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Amplification of over 100 kbp DNA from Single Template Molecules in Femtoliter Droplets

  • Hiroshi Ueno
    Hiroshi Ueno
    Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
    More by Hiroshi Ueno
  • Hiroki Sawada
    Hiroki Sawada
    Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
  • Naoki Soga
    Naoki Soga
    Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
    More by Naoki Soga
  • Mio Sano
    Mio Sano
    Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
    More by Mio Sano
  • Seia Nara
    Seia Nara
    Department of Life Science, College of Science, Rikkyo University, Tokyo 171-8501, Japan
    More by Seia Nara
  • Kazuhito V. Tabata
    Kazuhito V. Tabata
    Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
  • Masayuki Su’etsugu
    Masayuki Su’etsugu
    Department of Life Science, College of Science, Rikkyo University, Tokyo 171-8501, Japan
  • , and 
  • Hiroyuki Noji*
    Hiroyuki Noji
    Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
    *Email: [email protected]
Cite this: ACS Synth. Biol. 2021, 10, 9, 2179–2186
Publication Date (Web):August 18, 2021
https://doi.org/10.1021/acssynbio.0c00584

Copyright © 2021 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

Reconstitution of the DNA amplification system in microcompartments is the primary step toward artificial cell construction through a bottom-up approach. However, amplification of >100 kbp DNA in micrometer-sized reactors has not yet been achieved. Here, implementing a fully reconstituted replisome of Escherichia coli in micrometer-sized water-in-oil droplets, we developed the in-droplet replication cycle reaction (RCR) system. For a 16 kbp template DNA, the in-droplet RCR system yielded positive RCR signals with a high success rate (82%) for the amplification from single molecule template DNA. The success rate for a 208 kbp template DNA was evidently lower (23%). This study establishes a platform for genome-sized DNA amplification from a single copy of template DNA with the potential to build more complex artificial cell systems comprising a large number of genes.

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Cell-free synthetic biology has seen rapid growth. (1−3) This trend has become obvious after the establishment of key technologies of in vitro reconstitution and cell-free technologies, such as cell-free transcription and translation systems (4,5) and DNA replication systems. (6−8) On the basis of these cell-free platforms, several cell-free systems have been developed, such as phage genome replication, DNA segregation, gene circuits, signal transduction/sensing systems, and metabolic and energy transduction systems. (9−11) One of the beneficial features of cell-free synthetic biology is the circumvention of unavoidable limitations posed on cell-based technologies and the need to meet the physiological requirements of host cells. Another beneficial feature is that it allows the experimental investigation of biomolecular systems reconstituted from purified components without unidentified factors. Thus, this approach enables the identification of the components responsible for the biomolecular function of interest as well as a detailed analysis of the functions of biomolecular systems under defined conditions. (12) From an engineering point of view, the open nature of cell-free systems enables the rational design of biomolecular systems, rapid prototyping of functional biomolecules/biosystems, (13) and the implementation of microdevices for high-throughput analysis/screening. (14,15)
One of the ultimate goals of cell-free synthetic biology is the bottom-up reconstitution of autonomously self-replicating systems, that is, autonomous artificial cells. (16) Implementation of in vitro DNA replication systems into cell-sized reactors is a primary step to realize such a self-replicating molecular system. Many studies have attempted to accommodate DNA replication systems in microreactors. In this regard, polymerase chain reaction-based amplification systems have been extensively studied, in which template DNA, substrates, enzymes, and other components (primers, chemicals, and ions) are encapsulated in water-in-oil (w/o) droplet or liposomes. (17−21) One of the pioneering achievements in this area is the development of synthetic vesicle systems that entrap synthetic catalysts, precursors of lipid molecules, and polymerase chain reaction components. (18−20) Thermal cycle treatment and the subsequent activation of lipid precursors by catalysts have been shown to enhance the growth and fission of vesicles. (18−20,22) Isothermal amplification systems have also been developed, (23) and implemented in microcompartments. (24) By coupling in vitro transcription and translation systems, RNA/DNA replication catalyzed by polymerases encoded in RNA/DNA was also implemented in w/o droplet or liposomes. (25,26) Such replication by self-encoded polymerases was studied in evolutional experiments. (16,23,25)
Accordingly, the microcompartmentalization of DNA amplification systems has shown significant progress. However, amplification of DNA templates with a size over 100 kbp, close to the size of minimal genome for free-living cells, around 500 kbp (27) in microcompartments remains to be achieved. Considering that the genome size for self-sustaining cells is thought to be in the range of hundreds kbp to encode hundreds of genes, (28,29) the reconstitution of amplification systems for such long DNAs in microreactors is a requisite for artificial cell construction research. Although a wide variety of DNA amplification methods exist, (7,8,30,31)in vitro amplification methods for genome-sized DNA are very limited. In addition, most DNA amplification reactions are based on a rolling-cycled reaction and are basically nonrecursive; the structure of template DNA is not maintained in the produced DNA molecules. Regarding this, there are several advancements in recent studies; Okauchi et al. reported a cycled DNA replication system based on a rolling-circle amplification termed repetitive sequence replication. (23) Libicher et al. also reported similar DNA replication through rolling-circle amplification. (32) However, there are still challenges in the development of recursive and efficient replication systems of over hundreds kbp DNA.
Recently, we reported the replication cycle reaction (RCR), (6) which is a fully reconstituted version of the chromosome replication machinery complex of Escherichia coli (Figure 1a). Although minichromosome replication has been reconstituted in vitro since more than 30 years ago, (33) the stably cycled replication of genome-sized DNA has not been achieved. RCR amplifies a circular DNA with an oriC sequence. The reaction cycle number in a single assay reached 1010 rounds of replication, and the error rate was shown to be significantly low, on the order of 10–8 per base per replication cycle. A genome-sized DNA over 1 Mbp was also successfully amplified in a tube. (34) Thus, RCR is expected to be a platform technology for in vitro amplification system of genome-sized DNA and has great potential as a platform for the reconstitution of autonomous artificial cells, although it should be challenging to self-produce all components of RCR in the right quantities and stoichiometries.

