Deoxyribonucleic acid (DNA) nanotechnology is a growing ﬁeld with potential intracellular applications. In this work, we use an Escherichia coli cell-free transcription–translation (TXTL) system to assay the robustness of DNA nanotubes in a cytoplasmic environment. TXTL recapitulates physiological conditions as well as strong linear DNA degradation through the RecBCD complex, the major exonuclease in E. coli. We demonstrate that chemical modiﬁcations of the tiles making up DNA nanotubes extend their viability in TXTL for more than 24 h, with phosphorothioation of the sticky end backbone being the most effective. Furthermore, we show that a Chi-site double-stranded DNA, an inhibitor of the RecBCD complex, extends DNA nanotube lifetime signiﬁcantly. These complementary approaches are a ﬁrst step toward a systematic prototyping of DNA nanostructures in active cell-free cytoplasmic environments and expand the scope of TXTL utilization for bioengineering. Key words: DNA nanostructure; in vitro systems; cell-free systems; DNA nuclease; synthetic biology. drug delivery, intracellular scaffolding and synthetic-cell design 1. Introduction (4–7). The diversity of DNA nanostructures, reported in the liter- Several attempts to transition deoxyribonucleic acid (DNA) ature, illustrates the benefits of working with this versatile self- nanotechnology from ideal test tube conditions to living sys- assembling material. Synthetic-DNA nanotubes assemble from tems have been carried out in recent years (1). Interfacing DNA DNA tiles via programed interactions of the tile sticky ends nanostructures with biology, however, faces many challenges, a (8, 9). Their geometry and mechanical properties are close to major one being conferring resilience to the nanostructures for those of actin filaments and microtubules, suggesting that these in vivo operation. In this work, we engineered synthetic DNA nanotubes could serve as an artificial cytoskeleton in synthetic nanostructures and developed methods to increase their life- cells (8). They could operate as scaffolds to localize components, time in an all Escherichia coli cell-free transcription–translation actively transport material and influence cell morphology. (TXTL) system. TXTL, used as an active cytoplasmic environ- Further, DNA nanotubes tagged with fluorophores are easily ment (2, 3), recapitulates the physiological conditions found in observed with epifluorescence microscopy (Figure 1a), making cells (Figure 1b), as well as harsh linear DNA degradation characterization easier than for other nanometer-scale DNA through the RecBCD complex. structures. Synthetic DNA can be designed to fold into unique nano- Despite reports of DNA nanostructure stability in cell lysate scale structures, which can perform specific tasks, such as and serum (10), we found that these nanotubes degrade within Submitted: 20 October 2017; Received (in revised form): 9 December 2017; Accepted: 22 December 2017 V C The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact email@example.com Downloaded from https://academic.oup.com/synbio/article-abstract/3/1/ysy001/4840637 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2| Synthetic Biology, 2018, Vol. 3, No. 1 Figure 1. (a) DNA nanotubes self-assemble from tiles composed of 5 ssDNA oligomers. Complementary regions on the oligomers bind together to form a DNA tile; at each corner of the DNA tile there is a single-stranded domain (sticky end) serving as programmable binding sites for other tiles. Formation of tubular structures occurs due to the angle at which tiles bind via complementary sticky ends (8) (left). A representative ﬂuorescence microscopy image of DNA nanotubes labeled with Cy 3, in which the nanotubes are in TAE buffer and adhered to the surface of the microscope coverslip for optimal imaging (right). (b) Overview of the all E. coli cell-free system used in this work (2). a few hours in active E. coli TXTL. This