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Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation

Zeljka Trepotec1, Johannes Geiger2, Christian Plank2,3

  • 1Department of Pediatrics, Ludwig-Maximilian-University of Munich, 80337 Munich, Germany.

RNA (New York, N.Y.)
|January 17, 2019
PubMed

Insights

Segmented poly(A) tails reduce plasmid recombination in E. coli, improving messenger RNA (mRNA) performance. This novel approach enhances mRNA half-life and translation efficiency without negatively impacting protein expression.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • RNA Therapeutics

Background:

  • Messenger RNA (mRNA) therapeutics are advancing clinically, with the poly(A) tail being a crucial structural element.
  • Conventional methods of poly(A) tail addition can lead to plasmid recombination in E. coli, shortening the tail and affecting mRNA function.
  • Plasmid instability during production impacts the yield and quality of mRNA for therapeutic applications.

Purpose of the Study:

  • To investigate the efficacy of a segmented poly(A) tail approach in mitigating plasmid recombination during mRNA production.
  • To evaluate the impact of segmented poly(A) tails on mRNA half-life, protein expression, and translational efficiency.
  • To compare the performance of segmented poly(A) tails against traditional homogeneous poly(A) tails.

Main Methods:

  • Development and implementation of a segmented poly(A) tail strategy, comprising multiple adenosine-rich elements separated by spacer sequences.
  • Assessment of plasmid recombination rates in E. coli using segmented versus homogeneous poly(A) tails.
  • Quantification of mRNA half-life and protein expression levels derived from constructs with different poly(A) tail designs.

Main Results:

  • Segmented poly(A) tails significantly reduced plasmid recombination in E. coli compared to homogeneous poly(A) tails.
  • This reduction in recombination was independent of the coding sequence and did not negatively affect mRNA half-life or protein expression.
  • Optimizing spacer length within the segmented poly(A) tail further enhanced translation efficiencies and reduced recombination.

Conclusions:

  • Segmented poly(A) tails offer a superior alternative to homogeneous poly(A) tails for robust mRNA production.
  • This strategy enhances plasmid stability and improves overall mRNA performance, including translational efficiency and half-life.
  • The findings support the potential of segmented poly(A) tails for advancing mRNA-based therapeutics.
Keywords:
mRNA therapeuticsplasmid recombinationpoly(A) tailtranscript therapy

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