Improvement of DNA Minicircle Production by Optimization of the Secondary Structure of the 5#-UTR of Para Resolvase
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Improvement of DNA minicircle production by optimization of the secondary structure of the 5#-UTR of ParA resolvase The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Šimčíková, Michaela et al. “Improvement of DNA Minicircle Production by Optimization of the Secondary Structure of the 5#- UTR of ParA Resolvase.” Applied Microbiology and Biotechnology 100.15 (2016): 6725–6737. As Published http://dx.doi.org/10.1007/s00253-016-7565-x Publisher Springer Berlin Heidelberg Version Author's final manuscript Citable link http://hdl.handle.net/1721.1/104377 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike Detailed Terms http://creativecommons.org/licenses/by-nc-sa/4.0/ Appl Microbiol Biotechnol (2016) 100:6725–6737 DOI 10.1007/s00253-016-7565-x APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase Michaela Šimčíková1,2,3 & Cláudia P. A. Alves1 & Liliana Brito1 & Kristala L. J. Prather2,3,4 & Duarte M. F. Prazeres1,2,3 & Gabriel A. Monteiro1,2,3 Received: 21 March 2016 /Revised: 8 April 2016 /Accepted: 16 April 2016 /Published online: 5 May 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract The use of minicircles in gene therapy applications the optimized 5′-UTR. Interestingly, the amount of this tran- is dependent on the availability of high-producer cell systems. script was 2.6–3-fold higher when compared with the tran- In order to improve the performance of minicircle production script generated from the original sequence, highlighting that in Escherichia coli by ParA resolvase-mediated in vivo re- 5′-UTR affects the level of the transcript. A Western blot anal- combination, we focus on the 5′ untranslated region (5′- ysis confirmed that E. coli synthesized higher amounts of UTR) of parA messenger RNA (mRNA). The arabinose- ParA with the new 5′-UTR (∼1.8 ± 0.7-fold). Overall, these inducible PBAD/araC promoter controls ParA expression and results show that the improvements made in the 5′-UTR can strains with improved arabinose uptake are used. The 27- lead to a more efficient translation and hence to faster and nucleotide-long 5′-UTR of parA mRNA was optimized using more efficient minicircle generation. a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of Keywords Minicircle . parA resolvase . 5′-UTR . 8.6–14.9 kcal/mol in the change in Gibbs free energy upon Ribosome binding site . mRNA secondary structure . assembly of the 30S ribosome complex with the mRNA sub- Translational initiation rate sequences, 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 Introduction expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2 h. However, when The low efficacy of plasmids as gene transfer vectors in gene the parA gene was inserted in the bacterial chromosome, a therapy and DNA vaccination applications has been attributed faster and more effective recombination was obtained with to several reasons, including (i) low cell and nucleus uptake (Kreiss et al. 1999; Dean et al. 2005), (ii) degradation by endogenous nucleases during cytoplasm trafficking Electronic supplementary material The online version of this article (Lechardeur et al. 1999; Azzoni et al. 2007), and (iii) silencing (doi:10.1007/s00253-016-7565-x) contains supplementary material, which is available to authorized users. of transgene expression (Faurez et al. 2010;Luetal.2012). As a result, plasmid-mediated transgene expression is usually * Gabriel A. Monteiro short-lived and gene delivery falls short of its goal. [email protected] Furthermore, and although plasmids have a very good safety record in the clinic, concerns regarding the induction of un- 1 iBB—Institute for Bioengineering and Biosciences, Department of wanted immunogenic reactions (Zhao et al. 2004) and inte- Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, gration into the host genomic DNA (Wang et al. 2004)are Av. Rovisco Pais, 1049-001 Lisbon, Portugal recurrent. These events have been linked in part to the pres- 2 MIT-Portugal Program, Cambridge, MA, USA ence in the plasmid vector backbone of DNA sequences such 3 MIT-Portugal Program, Lisbon, Portugal as promoters, un-methylated cytosine-phosphate-guanine 4 Department of Chemical Engineering, Massachusetts Institute of (CpG) motifs, or selection markers, which are mostly of bac- Technology, Cambridge, MA, USA terial origin. For example, bacterial DNA sequences have 6726 Appl Microbiol Biotechnol (2016) 100:6725–6737 been shown to play a role in the silencing of plasmid-mediated researchers showed that complete recombination could be transgene expression (Chen et al. 2004; Chen et al. 2008). achieved if each parental plasmid encoded its own Additionally, CpG motifs of bacterial origin found in most recombinase (Chen et al. 2003;Jechlingeretal.2004; Chen plasmid vectors are known to drive inflammatory