deficiency renders DNA 0.9 mM CTP and UTP, 0.2 mg/ml tRNA, 0.26 mM coenzyme A, nanotubes impractical for both synthetic-cell and intracellular 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid, 1 mM sper- applications that require operational viability for timescales midine, 30 mM 3-PGA, 2% PEG8000, 10–15 mM maltose or exceeding 10 h (1). Previous reports on the stability of DNA ori- 20–40 mM maltodextrin, 1.5–3 mM of each of the 20 amino acids, gami in cell lysates incorporated sodium dodecyl sulfate (SDS) 40–120 mM K-glutamate and 2–7 mM Mg-glutamate. Plasmid and deoxycholic acid (DCA), rendering the lysate inactive (10). DNA concentration range from 0 to 2 nM while DNA nanotube We found that SDS and DCA inactivate TXTL as well, suppress- concentration was kept at 200 nM. Chi6 dsDNA were used at 0 or ing GFP expression (Supplementary Figure S1). This suggests 10 mM. The TXTL system used in this work is commercially that cellular lysates, which include SDS and DCA are not a good available from Arbor Biosciences, under the name myTXTL. model system for a live cytoplasmic environment. Herein, we explore the utility of modifying oligonucleotide 2.2 Fluorescence anisotropy assays strands to improve the resilience of DNA nanotubes and of Anisotropy and deGFP expression experiments were carried out modifying the composition of E. coli cell-free extract. We were in 2 ml reactions assembled using a Labcyte Echo Liquid Handler able to increase the lifetime of nanotubes incubated in TXTL to 550 in Costar 96 well, V-bottom plates. Kinetic experiments more than 24 h by combining two approaches: inclusion of Chi- were carried out at 29 C or room temperature in a Biotek containing double-stranded DNA (dsDNA) as a decoy for the Synergy Neo2. To monitor GFP fluorescence, monochromators complex RecBCD (11) and phosphorothioate (PS) bonding were set to excitation 485 nm and emission 528 nm. Parallel and between bases of sticky ends of the tiles. By achieving lifetimes perpendicular polarization was measured using a filter cube of over 24 h, we approach the ability to integrate tile-based DNA with an excitation filter of 540 nm and emission filter of 590 nm. nanotubes with intracellular applications and gene expression The dichroic mirror in the filter cube had a cut-off of 550 nm. systems. Our results suggest that two dominant contributors to degradation of DNA in E. coli cell extract are RecBCD and deoxy- ribonucleases (DNases) (12). 2.3 Nanotube assembly Lyophilized DNA oligonucleotides were purchased from 2. Materials and methods Integrated DNA Technologies (Coralville, IA, USA), resuspended in water, quantitated by UV absorbance at 260 nm using a 2.1 TXTL preparation and reactions Thermo Scientific Nanodrop 2000c Spectrophotometer, and The all E. coli cell-free TXTL system was prepared using BL21 stored at 20 C. All samples were stored or mixed using DNA Rosetta two competent cells from Novagen using procedures Lo-bind tubes (# 022431021). Nanotubes were annealed at either 3 or 1.8 lM tile concen- described previously (2, 3, 13, 14). Reactions are composed of 33% crude lysate with the remaining 67% being amino acids, tration by mixing each tile strand at 3 or 1.8 lM (final concentra- salts, an energy buffer and other cofactors. A typical TXTL reac- tion), in Tris-Acetate-EDTA (TAE) and 12.4 mM MgCl . Nanopure tion is composed of 50 mM HEPES pH 8, 1.5 mM ATP and GTP, water was added to achieve the appropriate concentration of Downloaded from https://academic.oup.com/synbio/article-abstract/3/1/ysy001/4840637 by Ed 'DeepDyve' Gillespie user on 16 March 2018 M.A. Klocke et al. | 3 components. All nanotube designs, except those which were to Variants of the tile-based DNA nanotubes degrade within a be ligated, were annealed using an Eppendorf Mastercycler PCR few hours after addition into TXTL reactions (Figure 2a). We machine by heating the sample to 90 C, and cooling it to 25 C used