Figure 1

Figure 1. In-droplet RCR. (a) Scheme of RCR. The RCR system is a fully reconstituted genome replisome from E. coli, which comprises 14 purified proteins, including DNA polymerase III holoenzyme, DnaA, and DnaG proteins and chemicals such as dNTPs and rNTPs. Unlike other amplification reactions of DNA amplification such as rolling circle amplification, the RCR system duplicates supercoiled circular DNA molecules with the exact same topology. RCR requires the oriC sequence on template DNAs for initiation complex formation. RCR proceeds in four steps: initiation complex formation, replication fork progression, Okazaki fragment maturation, and decatenation. (b) Experimental scheme. The reaction mixture of RCR including Alexa 647 dye was emulsified with mineral oil and a surfactant mix (3% ABIL EM 90 and 0.05% Triton X-100). The emulsion mixture was incubated at 30 °C for 3 h for isothermal amplification. For imaging of isothermal amplification of DNA with a fluorescence confocal microscope, the emulsion mixture was stained with an intercalator, SYBR Green I, by mixing with mineral oil pre-equilibrated with SYBR Green I solution.

This work principally aimed to test the performance of RCR in a microcompartment condition, that is, in-droplet RCR. In particular, we focused on the “success rate” of the amplification, the efficiency with which in-droplet RCR initiates DNA amplification from a single molecule of template. For efficient and robust selection/evolution of artificial cells and protocell models against inevitably emerging parasites, it is desirable to encapsulate a single entity of genetic material in each cell/reactor. (16,25) Thereby, it is crucial to achieve a high success rate of DNA replication from a single molecule of DNA for the propagation of genetic materials to descent cells. In addition, a high success rate is requisite to retain high genetic diversity in the population of the cells. However, recursive and isothermal DNA amplification methods were not well characterized in terms of the success rate of DNA amplification in microcompartments. We tested in-droplet RCR amplification of circular DNAs with the length of 16 kbp or 208 kbp DNA. The success rate was around 82% for in-droplet RCR of 16 kb DNA. Although the success rate of RCR for 208 kbp DNA was significantly lower, 23%, the recursive and isothermal replication of over 200 kbp DNA was demonstrated, posing the possibility of reconstituting the autonomous artificial cell with genome-sized DNA. It was also found that in-droplet RCR is more robust than conventional in-tube RCR against the interference effect of linear template DNA, giving clues for further improvement of RCR in microcompartment conditions.

Results and Discussion

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Experimental Scheme of In-Droplet RCR

The reaction mixture of the RCR contains 14 purified proteins, including DNA polymerase III holoenzyme, DnaA and DnaG proteins from E. coli, in addition to small chemicals such as dNTPs and rNTPs. The RCR system specifically recognizes supercoiled circular DNA with the oriC sequence as the template DNA for replication. (6) RCR proceeds in four steps: initiation complex formation, replication fork progression principally driven by DnaB helicase, and DNA polymerase, Okazaki fragment maturation, and decatenation, where two DNA molecules catenated through replication fork progression are separated by the activity of DNA topoisomerases (Topo III and Topo IV proteins) (Figure 1a).
The assay mixtures for in-droplet and in-tube RCR in the present study were prepared principally according to the process described in the original report on RCR. (6) In this study, plasmid DNAs carrying the oriC sequence with different lengths (16 kbp and 208 kbp) were used.
Figure 1b shows the experimental scheme of the in-droplet RCR. The RCR solution was stained with the fluorescence marker dye Alexa 647 and emulsified by mixing with mineral oil with surfactant mix: 3% ABIL EM 90 and 0.05% Triton X-100 for the stabilization of water-in-oil (w/o) droplets. The emulsion mixture was then subjected to isothermal amplification by incubation at 30 °C for 3–5 h.
RCR is susceptible to intercalators. DNA molecules in the droplets were stained with an intercalator, SYBR Green I, after the isothermal reaction by adding mineral oil that was pre-equilibrated with SYBR Green I solution. In each experiment, a sample mixture of RCR reaction was injected into one of the flow channels prepared on a flow cell chamber for microscopic observation. Other flow channels were loaded with control samples: (−DNA and +RCR enzyme) and (+DNA and −RCR enzyme) for comparison purposes. RCR amplification was confirmed from the fluorescence signal of the SYBR green from droplets observed with a confocal fluorescence microscope.

In-Droplet RCR for 16 kbp DNA

Figure 2a shows the fluorescence image of droplets after RCR amplification of 16 kbp DNA. While no bright fluorescence from the DNA intercalator was observed in control samples: (−DNA, +RCR enzyme) and (+DNA, −RCR enzyme), the droplets of RCR mixture, (+DNA, +RCR enzyme) showed a clear fluorescence signal. In addition, we confirmed the DNA amplification of in-droplet RCR by performing electrophoresis analysis with droplets collected by centrifugation. DNA molecules were recovered after phenol–chloroform extraction and subjected to agarose gel electrophoresis. As a reference, we also extracted DNA from an in-tube RCR mixture that was emulsified after the reaction and subjected to the phenol–chloroform extraction of the exact same procedures for in-droplet RCR. Figure 2b shows a result of agarose gel electrophoresis of in-droplet RCR and in-tube RCR. During the agarose gel electrophoresis for DNA of several tens of kbp in length, supercoiled circular DNA migrates faster than linear or nicked DNA, as marked in Figure 2b. The electrophoresis result showed similar migration patterns between in-droplet RCR and in-tube RCR: the quantity of total produced DNA as well as the quality of the product, that is, the population of supercoiled circular DNA in the total production. However, it should be noted that the RCR enzyme cocktail has lot-to-lot variation, resulting in the different quantity and quality of DNA product in some cases. In what follows, the same lot of RCR enzyme cocktail mixture was used throughout unless otherwise mentioned.

