A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Zhang, Yi; Kurosawa, Kanako; Nishiura, Daisuke; Tei, Mika; Tsudome, Mikiko

doi:10.3791/60945

Cited by 3 publications

(4 citation statements)

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“…Thus, the batch CFPS can be carried out overnight, and only endpoint single-frame imaging is required (S1 Movie). The previous studies have already proved that every single purified template DNA molecule in FemDA can trigger the CFPS reaction [13,21] since the input concentration (lower than one molecule per droplet on average) of template DNA was consistent with the output ratio of fluorescent droplets (denoted as P positive , which is equal to the number of positive droplets divided by the total number of droplets) over the array. In this study, the situation is slightly different from the previous ones as the intact single DNA molecules always coexist with the interferential DNA digest fragments even after partitioning the CFPS solution into droplets.…”

Section: Plos Onementioning

confidence: 85%

“…A microchamber array was fabricated on a cover glass via the previously established microfabrication process [21]. The resulting diameter and depth of each microchamber were 4 μm and 3 μm, respectively.…”

Section: Microfabricationmentioning

confidence: 99%

“…A 60× or 100× objective lens (CFI Apo TIRF, NA = 1.49, Nikon) and a CCD camera (Andor iXon3 DU-897) was used for imaging. The exposure time was 100 ms. An automatic image analysis was carried out as previously reported [21]. Histogram plot and Gaussian fitting of the fluorescence intensities were done by KaleidaGraph (Synergy).…”

Section: Imaging and Data Analysismentioning

confidence: 99%

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A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

et al. 2020

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Restriction endonucleases play a central role in the microbial immune system against viruses and are widely used in DNA specific cleavage, which is called restriction digestion, for genetic engineering. Herein, we applied digital cell-free protein synthesis as an easy-to-use orthogonal readout means to assess the restriction digest efficiency, a new application of digital bioassays. The digital counting principle enabled an unprecedentedly sensitive trace analysis of undigested DNA at the single-molecule level in a PCR-free manner. Our approach can quantify the template DNA of much lower concentrations that cannot be detected by ensemble-based methods such as gold-standard DNA electrophoresis techniques. The sensitive and quantitative measurements revealed a considerable variation in the digest efficiency among restriction endonucleases, from less than 70% to more than 99%. Intriguingly, none of them showed truly complete digestion within reasonably long periods of reaction time. The same rationale was extended to a multiplexed assay and applicable to any DNA-degrading or genome-editing enzymes. The enzyme kinetic parameters and the flanking sequence-dependent digest efficiency can also be interrogated with the proposed digital counting method. The absolute number of residual intact DNA molecules per microliter was concluded to be at least 107, drawing attention to the residual issue of genetic materials associated with the interpretation of nucleases’ behaviors and functions in daily genetic engineering experiments.

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Section: Plos Onementioning

confidence: 85%

Section: Microfabricationmentioning

confidence: 99%

Section: Imaging and Data Analysismentioning

confidence: 99%

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A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

et al. 2020

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“…When nanomolar or sub-micromolar concentrations are required for stable oligomerization, the volume of the reactor must be reduced to attoliters (10 −18 L) or sub-attoliters. It presents a technical challenge to microfabrication as the resolving power of traditional top-down lithography we applied before would become insufficient ( 71 ).…”

Section: Discussionmentioning

confidence: 99%

A sequential one-pot approach for rapid and convenient characterization of putative restriction-modification systems

Zhang,

Takaki,

Yoshida-Takashima

et al. 2023

mSystems

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With the advance of high-throughput sequencing, the molecular basis of coevolutionary interaction between viruses and host microorganisms is predominantly elucidated by in silico genomic analyses, which revealed potential communication of genetic materials related to microbial immune systems such as the restriction-modification (R-M) system. However, the sequence-dependent information is often insufficient to output a conclusive argument without biochemical characterizations, particularly for homologs of rare genes considered less accurately annotated. We proposed a 1-day and one-pot workflow covering in vitro protein synthesis and enzymatic assays to confirm the exact function of putative R-M genes only with manual pipetting operations of microliter-scale liquids. The proof-of-demonstration experiments mainly focused on a series of putative R-M enzymes from our recently found deep-sea temperate bacteriophage and its host bacterium. Two new restriction endonucleases and two new methyltransferases with respective unambiguous substrate specificities, superior catalytic performance, or unique sequence preferences were quickly identified. A frequent discrepancy between sequence similarity search and single-molecule methylation-sensitive sequencing toward the prediction of recognition motifs can get settled with the established direct biochemical characterization. The proposed approach under the cell-free one-pot concept allows for preliminary characterizations of diverse categories (e.g., Types I, II, and III) of putative R-M systems at most laboratories with minimum equipment and time costs. IMPORTANCE The elucidation of the molecular basis of virus-host coevolutionary interactions is boosted with state-of-the-art sequencing technologies. However, the sequence-only information is often insufficient to output a conclusive argument without biochemical characterizations. We proposed a 1-day and one-pot approach to confirm the exact function of putative restriction-modification (R-M) genes that presumably mediate microbial coevolution. The experiments mainly focused on a series of putative R-M enzymes from a deep-sea virus and its host bacterium. The results quickly unveiled unambiguous substrate specificities, superior catalytic performance, and unique sequence preferences for two new restriction enzymes (capable of cleaving DNA) and two new methyltransferases (capable of modifying DNA with methyl groups). The reality of the functional R-M system reinforced a model of mutually beneficial interactions with the virus in the deep-sea microbial ecosystem. The cell culture-independent approach also holds great potential for exploring novel and biotechnologically significant R-M enzymes from microbial dark matter.

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A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Cited by 3 publications

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A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

A sequential one-pot approach for rapid and convenient characterization of putative restriction-modification systems

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