title: Improvement of DNA minicircle production by optimization of the secondary structure of the 5'-UTR of ParA resolvase.
abstract: The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5' untranslated region (5'-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5'-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6-14.9 kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48-817-fold) translation initiation rate. No effect of the 5'-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2 h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5'-UTR. Interestingly, the amount of this transcript was 2.6-3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5'-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5'-UTR (∼1.8 ± 0.7-fold). Overall, these results show that the improvements made in the 5'-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.|MIT-Portugal Program, Cambridge, MA, USA.|MIT-Portugal Program, Lisbon, Portugal.
authors.forename: Michaela
authors.initials: M
authors.lastname: Šimčíková
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.
authors.forename: Cláudia P A
authors.initials: CPA
authors.lastname: Alves
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.
authors.forename: Liliana
authors.initials: L
authors.lastname: Brito
authors.affiliation: MIT-Portugal Program, Cambridge, MA, USA.|MIT-Portugal Program, Lisbon, Portugal.|Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: Kristala L J
authors.initials: KLJ
authors.lastname: Prather
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.|MIT-Portugal Program, Cambridge, MA, USA.|MIT-Portugal Program, Lisbon, Portugal.
authors.forename: Duarte M F
authors.initials: DMF
authors.lastname: Prazeres
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal. gabmonteiro@tecnico.ulisboa.pt.|MIT-Portugal Program, Cambridge, MA, USA. gabmonteiro@tecnico.ulisboa.pt.|MIT-Portugal Program, Lisbon, Portugal. gabmonteiro@tecnico.ulisboa.pt.
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Monteiro
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D051777:AraC Transcription Factor / Q000235:genetics; D004270:DNA, Circular / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D045522:Recombinases / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D020121:5' Untranslated Regions; D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D004270:DNA, Circular; D029968:Escherichia coli Proteins; C541712:ParA protein, E coli; D012333:RNA, Messenger; D045522:Recombinases
keywords: 5′-UTR; Minicircle; Ribosome binding site; Translational initiation rate; mRNA secondary structure; parA resolvase