Figure 2

Figure 2. In-droplet RCR for 16 kbp plasmid DNA. (a) After 3 h the in-droplet RCR reaction of 16 kbp template DNA, fluorescence images of the droplets were obtained with a confocal fluorescence microscope. The upper row shows images of the water-in-oil droplet marker, Alexa 647 (red). The lower row shows images of the DNA marker SYBR Green I (green). Scale bar, 20 μm. (b) Electrophoresis analysis of in-tube and in-droplet RCR product using the same 16 kbp template DNA (92 pg/μL). After 3 h RCR reaction, DNA molecules were recovered after phenol–chloroform extraction and subjected to 0.5% agarose gel electrophoresis. The 2.5 kb DNA Ladder (Takara Bio) was used as the size marker. Lanes 1 and 2, 2 and 10 μL of recovered DNA after in-tube RCR; lanes 3 and 4, 2 and 10 μL of recovered DNA after in-tube RCR without RCR enzyme; lanes 5 and 6, 2 and 10 μL of recovered DNA after in-droplet RCR; lanes 7 and 8, 2 and 10 μL of recovered DNA after in-droplet RCR without RCR enzyme; lane 9, template 16 kbp DNA. Arrows indicate the migration positions for supercoiled DNA, linearized DNA, and nicked open circular DNA.

In-Droplet RCR for 208 kbp DNA

In-droplet RCR with the 208 kbp plasmid DNA was conducted similarly to the 16 kbp DNA. The reaction products were analyzed using a confocal fluorescence microscope and an electrophoresis. Figure 3a shows the fluorescence image of the droplet after RCR. Microscopic analysis of in-droplet RCR for 208 kbp DNA showed an evident fluorescence signal of SYBR green; however, the intensity was lower than that of RCR for 16 kbp DNA, suggesting a lower yield of reaction products. In addition, droplets showed nonhomogeneous fluorescence. This occurs because such long DNA molecules undergo slow diffusion, giving fluorescence spot/clusters. When observed in enhanced contrast, the control samples: (−DNA, +RCR enzyme) and (+DNA, −RCR enzyme) also showed some fluorescence spots. These spots were dimmer and smaller than the droplets of RCR samples. The dim fluorescent spots observed in the control sample were probably enzyme aggregations stained with SYBR green because (−DNA, +RCR enzyme) showed more background spots. Another origin of the signal of the control samples should be template DNA molecules.

Figure 3

Figure 3. In-droplet RCR for 208 kbp plasmid DNA. (a) After 3 h in-droplet RCR reaction of 208 kbp template DNA, fluorescence images of the droplets were obtained. The upper row shows images of the water-in-oil droplet marker, Alexa 647 (red). The lower row shows images of the DNA marker SYBR Green I (green). Scale bar, 10 μm. (b) Electrophoresis analysis of in-tube and in-droplet RCR product using the same 208 kbp template DNA (5 ng/μL). After 3 h RCR reaction, DNA molecules were recovered after phenol–chloroform extraction, and 20 μL of the recovered solution was subjected to 0.5% agarose gel electrophoresis. The 2.5 kb DNA Ladder (Takara Bio) was used as the size marker. Lane 1, after in-tube RCR; lane 2, after in-tube RCR without RCR enzyme; lane 3, after in-droplet RCR; lane 4, after in-droplet RCR without RCR enzyme; lane 5, template 208 kbp DNA. Arrows indicate the migration positions for supercoiled DNA, linearized DNA (or concatemer DNA), and nicked open circular DNA (well).

Figure 3b shows the agarose gel electrophoresis analysis of DNA extracted from in-droplet RCR (lane 3) and DNA from in-tube RCR (lane 1) prepared the same as the in-droplet RCR sample. Supercoiled DNA was found between the loading well and the band at the separation limit of linear DNA, as indicated. The migration distance of supercoiled DNA of over 100 kbp is shorter than that of linear DNA with a corresponding molecular weight. (6) In comparison with the control in-tube RCR sample, in-droplet RCR produced more DNA in both supercoiled form and linear form, showing that emulsification enhances the RCR reaction.

In-Droplet RCR under Digital Condition

The number of template DNA molecules encapsulated in a droplet should be proportional to the droplet volume. Therefore, most of the large microdroplets (φ ≥ 5 μm) showed distinct fluorescence signals, whereas many small microdroplets (φ < 5 μm) remained dark (Figure 4). For quantitative analysis of the probability of RCR-positive, the fluorescence intensity was analyzed. After identifying the droplet position from the fluorescence marker (Alexa 647), the fluorescence intensity of the DNA intercalator (SYBR Green I) was quantitatively analyzed. Figure 5 panels a and b show the histograms of fluorescence intensity of droplets from in-droplet RCR of 16 kbp and 208 kbp DNA. The leftmost peak corresponding to the dark droplets for 16 kb DNA is distinctively separated from the second peak with a much lower height representing the RCR-positive droplets. However, the fraction of RCR-positive droplets for 208 kbp DNA is not well separated from the main peak for RCR-negative droplets. Therefore, to define the threshold to discriminate positive droplets from negative ones, we measured the fluorescence histogram of control sample mix (−DNA, +RCR Enzyme) prepared in another flow channel on the same flow cell chamber for RCR positive samples. As shown in Supporting Information, Figure S1, the histograms of the control samples were principally Gaussian, however, slightly tailing on the right sides corresponding to the fluorescent spots found in the control samples. To get rid of such background signals, we defined the threshold as mean ±5 × standard deviation (s.d.) of negative control samples. Although the background signal was not completely removed with the threshold, the population of the false-positive droplets was only 1–2% of the population and essentially did not affect the estimation of the population of RCR-positive droplets.