fluorescence anisotropy to monitor in real time the degra- over a 6 h period. Nanotubes which were to be ligated were dation of nanotubes labeled with Cy3 (Ex/Em 540/590 nm). annealed by heating the sample to 90 C, and cooling it to 25 C Fluorescence anisotropy relies on the change in rotational diffu- over a 54 h period. sion of molecules. The difference in fluorescence polarization of Nanotubes were ligated at 1.2 lM using New England Biolabs the parallel and perpendicular intensities is measured and fluo- (NEB) T4 DNA Ligase (# M0202S; 20 000 units) at 1.0 units/ll and rescence anisotropy is calculated as r ¼ (I I )/ parallel perpendicular NEB T4 DNA Ligase buffer (# B0202S, 10, 10 mM MgCl ,50 mM (I ). Larger molecules have larger fluorescence anisotropy sig- 2 total Tris-HCl, 1 mM ATP, 10 mM DTT, pH 7.5 at 25 C) at 1. nals. Consequently, the degradation of DNA nanotubes is Nanotubes were incubated at room temperature for at least observed as a decrease of fluorescence anisotropy. In TAE buf- 2 days in ligation solution preceding experiments. fer, i.e. in the absence of degradation, there is no significant change in anisotropy of DNA structures and tiles as long as DNA adsorption is inhibited using a poly-T ssDNA molecule 2.4 Fluorescence microscopy (Supplementary Figure S10). Anisotropy data were fitted using a Nanotube samples were imaged using an inverted microscope simple exponential decay model: (Nikon Eclipse TI-E) with 60/1.40 NA oil immersion objectives. Samples containing nanotubes were imaged at 200 nM tile concen- t=b b b e 1 2 tration in E. coli cell extract with either 0 or 10 lMChi6 dsDNA (11). Samples were imaged in chambers made using a Bio-Rad frame- The time constant b is reported in Supplementary Table S4 seal (# SLF0601; size: 15 15 mm, 65 ll) placed on Fisherbrand for all variations of nanotubes, temperature and Chi concentra- microscope cover glass (# 12-545E No. 1, thickness¼ 0.13–0.17 mm; tion. To quickly assess nanotube robustness in TXTL, we first size: 50 22 mm); VWR Micro Slides (Plain, Selected, Pre-cleaned, tested two nanotube variants in which tiles have 8 base-long 25 75 mm, 1.0 mm thick, # 48300-025) were placed on the frame sticky ends, with and without a single stranded extension (toe- seal to complete the imaging chamber. Each sample chamber was hold) (Figure 2 and Supplementary Figure S7a). The rapid degra- imaged throughout the duration of the experiments. The fluores- dation of the DNA nanotubes observed by fluorescence cence microscopy image in Figure 1a was prepared as described by microscopy was confirmed by fluorescence anisotropy. The Rothemund et al. (8). The sample preparation method described by decrease in anisotropy begins immediately, suggesting diges- Rothemund et al. (8) results in images of distinct, individual nano- tion of the DNA nanotubes begins within 5 min of their addition tubes in one focal plane, as the nanotubes are adhered to the sur- to the extract (Figure 2b). In general, we observe that the aniso- face of the coverslip. In contrast, imaging DNA nanotubes in tropy signal of nanotubes incubated in TXTL decreases reaching crowded environments, such as TXTL, results in bundling of nano- a steady-state comparable to that of a control sample of DNA tubes not seen in non-crowded buffers in which the nanotubes are nanotubes incubated with DNAse I at room temperature in TAE annealed (Figures 1a and 2a and c)(15). Nanotubes labeled with buffer (Supplementary Figure S10). Nanotubes assembled from Cy3 fluorescent molecule were imaged using a Cy3 filter cube tiles without a toehold (8bNT) were slightly more robust than (Semrock Brightline—Cy3-404 C-NTE-ZERO). Exposure time was set the toeholded variant based on microscopy data. The effect was to 30 ms. Samples for the 29 C experiments were incubated at 29 C insignificant in anisotropy assays. We hypothesize that the toe- on a Nikon Tokai Hit Thermo Plate (# MATS-VAXKW-D). hold provides an easier access to RecBCD for degradation. 