Autonomous ribosome biogenesis in vitro

Abstract

Ribosome biogenesis is pivotal in the self-replication of life. In Escherichia coli, three ribosomal RNAs and 54 ribosomal proteins are synthesized and subjected to cooperative hierarchical assembly facilitated by numerous accessory factors. Realizing ribosome biogenesis in vitro is a critical milestone for understanding the self-replication of life and creating artificial cells. Despite its importance, this goal has not yet been achieved owing to its complexity. In this study, we report the successful realization of ribosome biogenesis in vitro. Specifically, we developed a highly specific and sensitive reporter assay for the detection of nascent ribosomes. The reporter assay allowed for combinatorial and iterative exploration of reaction conditions for ribosome biogenesis, leading to the simultaneous, autonomous synthesis of both small and large subunits of ribosomes in vitro through transcription, translation, processing, and assembly in a single reaction space. Our achievement represents a crucial advancement toward revealing the fundamental principles underlying the self-replication of life and creating artificial cells.

Fig. 1. Screening orthogonal oSD·oASD pairs with two-sided orthogonality in vitro.

A Four types of orthogonalities. SD, Shine–Dalgarno sequence; ASD, anti-Shine–Dalgarno sequence; oSD, orthogonal SD; oASD, orthogonal ASD. B Experimental scheme to screen oSDs that do not interact with native ribosomes in cell extracts. Fluorescence was detected using fluorescence microplate readers. C oSD selection. Either a WT-SD–sfGFP or an oSD–sfGFP reporter (named a, b, c, d, or1, or4, and j) was mixed with S12 or S30 cell extracts. NC, negative control without a reporter. Mean ± SD (n = 3). **, p < 0.01; n.s., not significant; one-way ANOVA with Dunnett’s test against NC. D Further oSD selection. Either a WT-SD–LacZ or an oSD–LacZ reporter (b, or1, or4, and j) was mixed with S12 cell extracts. a.u., arbitrary unit. Mean ± SD (n = 3). One-way ANOVA with Dunnett’s test against NC. E Experimental scheme to screen oSD·oASD pairs with two-sided orthogonality in cell extracts. Cell extracts were prepared using BL21 Star^TM (DE3) lacZ::frt expressing an artificial rRNA operon with WT-ASD or oASD (b, or1, or or4) and C1192U spectinomycin resistance (SpcR). F Screening oASDs that do not interact with the WT-SD–LacZ reporter. The cell extracts were mixed with the WT-SD–LacZ reporter and spectinomycin. Mean ± SD (n = 3). *, p < 0.05; two-tailed Welch’s t-test. G Screening oSD·oASD pairs with two-sided orthogonality. The cell extract was mixed with the cognate oSD–LacZ reporter and spectinomycin. Mean ± SD (n = 3). ***, p < 0.001; two-tailed Welch’s t-test. Three biological replicates were used in all experiments. Source data are provided as a Source Data file.

Fig. 2. Highly sensitive detection of the artificial ribosome translational activity.

A Comparison between a conventional bulk assay and a femtoliter droplet assay. B Detection of the translational activity of the artificial ribosomes using the droplet assay. We prepared two types of S12 cell extracts; one contained native ribosomes and 1.2 μM of artificial ribosomes with or1-oASD and C1192U spectinomycin resistance (SpcR) and the other only native ribosomes. The cell extract containing the artificial ribosomes was diluted by the control cell extract at the indicated ratio. The cell-free transcription and translation in droplets were carried out in the presence of the or1-oSD–LacZ reporter and spectinomycin. In the scatter plots, the vertical and horizontal axes indicate the mean fluorescence intensity and the ID of each droplet, respectively. Droplets exceeding the threshold (mean fluorescence intensity ≥ 22) are indicated in red. The threshold was set at a value that none of the droplets in the negative control exceeded. PC, positive control without dilution; NC, negative control using only the control cell extract; a.u., arbitrary unit. Source data are provided as a Source Data file.

Fig. 3. SSU biogenesis in vitro.

