Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life.
RNA
; 25(4): 507-518, 2019 04.
Article
em En
| MEDLINE
| ID: mdl-30647100
ABSTRACT
Extensive research in the past decade has brought mRNA closer to the clinical realization of its therapeutic potential. One common structural feature for all cellular messenger RNAs is a poly(A) tail, which can either be brought in cotranscriptionally via the DNA template (plasmid- or PCR-based) or added to the mRNA in a post-transcriptional enzymatic process. Plasmids containing poly(A) regions recombine in E. coli, resulting in extensive shortening of the poly(A) tail. Using a segmented poly(A) approach, we could significantly reduce recombination of plasmids in E. coli without any negative effect on mRNA half-life and protein expression. This effect was independent of the coding sequence. A segmented poly(A) tail is characterized in that it consists of at least two A-containing elements, each defined as a nucleotide sequence consisting of 40-60 adenosines, separated by a spacer element of different length. Furthermore, reducing the spacer length between the poly(A) segments resulted in higher translation efficiencies compared to homogeneous poly(A) tail and reduced recombination (depending upon the choice of spacer nucleotide). Our results demonstrate the superior potential of segmented poly(A) tails compared to the conventionally used homogeneous poly(A) tails with respect to recombination of the plasmids and the resulting mRNA performance (half-life and translational efficiency).
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Plasmídeos
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Poli A
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Biossíntese de Proteínas
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RNA Mensageiro
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Engenharia Genética
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article