2.5 Gel electrophpresis 3.2 Nanotube tile structure influences degradation rates Ligated DNA nanotubes were diluted to 180 nM in pure water. Nanotube tile structure, for example sticky end length or inclu- To denature the nanotubes, the nanotubes were mixed 1:1 with sion of additional binding sites, can be designed to suit different 8 M urea and then heated to 90 C for 5 min. Denatured nanotube purposes. To thoroughly characterize degradation of nanotubes solution was mixed 1:1 with gel loading buffer II (Ambion, with different tile structures in TXTL, we worked with four dif- AM8547) and loaded into a 1.0 mm 12% polyacrylamide denatur- ferent tile variants. Increasing the length of the sticky ends ing gel. As a control, we used a 10 base DNA ladder (Invitrogen). results in an increased melting temperature of nanotubes. We Gel electrophoresis was done at 100 V for 90 min, followed by considered nanotubes with either 5-base (5b) or 8-base (8b) 20 min of gel staining in 1 TBE with 0.2 Syber Gold sticky ends (Supplementary Figures S2 and S3). For each case, (Invitrogen, S11494). Imaging was done using a ChemiDoc MP we considered an additional tile variant that includes a toehold Imaging System (Bio-Rad, Universal Hood III). region on one of the sticky ends (Supplementary Figure S4). The toehold region can be used to link other ligands to the nano- 3. Results and discussions tubes (17), or to incorporate strand displacement reactions at the sticky ends (18). We examined degradation of 5b and 8b 3.1 Nanotubes degrade rapidly within active E. coli TXTL nanotubes both with (5bT, 8bT) and without (5bNT, 8bNT) We investigated degradation of DNA nanotubes in the all E. coli toeholds. TXTL system (2)(Figure 1) using fluorescence microscopy and flu- Of the four designs studied, the 8b nanotubes outlast the 5b orescence anisotropy. Because TXTL includes all the components nanotubes at both room temperature and 29 C(Figure 2 and required for protein synthesis in vitro in physiological conditions, Supplementary Figures S5–S7). At room temperature, 8b nano- other cellular functions such as linear DNA degradation proceed tubes degrade in under 3 h (Figure 2a), while 5bT and 5bNT as well due to the remaining cellular machinery present in the nanotubes degrade in under an hour (Supplementary lysate. In the E. coli TXTL, the major enzyme responsible for linear Figure S5a). Degradation occurs more quickly at 29 C, with 8bNT DNA degradation is the RecBCD enzyme complex (11, 16). nanotubes degrading within 3 h (Supplementary Figure S7 and Downloaded from https://academic.oup.com/synbio/article-abstract/3/1/ysy001/4840637 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4| Synthetic Biology, 2018, Vol. 3, No. 1 Figure 2. Enhancing the stability of DNA nanotubes in TXTL with Chi-site DNA. (a) Fluorescence microscopy kinetics of 8-base DNA nanotubes with (8bT) and without (8bNT) a toehold site incubated in E. coli TXTL reactions with no double stranded Chi-site DNA present. Bundling of DNA nanotubes is a result of the crowded TXTL environment. (b) Fluorescence anisotropy kinetics of a single stranded DNA oligo (SE3ss) and 8-base DNA nanotubes with (8bT) and without (8bNT) a toehold site incu- bated in E. coli TXTL reactions with no double stranded Chi-site DNA present. Solid lines represent the prediction of a ﬁtted exponential decay model. (c) Fluorescence microscopy kinetics of 8-base DNA nanotubes with (8bT) and without (8bNT) a toehold site incubated in E. coli TXTL reactions with 10 mM of double stranded Chi-site DNA. (d) Fluorescence anisotropy kinetics of single stranded DNA oligo (SE3ss) and 8-base DNA nanotubes with (8bT) and without (8bNT) a toehold site incubated in E. coli TXTL reactions with 10 mM of double stranded Chi-site DNA (Chi6). Solid lines are the prediction of a ﬁtted exponential decay model (Supplementary Table S4). All the reactions