A Experimental scheme for SSU biogenesis in vitro. B Exploring optimal conditions for the first reaction using a simplex-lattice design. In the first reaction, the concentrations of the native ribosomes, the artificial rRNA operon with or1-oASD and C1192U spectinomycin resistance (SpcR), and 21 SSU r-protein genes were 0–100, 0–3, and 0–0.5 nM each, respectively. The second reaction was conducted with the or1-oSD–LacZ reporter and spectinomycin using the droplet assay. The data represent the mean fluorescence intensity of droplets. a.u., arbitrary unit. C Follow-up optimization of the first reaction. In the first reaction, the concentrations of the native ribosomes, the artificial rRNA operon, and 21 SSU r-protein genes were 0–240, 0–0.9, and 0–0.15 nM each, respectively. The second reaction was conducted with the or1-oSD–LacZ reporter and spectinomycin using the droplet assay. The optimal reaction condition was a native ribosome concentration of 120 nM. However, in the following experiments, we selected suboptimal reaction conditions with 80 nM native ribosomes to reduce reagent costs. D Successful detection of the nascent artificial SSU translational activity using the bulk assay under the optimized reaction condition. In the first reaction, the concentrations of the native ribosomes, the artificial rRNA operon, and 21 SSU r-protein genes were 80, 0.3, and 0.05 nM each, respectively. The second reaction was conducted with the or1-oSD–LacZ reporter and spectinomycin using the bulk assay. Mean ± SD (n = 3, biological replicates). ****, p < 0.0001; **, p < 0.01; n.s., not significant; one-way ANOVA with Dunnett’s test against the negative control without native ribosomes. The p-values showing p  <  0.0001 and p  >  0.9999 are p  =  1.0 × 10⁻⁵ and p  =  9.9999 × 10⁻¹, respectively. Source data are provided as a Source Data file.

Fig. 4. LSU biogenesis in vitro.

A Experimental scheme for LSU biogenesis in vitro. B Exploring optimal conditions for the first reaction. In the first reaction, the concentrations of the native ribosomes, artificial rRNA operon with A2058U clindamycin resistance (CldR), and 33 LSU r-protein genes were 80, 0.6–1.5, and 0–0.015 nM each, respectively. The second reaction was conducted with the WT-SD–LacZ reporter and clindamycin using the bulk assay. a.u., arbitrary unit. C Reproducible detection of the nascent artificial LSU translational activity under the optimized reaction condition. In the first reaction, the concentrations of the native ribosomes, the artificial rRNA operon, and 33 LSU r-protein genes were 80, 0.9, and 0.01 nM each, respectively. The second reaction was conducted with the WT-SD–LacZ reporter and clindamycin using the bulk assay. Mean ± SD (n = 3, biological replicates). ****, p < 0.0001; n.s., not significant; one-way ANOVA with Dunnett’s test against the negative control without native ribosomes. The p-value showing p  <  0.0001 is p  =  1.0 × 10⁻⁵. D Improvement of the nascent LSU-derived fluorescence signal. The experimental condition was the same as Fig. 4C except that an improved LacZ reporter with a modified 5′UTR sequence was used instead of the WT-SD–LacZ reporter. The signal-to-noise ratio was improved because the background translation by the native ribosomes was sufficiently suppressed by 1.5 mM clindamycin (Supplementary Fig. 7A). Mean ± SD (n = 3, biological replicates). *, p < 0.05; two-tailed Welch’s t-test. Although the experimental conditions in lane 1 in Fig. 4C and lane 3 in Fig. 4D were identical, they showed different values because these experiments were performed on different days. Source data are provided as a Source Data file.

Fig. 5. Synthesis of ribosomes in vitro.

A Scheme showing in vitro synthesis of both SSU and LSU in a single reaction space. B Successful detection of the nascent artificial SSU and LSU translational activity in the bulk assay. In the first reaction, the concentrations of the native ribosomes, artificial rRNA operon with or1-oASD, C1192U spectinomycin resistance (SpcR), and A2058U clindamycin resistance (CldR), and 54 r-protein genes were 80, 0.9, and 0.01 nM each, respectively. The second reactions using the bulk assay were conducted with the or1-oSD–LacZ reporter and spectinomycin for the nascent artificial SSU, the improved LacZ reporter and clindamycin for the nascent artificial LSU, the or1-oSD–LacZ reporter, spectinomycin, and clindamycin for the nascent artificial ribosomes. a.u., arbitrary unit. Mean ± SD (n = 3, biological replicates). **, p < 0.01; n.s., not significant; two-tailed Welch’s t-test. C Successful detection of the nascent artificial ribosome translational activity. The nascent artificial SSU (SpcR) and LSU (CldR) with streptavidin-binding aptamer (Sb-aptamer) were synthesized by the in vitro SSU and LSU biogenesis, respectively, and purified using streptavidin resin under the subunit dissociation condition (1 mM Mg²⁺), as described in Supplementary Fig. 9C. We observed translational activity under the double-antibiotic condition only when we mixed the purified nascent artificial SSU and LSU. NC, negative control prepared using the same production and purification procedure without expressing the artificial rRNA operon with Sb-aptamer. Violin plot represents the mean fluorescence intensity values of droplets from three independent experiments. ****, p < 0.0001; one-way ANOVA with Dunnett’s test against NC. Scale bars = 10 μm. The p-value showing p  <  0.0001 is p  =  1.0 × 10⁻⁵. Source data are provided as a Source Data file.