Figure 4

Figure 4. Digital in-droplet RCR for 16 kbp DNA. Fluorescence image of digital in-droplet RCR. Red indicates the fluorescence image of the w/o droplet marker (Alexa 647). Green indicates the image of amplified DNA molecules stained with SYBR Green I. Dotted circles indicate the examples of RCR-negative droplets and dark droplets. Scale bar, 10 μm.

Figure 5

Figure 5. Histograms of fluorescence intensity of droplets after in-droplet RCR. Histogram of fluorescence intensity of droplets after RCR amplification of 16 kbp (125 pg/μL template) (a) and 208 kbp DNA (5 ng/μL template) (b). The red triangle indicates the position of the threshold for the identification of RCR-positive droplets, which is defined as mean ±5 × s.d. of the negative control peak (see Figure S1) that corresponds to dark droplets. N indicates the total number of droplets in each data set.

The probability of RCR-positive droplet for RCR of 16 kbp or 208 kbp was plotted against the diameter of the droplets (Figure 6a,b). We investigated six concentrations of template DNA for 16 kbp (Figure 6a shows three of them for simplicity) and two concentrations for 208 kbp. As expected, the probability of RCR-positive droplet increased with the diameter. For quantitative analysis, we define the success rate of RCR as the probability of RCR amplification from a single molecule DNA, and we formulate the success rate as a function of the droplet diameter and the concentration of template DNA as below. The mean number of template DNA molecules per droplet is represented as follows:
(1)
where c is the concentration of DNA, v is the volume of the emulsion, NA is the Avogadro constant, and d is the diameter of the droplet. The number of DNA molecules contained in each emulsion should obey the Poisson distribution. The probability of containing k molecules of DNA in one microreactor P(k) is represented as follows:
(2)
A positive reactor should contain one or more template DNA (x ≥ 1) molecules and is represented as follows:
(3)
Then, the success rate of RCR amplification is estimated as the ratio of effective concentration of DNA molecules that lead RCR amplification, c′ against the actual concentration of DNA, c as follows:
(4)
The experimental data points were fitted with eq 3 to determine c′ (solid lines in Figure 6a,b). The mean success rate of in-droplet RCR of 16 kbp was 81.8% (Figure 6c). This means that the RCR amplifies DNA from a single copy of 16 kbp DNA with a high success rate in microdroplets. On the other hand, the mean success rate for RCR of 208 kbp was significantly low, only 22.9%. The large scattering of data points for the large droplets in 208 kbp RCR is due to the small numbers of droplets (N < 10) (Figure S4) that causes relatively large Poisson noise (1/√N), >30%.

Figure 6

Figure 6. Success rate of digital in-droplet RCR. (a) Probability of RCR-positive droplets plotted against the diameter of droplets obtained at three different concentrations of template 16 kbp DNA: 62.5, 125, and 250 pg/μL (6 pM, 12 pM, 24 pM). The dots indicate the experimental data points. Solid lines indicate the theoretical probability estimated from the fitting (see eq 3). (b) Probability of RCR-positive droplets plotted against the diameter of droplets obtained at two different concentrations of template 208 kbp DNA: 1.5 ng/μL and 5 ng/μL (11 pM, 37 pM). (c) Success rate of RCR amplification for RCR of 16 kbp and 208 kbp template DNA.

Note that the data points with lower values of RCR-positive droplets tend to drop above the fitting line. This is at least partly attributable to the above-mentioned effect of false-positive droplets. However, because we estimate that such a false-positive droplet is around 1–2% of total, another mechanism should exist. One possible explanation is the surface effect; micrometer-sized droplets might attract and/or capture DNA on their inner surface, enhancing the encapsulation efficiency. The exact mechanism remains to be elucidated.
We then analyzed the yield of the RCR products. Figure 7 shows the means and standard deviations of fluorescence intensity of RCR-positive droplets with indicated diameters. The plot showed that the mean signal of RCR with 16 kbp DNA was around 800 arbitrary units (a.u.), independent of the droplet size, that is, from 5 to 10 μm. This means that the concentration of the DNA product from RCR was constant irrespective of droplet volume, while the total copy number of produced DNA molecules should increase with droplet volume. This suggests that the yield of RCR products is limited by the total amount of chemical resources, nucleotides in a droplet. We attempted to estimate the concentration of produced DNA in droplets according to the calibration curve (Figure S2). The calculated value was 174 ng/μL DNA. Since the total nucleotide concentration was 130 ng/μL, this value is obviously an overestimation for unknown reasons. However, it is highly likely that the RCR for the 16 kbp DNA mostly reached the upper limit. The yield of RCR with the 208 kbp DNA was evidently lower, between 100 and 200 a.u. (Figure 7). Nevertheless, the size of the droplets did not significantly affect the distributions again, suggesting that mechanisms other than the shortage of nucleotides could limit the yield of RCR products for the 208 kbp DNA template.