responses. et al. 2005; Mayrhofer et al. 2005). Complete recombination While this property can be advantageously explored when the of parental plasmid from the chromosome is also feasible if generation of an adaptive response to a given antigen is being expression is driven by more than one gene copy, as demon- sought (e.g., in the case of DNA vaccines, Chen et al. 2011), strated by the use of ten copies of the ϕC31 integrase in the such inflammatory responses may be strong enough to cause E. coli strain ZYCY10P3S2T (Kay et al. 2010). severe adverse effects (Zhao et al. 2004). Thus, the removal of The application of minicircles in the fields of DNA vacci- CpG motifs from a plasmid vector can substantially improve nation and gene therapy is currently hampered by the lack of both safety and the duration of the expression of the encoded technologies capable of producing the gram-kilogram therapeutic function (Yew et al. 2002). Additionally, the anti- amounts of minicircles required for pre-clinical and clinical biotic selection marker used to select plasmids during produc- trials. For example, the best volumetric productivities of tion may in some cases increase the risk of spread of antibiotic minicircles obtained during the cell culture step reported in resistance traits to environmental microbes (EMA 2001; the literature are still only of the order of 0.5–9.0 mg/L USFDA 2007; Salyers et al. 2004). Finally, cryptic eukaryotic (Kreiss et al. 1998;Chenetal.2005; Mayrhofer et al. 2008; transcription signals naturally occurring in plasmid backbones Kay et al. 2010;Gasparetal.2014). This offers a striking may give rise to deviant transcripts and unanticipated RNA contrast with the developments observed in plasmid processing that alter gene activity (Bert et al. 2000;Lempetal. manufacturing, which have pushed fermentation productiv- 2012). Although essential for selection and replication in mi- ities up to 2.2–2.6 g/L (Carnes et al. 2011; Gonçalves et al. crobial host cells (e.g., Escherichia coli)duringmanufactur- 2014). The low performance of the fermentation processes ing, most of the sequences described above are superfluous in used to produce minicircle could be ascribed to (i) low expres- terms of the eukaryotic gene expression that is at the heart of sion of recombinases (Bigger et al. 2001; Jechlinger et al. gene therapy and DNA vaccination applications. 2004), (ii) low recombinase activity in cells reaching station- Minicircles are plasmid-derived, covalently closed double- ary phase (Kay et al. 2010), and (iii) susceptibility of stranded DNA molecules that do not contain the bacterial recombinases to changes in pH, temperature, and metabolite elements required for selection and amplification. Since they accumulation (Chen et al. 2005; Simcikova et al. 2014). comprise exclusively the eukaryotic expression cassette, One aspect that has not been examined in the context of minicircles exhibit increased transfection efficiency and trans- minicircle production is the role of messenger RNA (mRNA) gene expression when compared to their parental plasmids on secondary structure on the expression of the recombinase. an equimolar basis (Darquet et al. 1997; Darquet et al. 1999; This could be an important bottleneck since a number of au- Kreiss et al. 1998; Mayrhofer et al. 2008; Mayrhofer et al. thors have gathered evidence that points to the fact that gene 2009). The typical minicircle production scheme encompasses expression in E. coli is inversely related to the stability of the an in vivo induced recombination event between two direct secondary structure of their ribosome binding sites (RBS) repeats within a parental plasmid. This event originates two (e.g., de Smit and van Duin 1990; Pfleger et al. 2005; Cèbe supercoiled plasmid DNA molecules: (i) a minicircle that and Geiser 2006; Salis et al. 2009). The main goal of this study carries the eukaryotic expression cassette and (ii) a replicative was to improve the performance of minicircle production by miniplasmid that contains the bacterial elements required for ParA resolvase-mediated in vivo recombination by focusing amplification. Various recombinases acting under the regula- attention on the RBS and surrounding sequence of the mRNA tion of inducible promoters have been used to catalyze the of ParA (Smith and Schleif 1978). We reasoned that by chang- excision and ligation of the minicircle sequences from paren- ing the nucleotide sequence of this 5′ untranslated region (5′- tal plasmids. For example, the phage λ integrase (Darquet UTR), potential hairpins that might be causing ribosomes to et al. 1997; Darquet et al. 1999;Kreissetal.1998)isinduced stall could be destabilized, thus increasing translation of by a thermal shift to 42 °C from the cI857 promoter, whereas mRNA and ParA levels and ultimately improving recombina- Cre recombinase (Bigger et al. 2001), ϕC31 integrase (Chen tion of parental plasmids into minicircles.