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Secuencias Repetitivas de Ácidos Nucleicos
/
Expresión Génica
/
Técnicas Genéticas
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Genes Dev
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2015
Tipo del documento:
Article
País de afiliación:
Estados Unidos