were incubated at room temperature. Table S4), and 5bT, 5bNT and 8bT nanotubes degrading in under robustness in TXTL (Supplementary Table S4). Increasing the liga- an hour (Supplementary Figures S6 and S7, Table S4). tion efficiency could lead to even greater improvement in the resil- ience of the DNA nanotubes in TXTL. Nucleases, such as DNase I, are inhibited by PS bonded DNA. 3.3 Nanotube lifetime increased in presence of Chi and PS bonds connect the sugar-phosphate backbone of one base to chemical modification of the tiles another, much as a phosphodiester bond does in native DNA, with We hypothesized that by adding double stranded (ds) DNA mole- sulfur replacing the native oxygen (Supplementary Figure S4b). 0 0 cules containing six repeats of the Chi sequence (5 -GCTGGTGG-3 ) Exonucleases are inhibited by PS bonds located at the ends of to TXTL reactions, we would see an increase in DNA nanotube life- DNA, while endonucleases are inhibited by incorporating PS bonds time (11). We recently demonstrated that Chi6 inhibits the degra- into the entire backbone of a DNA structure (20). dation of linear dsDNA in TXTL by sequestering RecBCD, with the Because the 8bNT nanotube design is the most stable in optimal concentration of Chi6 for protein expression being 2–4 mM. TXTL, we designed 8bNT tiles with PS bonds between the 8 We choose to use 10 mM Chi6 in order to maximize nanotube bases of the sticky ends of the tiles (Figure 3d). By modifying the stability at the cost of slightly depressed protein expression sticky ends, we expected to impede nanotube degradation by (Supplementary Figure S11). RecBCD and other exonucleases. The lifetimes of PS bonded At room temperature, 8bT and 8bNT nanotubes last up to DNA nanotubes was more than double that of unmodified 8bNT 10 h in the presence of 10 mM Chi6 in the cell-free extract nanotubes, as observed by fluorescence microscopy and fluo- (Figure 2c). Anisotropy assays indicate that the nanotube degra- rescence anisotropy (Figure 3e and f). At room temperature, dation is not complete, evidenced by the fact that the absolute 8bPS tubes lasted up to 10 h without the Chi6 DNA, with aggre- values of anisotropy for 8bT and 8bNT nanotubes with 10 mM gates of degraded tubes visible at 10 h. In the presence of the Chi do not converge with those of the 8bT and 8bNT in the Chi6 DNA, at room temperature, 8bPS nanotubes remained at absence of Chi (Figure 2d). This suggests that aggregates not least 24 h in TXTL. At 29 C, 8bPS nanotubes degraded in under visible in microscopy experiments might remain for time peri- 3 h without the Chi6 DNA and between 3 and 6 h with the Chi6 ods greater than 10 h. DNA (Supplementary Figure S9). Robustness of nanotubes under different environmental stres- sors, such as chemical or thermal stress, is improved with ligation (19). Ligation seals the breaks, or nicks, in the sugar-phosphate 4. Summary and conclusions backbone found at the four corners of inter-tile junctions DNA nanotubes are rapidly degraded in the TXTL environment. (Figure 3a). Although the ligation efficiency for the 5 end of strand Incorporating Chi DNA into the cell-free system extends the SE2 was lower than that of the other strands, we verified ligation of lifetime of the DNA nanotubes, confirming the previous obser- all four corner nicks via denaturing gel (Supplementary Figure S12). Both the microscopy images (Figure 3b, Supplementary Figure S8), vations that RecBCD is the major actor responsible for degrada- and the time constants obtained from the fluorescence anisotropy tion (11). Modifications to the DNA tiles themselves also affect observations (Figure 3c) suggest that ligation enhances nanotube the lifetime of the DNA nanotubes. Lengthening the tile sticky Downloaded from https://academic.oup.com/synbio/article-abstract/3/1/ysy001/4840637 by Ed 'DeepDyve' Gillespie user on 16 March 2018 M.A. Klocke et al. | 5 Figure 3. Enhancing the stability