Figure 7

Figure 7. Product yield of digital in-droplet RCR. Mean fluorescence intensity of positive droplets after RCR for 16 kbp and 208 kbp were calculated with the indicated diameters. The concentration of template DNA was 125 pg/μL for 16 kbp and 5 ng/μL for 208 kbp DNA.

As shown in Figures 2b and 3b, in-droplet RCR showed fine DNA amplification in quantity and quality in comparison to in-tube RCR. In some conditions, in-droplet RCR yielded evidently a higher quantity of product than in-tube RCR. It is known that RCR activity is dependent on the quality of template DNA, that is, the ratio of supercoiled circular DNA molecules against contaminating linear or nicked DNA. Considering this, we hypothesized that template DNA molecules in linear form interferes with the amplification of the supercoiled molecule and that the microcompartmentalization relieves this interference effect by physically separating the DNA molecule in droplets. To test this idea, we conducted a spike experiment of RCR for 16 kbp DNA, in which supercoiled circular DNA was subjected to the RCR reaction after being mixed with linear template DNA. The electrophoresis analysis showed that RCR activity was inhibited with the spiked linear DNA (Figure S3). Apparently, in-droplet RCR was more tolerant to the interference than in-tube RCR that was completely suppressed. Thus, the interference effect of linear DNA on RCR, and the protection from the interference by compartmentalization were confirmed, although the molecular mechanism for the interference effect by linear DNA is not clear. In this spike experiment, we used a different lot of RCR enzyme cocktail with lower activity than the principal RCR enzyme cocktail used in the above experiments. When the same spike experiment was conducted with the principal RCR enzyme cocktails, obvious interference was not observed. Thus, though it was shown that the in-droplet RCR was superior to in-tube RCR in terms of quantity, quality, and robustness against the template quality, more studies are required for elucidation of these mechanisms.

Conclusion

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In this study, RCR enabled the amplification of circular DNA with the length of 16 kb or 208 kbp from a single DNA template molecule in microdroplets. The success rate of RCR amplification from a single copy of template DNA was confirmed to be around 82% for 16 kbp DNA, and 23% for 208 kbp DNA. The production yield of RCR for 16 kbp DNA almost reached the upper limit determined by the total amount of nucleotide in the reaction mixture, although the RCR of 208 kbp showed lower production yield, about one-eighth of RCR for 16 kbp DNA. The lower efficiency of RCR for 208 kbp DNA can be at least partly attributed to the low quality of long template DNA that inevitably contains nicked or linear DNA. Regarding this point, we found microcompartmentalization can relieve the interference effect by linear DNA, posing clues for further improvement of RCR for long DNA under compartmentalization conditions. Thus, this method is expected to be a platform system for building artificial cells with the ability to replicate large DNA. Considering that RCR is a reconstituted system of genome replication from E. coli, the system would be applicable to elucidate the mechanism of genome replication and division of microorganisms. However, several technical challenges remain. The sizes of the droplets in this study were highly heterogeneous, which hampered more quantitative analysis. We recently developed a method to produce monodispersed micrometer-sized droplets/liposomes. (35) We believe that this technological advancement would allow for a more quantitative analysis of RCR. The end-point assay protocol in this study, that is, the staining of DNA after reaction, is another point to be addressed for more elaborate kinetic or real-time analysis.

Materials and Methods

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Chemicals, Proteins, and DNAs

Mineral Oil (Sigma-Aldrich, Japan), ABIL EM 90 (Evonik), Triton X-100 (Sigma-Aldrich, Japan), Alexa 647 (Thermo Fisher Scientific), SYBR Green I (Takara Bio), 6 × loading buffer (Takara Bio), and phenol–chloroform–isoamyl alcohol (Nacalai Tesque) were purchased from the respective suppliers. Proteins for RCR were prepared as previously reported. (6,36) As template DNA for RCR, two plasmids were prepared. For a model of genome-sized DNA, a 208 kbp plasmid (pMSR227-2) was prepared by inserting the fluorescent protein Venus sequence into the 205 kbp plasmid (pMSR227). (6) A midsized plasmid of 16 kbp was prepared as follows: three fragments, Lter11 (14.8 kbp), Cm cassette (0.75 kbp), and OLDT cassette (0.42 kbp) were prepared as described, (36) and assembled using the OriCiro Assembly Kit (Oriciro Genomics, Inc.). The assembly products were subjected to transformation of E. coli strain DH5α, and the plasmid was prepared from a chloramphenicol-resistant colony. Prepared DNA was quantified with intercalator dye, PicoGreen (Thermo Fisher Scientific). Because supercoiled circular DNA shows lower fluorescence, (37,38) the florescence intensity was measured after restriction enzyme digestion.

Protocol of RCR

The stock buffer solution and ×10 RCR buffer were prepared as reported. (6) The chemical contents of ×10 RCR buffer were 200 mM Tris-HCl pH 8.0, 1.6 M potassium acetate, 100 mM Mg (OAc) 2, 40 mM dithiothreitol (DTT), 40 mM creatine phosphate, 10 mM each NTP, 1 mM each dNTP, 0.5 μg/mL yeast tRNA, 2.5 mM NAD+, 100 mM ammonium sulfate, and 1 mM Tiron. The stock enzyme mix and ×5 enzyme mix were prepared as reported. (6) The contents of the ×5 enzyme mix were 2.5 mg/mL bovine serum albumin, 100 ng/mL creatine kinase, 0.5 mM ATP, 2 μM SSB4, 280 nM IHF2, 1 μM DnaG, 200 nM DnaN2, 70 nM Pol III*, 100 nM DnaB6 C6, 500 nM DnaA, 22 nM RNaseH, 140 nM ligase, 170 nM Pol I, 250 nM gyrase (GyrA2 B2), 25 nM Topo IV (ParC2 E2), 200 nM Topo III, 200 nM RecQ, and 300 nM Tus. The RCR reaction mixture was prepared by adding ×10 RCR buffer and ×5 Enzyme mix with Alexa 647 at 5 μM. After preincubation of the reaction mixture at 30 °C for 30 min, the reaction was started by injection of template DNA at the indicated concentration and incubated at 30 °C for 3 h.

