NAR Top Articles - Synthetic Biology and Chemistry
Amphiphilic block copolymers enhance the cellular uptake of DNA molecules through a facilitated plasma membrane transport
Chevre, R; Le Bihan, O; Beilvert, F; Chatin, B; Barteau, B; Mevel, M; Lambert, O; Pitard, B
Nucleic Acids Res. (2011) 39 (4): 1610-1622
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Amphiphilic block copolymers have been developed recently for their efficient, in vivo transfection activities in various tissues. Surprisingly, we observed that amphiphilic block copolymers such as Lutrol (R) do not allow the transfection of cultured cells in vitro, suggesting that the cell environment is strongly involved in their mechanism of action. In an in vitro model mimicking the in vivo situation we showed that pre-treatment of cells with Lutrol (R), prior to their incubation with DNA molecules in the presence of cationic lipid, resulted in higher levels of reporter gene expression. We also showed that this improvement in transfection efficiency associated with the presence of Lutrol (R) was observed irrespective of the plasmid promoter. Considering the various steps that could be improved by Lutrol (R), we concluded that the nucleic acids molecule internalization step is the most important barrier affected by Lutrol (R). Microscopic examination of transfected cells pre-treated with Lutrol (R) confirmed that more plasmid DNA copies were internalized...
TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain
Li, T; Huang, S; Jiang, WZ; Wright, D; Spalding, MH; Weeks, DP; Yang, B
Nucleic Acids Res. (2011) 39 (1): 359-372
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DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences...
Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes
Li, T; Huang, S; Zhao, XF; Wright, DA; Carpenter, S; Spalding, MH; Weeks, DP; Yang, B
Nucleic Acids Res. (2011) 39 (14): 6315-6325
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Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains...
Modular control of multiple pathways using engineered orthogonal T7 polymerases
Temme, K; Hill, R; Segall-Shapiro, TH; Moser, F; Voigt, CA
Nucleic Acids Res. (2012) 40 (17): 8773-8781
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Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a 'controller' plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions...
Chimeric TALE recombinases with programmable DNA sequence specificity
Mercer, AC; Gaj, T; Fuller, RP; Barbas, CF
Nucleic Acids Res. (2012) 40 (21): 11163-11172
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Site-specific recombinases are powerful tools for genome engineering. Hyperactivated variants of the resolvase/invertase family of serine recombinases function without accessory factors, and thus can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered zinc-finger proteins (ZFPs). However, imperfect modularity with particular domains, lack of high-affinity binding to all DNA triplets, and difficulty in construction has hindered the wide-spread adoption of ZFPs in unspecialized laboratories. The discovery of a novel type of DBD in transcription activator-like effector (TALE) proteins from Xanthomonas provides an alternative to ZFPs. Here we describe chimeric TALE recombinases (TALERs): engineered fusions between a hyperactivated catalytic domain from the DNA invertase Gin and an optimized TALE architecture. We use a library of incrementally truncated TALE variants to identify TALER fusions that modify DNA with efficiency and specificity comparable to zinc-finger recombinases in bacterial cells. We also show that TALERs recombine DNA in mammalian cells. The TALER architecture described herein provides a platform for insertion of customized TALE domains...
Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases
Perez-Pinera, P; Ousterout, DG; Brown, MT; Gersbach, CA
Nucleic Acids Res. (2012) 40 (8): 3741-3752
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Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies...
A multifunctional bioconjugate module for versatile photoaffinity labeling and click chemistry of RNA
Kellner, S; Seidu-Larry, S; Burhenne, J; Motorin, Y; Helm, M
Nucleic Acids Res. (2011) 39 (16): 7348-7360
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A multifunctional reagent based on a coumarin scaffold was developed for derivatization of naive RNA. The alkylating agent N3BC [7-azido-4-(bromomethyl)coumarin], obtained by Pechmann condensation, is selective for uridine. N3BC and its RNA conjugates are pre-fluorophores which permits controlled modular and stepwise RNA derivatization. The success of RNA alkylation by N3BC can be monitored by photolysis of the azido moiety, which generates a coumarin fluorophore that can be excited with UV light of 320 nm. The azidocoumarin-modified RNA can be flexibly employed in structure-function studies. Versatile applications include direct use in photo-crosslinking studies to cognate proteins, as demonstrated with tRNA and RNA fragments from the MS2 phage and the HIV genome. Alternatively, the azide function can be used for further derivatization by click-chemistry. This allows e.g. the introduction of an additional fluorophore for excitation with visible light.
Stimuli-responsive controlled-release system using quadruplex DNA-capped silica nanocontainers
Chen, CE; Pu, F; Huang, ZZ; Liu, Z; Ren, JS; Qu, XG
Nucleic Acids Res. (2011) 39 (4): 1638-1644
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A novel proton-fueled molecular gate-like delivery system has been constructed for controlled cargo release using i-motif quadruplex DNA as caps onto pore outlets of mesoporous silica nanoparticles. Start from simple conformation changes, the i-motif DNA cap can open and close the pore system in smart response to pH stimulus. Importantly, the opening/closing and delivery protocol is highly reversible and a partial cargo delivery can be easily controlled at will. A pH-switchable nanoreactor has also been developed to validate the potential of our system for on-demand molecular transport. This proof of concept might open the door to a new generation of carrier materials and could also provide a general route to use other functional nucleic acids/peptide nucleic acids as capping agents in the fields of versatile controlled delivery nanodevices.
Engineering synthetic TAL effectors with orthogonal target sites
Garg, A; Lohmueller, JJ; Silver, PA; Armel, TZ
Nucleic Acids Res. (2012) 40 (15): 7584-7595
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The ability to engineer biological circuits that process and respond to complex cellular signals has the potential to impact many areas of biology and medicine. Transcriptional activator-like effectors (TALEs) have emerged as an attractive component for engineering these circuits, as TALEs can be designed de novo to target a given DNA sequence. Currently, however, the use of TALEs is limited by degeneracy in the site-specific manner by which they recognize DNA. Here, we propose an algorithm to computationally address this problem. We apply our algorithm to design 180 TALEs targeting 20 bp cognate binding sites that are at least 3 nt mismatches away from all 20 bp sequences in putative 2 kb human promoter regions. We generated eight of these synthetic TALE activators and showed that each is able to activate transcription from a targeted reporter. Importantly, we show that these proteins do not activate synthetic reporters containing mismatches similar to those present in the genome nor a set of endogenous genes predicted to be the most likely targets in vivo. Finally, we generated and characterized TALE repressors comprised of our orthogonal DNA binding domains and further combined them with shRNAs...
DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency
Conrado, RJ; Wu, GC; Boock, JT; Xu, HS; Chen, SY; Lebar, T; Turnsek, J; Tomsic, N; Avbelj, M; Gaber, R; Koprivnjak, T; Mori, J; Glavnik, V; Vovk, I; Bencina, M; Hodnik, V; Anderluh, G; Dueber, JE; Jerala, R; DeLisa, MP
Nucleic Acids Res. (2012) 40 (4): 1879-1889
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Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli. This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures...
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