of DNA nanotubes in TXTL systems with Chi-site DNA and chemical modiﬁcations (ligation or phosphorothioate bonding of sticky ends). (a) Cartoon of ligation. (b) Fluorescence microscopy kinetics of 5-base DNA nanotubes with a ligation of the sticky ends of the tiles incubated in E. coli TXTL reac- tions with and without Chi-site DNA present. (c) Fluorescence anisotropy kinetics, and exponential decay model prediction (solid lines), of a single stranded DNA oligo (SE3ss) and 5-base DNA nanotubes with (S1245P) and without (5bNT) a ligation of the sticky ends of the tiles incubated in E. coli TXTL reactions with and without Chi- site DNA present. (d) Cartoon of phosphorothioation. (e) Fluorescence microscopy kinetics of 8-base DNA nanotubes with phosphorothioate bonded sticky ends of the tiles incubated in E. coli TXTL reactions with and without Chi-site DNA present. (f) Fluorescence anisotropy kinetics of a single stranded DNA oligo (SE3ss) and 8-base DNA nanotubes with (PSE) and without (8bNT) the phosphorothioation of the sticky ends of the tiles incubated in E. coli TXTL reactions with and without Chi-site DNA present. The predictions of our exponential decay model are shown as solid lines (Supplementary Table S4). All the reactions were incubated at room temperature. ends increases nanotube stability, and eliminating toehold as scaffolding, drug delivery or synthetic cell components, mak- overhangs increases the lifetime even further. PS bonding on ing the absence of characterization of these structures in cell- the sticky ends of the nanotubes was the most effective tile like environments a considerable impediment to these aims. modification, while ligating these same domains had a small We demonstrated that TXTL can be used as an experimental effect. platform to test the robustness of DNA nanostructures and to This study is limited to DNA nanotubes and does not con- engineer new ones with longer lifetimes in cytoplasmic envi- sider what the chemical modifications to the cell-free extract or ronment. This method could be used as a testbed to rapidly pro- to the DNA self-assembling components may have on other totype robust DNA nanostructures for in vivo applications. DNA structures, such as DNA origami (7) or crystals (21). We conjecture that more tightly packed assemblies such as origami might be stable for several days with appropriate chemical Supplementary data modifications. It is unclear if other modifications to the extract Supplementary Data are available at SYNBIO Online. could be used to prevent other nucleases from acting on DNA in the cytoplasmic environment as Chi DNA was used to specifi- Acknowledgements cally target RecBCD. Chi DNA is specific to RecBCD so it would not extend DNA nanotube lifetimes in cytoplasmic environ- We thank PWK Rothemund for discussions about DNA ments other than E. coli. However, in these cases we expect that nanotubes. it would still be possible to mitigate nanotube degradation by introducing or up-regulating the concentration of competitive binding sites for the corresponding enzymes. Funding While DNA nanostructures have a variety of potential appli- U.S. Department of Energy under Award Number [DE- cations in biological contexts, the stability of these structures SC0010595 to EF], which paid for reagents and supplies used has not been previously thoroughly demonstrated in cell-like at UC Riverside; the Human Frontier Science Program environments mimicking cytoplasmic conditions. Many DNA structures are designed with the intention for intracellular use, [RGP0037/2015 to V.N.]. Downloaded from https://academic.oup.com/synbio/article-abstract/3/1/ysy001/4840637 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6| Synthetic Biology, 2018, Vol. 3, No. 1 11. Marshall,R., Maxwell,C.S., Collins,S.P., Beisel,C.L. and Conﬂict of interest: Noireaux laboratory receives research Noireaux,V. 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