In-Droplet RCR

RCR reaction mixture of 4.5 μL was preincubated at 30 °C for 30 min, and 0.5 μL of the solution with template DNA was added. The reaction mixture was mixed with 70 μL of mineral oil with a surfactant mix: 3% ABIL EM 90 and 0.05% Triton X-100 for emulsification by pipetting multiple times. The resultant distribution of droplets was shown in the histogram (Figure S4). The emulsion mixture was incubated at 30 °C for 3 h for the RCR reaction for 16 kbp DNA, and 3–5 h for 208 kbp DNA. For staining with the DNA intercalator dye SYBR Green I, mineral oil was pre-equilibrated with SYBR Green I as follows: mineral oil with surfactant mix and aqueous solution of SYBR Green I were mixed at a volume ratio of 1:10, followed by incubation at 23 °C for 1 h. The oil-SYBR green mix was then centrifuged at 7000 rpm for 2 min. The supernatant oil was recovered using SYBR green oil. After the RCR reaction, the emulsion mixture was mixed with SYBR Green oil at a 1:2 volume ratio and incubated overnight at room temperature. The SYBR green stained emulsion was introduced into a flow cell chamber and observed with a confocal microscope (Ti-E, Nikon).

Image Analysis

Each image data point was composed of z-stacked fluorescence images of Alexa 647 and SYBR green. The images were analyzed automatically with in-house made macros on ImageJ (NIH). The position of droplets was determined from the fluorescence image of Alexa 647, and the region-of-interest (ROI) was defined. The fluorescence intensity of SYBR green in the corresponding ROI was analyzed for each z-section in order to determine the z-section with the maximum fluorescence intensity of the ROI as the section on the focal plane in the SYBR green channel. The mean fluorescence in the ROI was defined as the SYBR green intensity from the droplet.

Agarose Electrophoresis Analysis

A part of the emulsion mixture was centrifuged at 7000 rpm for 4 min to remove the oil solution in the upper layer. Eighteen microliters of 6× loading buffer (Takara) and 20 μL of phenol–chloroform–isoamyl alcohol (Nacalai Tesque) were added to the remaining aqueous phase and mixed several times by inversion. The mixture was centrifuged at 7000 rpm for 2 min. The aqueous sample from the supernatant was subject to electrophoresis on a 0.5% agarose gel. The gel was stained with SYBR Green I (Takara Bio) for image analysis. The samples of in-tube RCR were treated as exactly the same as in-droplet RCR; after in-tube RCR, the samples were mixed with mineral oil and detergents, and subject to the same treatment for in-droplet RCR.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.0c00584.

  • Histograms of fluorescence intensity of droplets for negative control, calibration curve of the fluorescence intensity of encapsulated DNA in droplets, electrophoresis analysis of in-droplet and in-tube RCR amplification with or without linearized template DNA, and size distribution of droplets of the RCR mixture (PDF)

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  • Corresponding Author
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    • Hiroshi Ueno - Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, JapanOrcidhttps://orcid.org/0000-0001-5331-4335
    • Hiroki Sawada - Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
    • Naoki Soga - Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
    • Mio Sano - Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
    • Seia Nara - Department of Life Science, College of Science, Rikkyo University, Tokyo 171-8501, Japan
    • Kazuhito V. Tabata - Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
    • Masayuki Su’etsugu - Department of Life Science, College of Science, Rikkyo University, Tokyo 171-8501, Japan
  • Author Contributions

    H.U. and H.S. contributed equally to this work. H.U., H.S., and M.S. performed experiments and analyzed data. N.S., S. N., K.V.T., and M.S. provided essential materials. M.S. and H.N. designed the research. H.U., H.S., and H.N. wrote the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank all members of our laboratory for helpful comments. This work was supported in part by Grant-in-Aid for Scientific Research on Innovation Areas (JP18H04817, JP19H05380 to H.U.), (JP17H06355 to K.V.T.), Grant-in-Aids for Scientific Research (S) (JP19H05624 to H.N.) from the Japan Society for the Promotion of Science, ImPACT Program of Council for Science, Technology, and Innovation, Japan Science and Technology Agency (to H.N., M.S., and K.V.T.), and JST CREST, Japan (JPMJCR19S4 to H.N. and JPMJCR18S6 to K.V.T. and M.S.).

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  • Abstract

    Figure 1

    Figure 1. In-droplet RCR. (a) Scheme of RCR. The RCR system is a fully reconstituted genome replisome from E. coli, which comprises 14 purified proteins, including DNA polymerase III holoenzyme, DnaA, and DnaG proteins and chemicals such as dNTPs and rNTPs. Unlike other amplification reactions of DNA amplification such as rolling circle amplification, the RCR system duplicates supercoiled circular DNA molecules with the exact same topology. RCR requires the oriC sequence on template DNAs for initiation complex formation. RCR proceeds in four steps: initiation complex formation, replication fork progression, Okazaki fragment maturation, and decatenation. (b) Experimental scheme. The reaction mixture of RCR including Alexa 647 dye was emulsified with mineral oil and a surfactant mix (3% ABIL EM 90 and 0.05% Triton X-100). The emulsion mixture was incubated at 30 °C for 3 h for isothermal amplification. For imaging of isothermal amplification of DNA with a fluorescence confocal microscope, the emulsion mixture was stained with an intercalator, SYBR Green I, by mixing with mineral oil pre-equilibrated with SYBR Green I solution.

    Figure 2

    Figure 2. In-droplet RCR for 16 kbp plasmid DNA. (a) After 3 h the in-droplet RCR reaction of 16 kbp template DNA, fluorescence images of the droplets were obtained with a confocal fluorescence microscope. The upper row shows images of the water-in-oil droplet marker, Alexa 647 (red). The lower row shows images of the DNA marker SYBR Green I (green). Scale bar, 20 μm. (b) Electrophoresis analysis of in-tube and in-droplet RCR product using the same 16 kbp template DNA (92 pg/μL). After 3 h RCR reaction, DNA molecules were recovered after phenol–chloroform extraction and subjected to 0.5% agarose gel electrophoresis. The 2.5 kb DNA Ladder (Takara Bio) was used as the size marker. Lanes 1 and 2, 2 and 10 μL of recovered DNA after in-tube RCR; lanes 3 and 4, 2 and 10 μL of recovered DNA after in-tube RCR without RCR enzyme; lanes 5 and 6, 2 and 10 μL of recovered DNA after in-droplet RCR; lanes 7 and 8, 2 and 10 μL of recovered DNA after in-droplet RCR without RCR enzyme; lane 9, template 16 kbp DNA. Arrows indicate the migration positions for supercoiled DNA, linearized DNA, and nicked open circular DNA.

    Figure 3

    Figure 3. In-droplet RCR for 208 kbp plasmid DNA. (a) After 3 h in-droplet RCR reaction of 208 kbp template DNA, fluorescence images of the droplets were obtained. The upper row shows images of the water-in-oil droplet marker, Alexa 647 (red). The lower row shows images of the DNA marker SYBR Green I (green). Scale bar, 10 μm. (b) Electrophoresis analysis of in-tube and in-droplet RCR product using the same 208 kbp template DNA (5 ng/μL). After 3 h RCR reaction, DNA molecules were recovered after phenol–chloroform extraction, and 20 μL of the recovered solution was subjected to 0.5% agarose gel electrophoresis. The 2.5 kb DNA Ladder (Takara Bio) was used as the size marker. Lane 1, after in-tube RCR; lane 2, after in-tube RCR without RCR enzyme; lane 3, after in-droplet RCR; lane 4, after in-droplet RCR without RCR enzyme; lane 5, template 208 kbp DNA. Arrows indicate the migration positions for supercoiled DNA, linearized DNA (or concatemer DNA), and nicked open circular DNA (well).

    Figure 4

    Figure 4. Digital in-droplet RCR for 16 kbp DNA. Fluorescence image of digital in-droplet RCR. Red indicates the fluorescence image of the w/o droplet marker (Alexa 647). Green indicates the image of amplified DNA molecules stained with SYBR Green I. Dotted circles indicate the examples of RCR-negative droplets and dark droplets. Scale bar, 10 μm.

    Figure 5

    Figure 5. Histograms of fluorescence intensity of droplets after in-droplet RCR. Histogram of fluorescence intensity of droplets after RCR amplification of 16 kbp (125 pg/μL template) (a) and 208 kbp DNA (5 ng/μL template) (b). The red triangle indicates the position of the threshold for the identification of RCR-positive droplets, which is defined as mean ±5 × s.d. of the negative control peak (see Figure S1) that corresponds to dark droplets. N indicates the total number of droplets in each data set.

    Figure 6

    Figure 6. Success rate of digital in-droplet RCR. (a) Probability of RCR-positive droplets plotted against the diameter of droplets obtained at three different concentrations of template 16 kbp DNA: 62.5, 125, and 250 pg/μL (6 pM, 12 pM, 24 pM). The dots indicate the experimental data points. Solid lines indicate the theoretical probability estimated from the fitting (see eq 3). (b) Probability of RCR-positive droplets plotted against the diameter of droplets obtained at two different concentrations of template 208 kbp DNA: 1.5 ng/μL and 5 ng/μL (11 pM, 37 pM). (c) Success rate of RCR amplification for RCR of 16 kbp and 208 kbp template DNA.

    Figure 7

    Figure 7. Product yield of digital in-droplet RCR. Mean fluorescence intensity of positive droplets after RCR for 16 kbp and 208 kbp were calculated with the indicated diameters. The concentration of template DNA was 125 pg/μL for 16 kbp and 5 ng/μL for 208 kbp DNA.

  • References

    ARTICLE SECTIONS
    Jump To

    This article references 38 other publications.

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      Garenne, D. and Noireaux, V. (2019) Cell-free transcription-translation: engineering biology from the nanometer to the millimeter scale. Curr. Opin. Biotechnol. 58, 1927,  DOI: 10.1016/j.copbio.2018.10.007
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      Laohakunakorn, N., Grasemann, L., Lavickova, B., Michielin, G., Shahein, A., Swank, Z., and Maerkl, S. J. (2020) Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology. Front. Bioeng. Biotechnol. 8, 213,  DOI: 10.3389/fbioe.2020.00213
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      Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., and Ueda, T. (2001) Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751755,  DOI: 10.1038/90802
    5. 5
      Garamella, J., Marshall, R., Rustad, M., and Noireaux, V. (2016) The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology. ACS Synth. Biol. 5, 344355,  DOI: 10.1021/acssynbio.5b00296
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      Su’etsugu, M., Takada, H., Katayama, T., and Tsujimoto, H. (2017) Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Res. 45, 1152511534,  DOI: 10.1093/nar/gkx822
    7. 7
      Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, E63,  DOI: 10.1093/nar/28.12.e63
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      Sakatani, Y., Yomo, T., and Ichihashi, N. (2018) Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination. Sci. Rep. 8, 13089,  DOI: 10.1038/s41598-018-31585-1
    9. 9
      Berhanu, S., Ueda, T., and Kuruma, Y. (2019) Artificial photosynthetic cell producing energy for protein synthesis. Nat. Commun. 10, 1325,  DOI: 10.1038/s41467-019-09147-4
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      Shin, J., Jardine, P., and Noireaux, V. (2012) Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS Synth. Biol. 1, 408413,  DOI: 10.1021/sb300049p
    11. 11
      Hurtgen, D., Mascarenhas, J., Heymann, M., Murray, S. M., Schwille, P., and Sourjik, V. (2019) Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System. ChemBioChem 20, 26332642,  DOI: 10.1002/cbic.201900299
    12. 12
      Liu, A. P. and Fletcher, D. A. (2009) Biology under construction: in vitro reconstitution of cellular function. Nat. Rev. Mol. Cell Biol. 10, 644650,  DOI: 10.1038/nrm2746
    13. 13
      Marshall, R., Maxwell, C. S., Collins, S. P., Jacobsen, T., Luo, M. L., Begemann, M. B., Gray, B. N., January, E., Singer, A., He, Y., Beisel, C. L., and Noireaux, V. (2018) Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System. Mol. Cell 69, 146157,  DOI: 10.1016/j.molcel.2017.12.007
    14. 14
      Tawfik, D. S. and Griffiths, A. D. (1998) Man-made cell-like compartments for molecular evolution. Nat. Biotechnol. 16, 652656,  DOI: 10.1038/nbt0798-652
    15. 15
      Zhang, Y., Minagawa, Y., Kizoe, H., Miyazaki, K., Iino, R., Ueno, H., Tabata, K. V., Shimane, Y., and Noji, H. (2019) Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array. Sci. Adv. 5, eaav8185,  DOI: 10.1126/sciadv.aav8185
    16. 16
      Ichihashi, N., Usui, K., Kazuta, Y., Sunami, T., Matsuura, T., and Yomo, T. (2013) Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment. Nat. Commun. 4, 2494,  DOI: 10.1038/ncomms3494
    17. 17
      Oberholzer, T., Albrizio, M., and Luisi, P. L. (1995) Polymerase chain reaction in liposomes. Chem. Biol. 2, 677682,  DOI: 10.1016/1074-5521(95)90031-4
    18. 18
      Shohda, K.-i., Tamura, M., Kageyama, Y., Suzuki, K., Suyama, A., and Sugawara, T. (2011) Compartment size dependence of performance of polymerase chain reaction inside giant vesicles. Soft Matter 7, 3750,  DOI: 10.1039/c0sm01463j
    19. 19
      Kurihara, K., Okura, Y., Matsuo, M., Toyota, T., Suzuki, K., and Sugawara, T. (2015) A recursive vesicle-based model protocell with a primitive model cell cycle. Nat. Commun. 6, 8352,  DOI: 10.1038/ncomms9352
    20. 20
      Matsuo, M., Kan, Y., Kurihara, K., Jimbo, T., Imai, M., Toyota, T., Hirata, Y., Suzuki, K., and Sugawara, T. (2019) DNA Length-dependent Division of a Giant Vesicle-based Model Protocell. Sci. Rep. 9, 6916,  DOI: 10.1038/s41598-019-43367-4
    21. 21
      Nakano, M., Komatsu, J., Matsuura, S., Takashima, K., Katsura, S., and Mizuno, A. (2003) Single-molecule PCR using water-in-oil emulsion. J. Biotechnol. 102, 117124,  DOI: 10.1016/S0168-1656(03)00023-3
    22. 22
      Tsugane, M. and Suzuki, H. (2018) Reverse Transcription Polymerase Chain Reaction in Giant Unilamellar Vesicles. Sci. Rep. 8, 9214,  DOI: 10.1038/s41598-018-27547-2
    23. 23
      Okauchi, H., Sakatani, Y., Otsuka, K., and Ichihashi, N. (2020) Minimization of Elements for Isothermal DNA Replication by an Evolutionary Approach. ACS Synth. Biol. 9, 17711780,  DOI: 10.1021/acssynbio.0c00137
    24. 24
      Sato, Y., Komiya, K., Kawamata, I., Murata, S., and Nomura, S. M. (2019) Isothermal amplification of specific DNA molecules inside giant unilamellar vesicles. Chem. Commun. (Cambridge, U. K.) 55, 90849087,  DOI: 10.1039/C9CC03277K
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      Matsumura, S., Kun, A., Ryckelynck, M., Coldren, F., Szilagyi, A., Jossinet, F., Rick, C., Nghe, P., Szathmary, E., and Griffiths, A. D. (2016) Transient compartmentalization of RNA replicators prevents extinction due to parasites. Science 354, 12931296,  DOI: 10.1126/science.aag1582
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    • Histograms of fluorescence intensity of droplets for negative control, calibration curve of the fluorescence intensity of encapsulated DNA in droplets, electrophoresis analysis of in-droplet and in-tube RCR amplification with or without linearized template DNA, and size distribution of droplets of the RCR mixture (PDF)


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