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Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements – Scientific Reports

Encapsulation of pDNA into the giant lipid vesicles

To determine the concentration of encapsulated pDNA in the three types of giant lipid vesicle, namely 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), DOPC/1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) (3:1 molar ratio), and DOPC/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (97:3 molar ratio), the giant lipid vesicles including the cell-free synthesis system without DFHBI-1T and YOYO-1-conjugated plasmid DNA were generated via the droplet transfer method. When the giant lipid vesicles were prepared using 20 ng/μL of pDNA, approximately 35 ng/μL of pDNA was encapsulated by all the vesicles. The concentration of encapsulated pDNA was not significantly different in the three types of giant lipid vesicles (Fig. 2a,b and Fig. S1a,c–g). Moreover, the encapsulated pDNA concentration in the giant lipid vesicles comprising DOPC/DOTAP (95:5 molar ratio and 99:1 molar ratio) was determined. The results showed that this concentration was not significantly different from those of the other components of the giant lipid vesicles (Fig. 2b). The fluorescence of YOYO-1 was not observed on the membranes of all giant lipid vesicles comprising DOPC/DOTAP (95:5 molar ratio), as shown in Fig. S1a. An electrostatic interaction between pDNA and membranes containing positively charged lipids (DOTAP) at 1–5 mol% did not occur because the CFPS system solution was present in the inner phase of the giant lipid vesicles.

Figure 2
figure 2

(a) Typical confocal images of the fluorescence intensities of oxazole yellow homodimer (YOYO-1)-conjugated sfCherry-producing plasmids under each membrane condition. (b) YOYO-1 analysis of plasmid DNA concentration. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)-containing lipid vesicle (blue), three independent experiments; 25 mol% 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS)-containing lipid vesicle (orange), three independent experiments; and 3 mol% 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP)-containing lipid vesicle (pink), three independent experiments; and 1-mol%-DOTAP-containing lipid vesicle (purple), three independent experiments; and 5-mol%-DOTAP-containing lipid vesicle (green), three independent experiments. Error bars indicate standard deviation (SD). All data of YOYO-1 fluorescence intensities was shown in Fig. S1.

When the giant lipid vesicles were prepared using 35 ng/μL of pDNA, the encapsulation concentration was higher compared with those prepared with 20 ng/μL of pDNA. The encapsulation concentration of pDNA in DOPC/DOTAP-containing vesicles was the highest among all. The encapsulated concentration varied among the three types of vesicles. The negatively charged pDNA is more efficiently encapsulated to the vesicles containing DOTAP, a positively charged phospholipid (Fig. 2a,b and Fig. S1h–j).

Observation of transcription and translation in the giant lipid vesicles

To observe the transcription and translation of E. Coli extract S30 system in the vesicles, the mRNAs encoding sfCherry followed by a tandem array of RNA aptamers binding to DFHBI-1T were synthesized using a cell-free synthesis system. The fluorescence intensity of DFHBI-1T correlates with the amount of synthesized mRNA, and thus can be defined as the transcriptional efficacy. The translation was confirmed based on the fluorescence intensity of sfCherry. First, changes in each fluorescence intensity caused by the transcription and translation were observed using a microplate reader. The maximum fluorescence intensity of DFHBI-1T was observed after 60 min incubation, which decreased over time. In contrast, the maximum fluorescence of sfCherry was observed after 240 min incubation (Fig. 3). Hence, the activation of transcription and translation was detected based on these increasing and decreasing fluorescence intensities. On the other hand, although the activation of the translation of PUREfrex (CFPS system of complete reconstruction) was later than that of E. coli extract S30 system, the decrease in fluorescence intensity of DFHBI-1T was not observed for 4 h (Fig. S2a). Therefore, the decrease in fluorescence intensity of DFHBI-1T in E. coli extract S30 system was caused by a digestion of mRNA owing to E. coli extract S30 solution that is not completely free of nuclease.

Figure 3
figure 3

Time-dependent change on fluorescence intensity of DFHBI-1T and sfCherry in bulk solution. Mean values (three independent experiments) of fluorescence intensity of DFHBI-1T (Green Square),), fluorescence intensity of sfCherry (Red Square). Data are expressed as the mean ± SD.

The activation of transcription and translation in the DOPC-containing vesicles was observed using CLMS. Due to the rapid initiation of transcription (starting from approximately 5 min), the observation of the initiation reaction was difficult and took 15 min to prepare the solutions of giant lipid vesicles containing cell-free transcription and translation system. Herein, the vesicles were observed after incubation for 30 min, 1 h, 2 h, and 4 h. For DOPC-containing vesicles, the fluorescence intensity of DFHBI-1T was the maximum at 30 min, which then decreased gradually. The DFHBI-1T fluorescence was rarely observed after 120-min incubation. In contrast, the sfCherry fluorescence was not observed after 30 min but was notable after 2 h of incubation. The fluorescence intensity of sfCherry increased gradually. Hence, using this method, the transcription and translation in giant lipid vesicles were observed (Fig. 4a).

Figure 4
figure 4

Comparison of transcription and translation inside giant lipid vesicles containing 20 ng/μL (final concentration) of plasmid DNA. (a) Typical confocal images of the fluorescence intensity change inside the lipid vesicles containing DOPC, DOPC/DOPS (75:25 molar ratio), or DOPC/DOTAP (97:3 molar ratio) on both leaflets. (b) DFHBI-1T fluorescence inside GUVs with DOPC, 25-mol%-DOPS or 3-mol%-DOTAP membrane in response to time changing shown in left (green). sfCherry fluorescence of GUVs with DOPC, 25-mol%-DOPS or 3-mol%-DOTAP membrane in response to time changing shown in right (red). DOPC vesicles; 30 min (n = 184 (184 vesicles), N = 2 (two independent experiments)), 60 min (n = 191, N = 2), 120 min (n = 259, N = 2), and 240 min (n = 247, N = 2). 25-mol%-DOPS vesicles; 30 min (n = 318, N = 3), 60 min (n = 322, N = 3), 120 min (n = 328, N = 3), and 240 min (n = 307, N = 3). 3-mol%-DOTAP vesicles; 30 min (n = 104, N = 5), 60 min (n = 102, N = 5), 120 min (n = 90, N = 5), and 240 min (n = 92, N = 5). (c) Time-dependent changes in fluorescence intensity of DFHBI-1T bound to messenger RNAs in the lipid vesicles. Comparison of the median value of DOPC-containing lipid vesicle (blue), 25-mol%-DOPS-containing lipid vesicle (orange), and 3-mol%-DOTAP-containing lipid vesicle (pink). Time-dependent changes in fluorescence intensity of sfCherry in the lipid vesicles. Comparison of the median value of DOPC-containing lipid vesicle (blue), 25-mol%-DOPS-containing lipid vesicle (orange), and 3-mol%-DOTAP-containing lipid vesicle (pink).

Next, to investigate the effect of varying surface charges of giant lipid vesicles on the activation of transcription and translation, we prepared three types of giant lipid vesicles (DOPC, DOPC/DOPS [3:1 molar ratio], and DOPC/DOTAP [97:3 molar ratio]). The maximum median values of DFHBI-1T fluorescence in these three types of vesicles were observed at 30 min, which decreased gradually after 30 min. When 20 ng/μL (final concentration) of pDNA was encapsulated into the three types of giant lipid vesicles, despite the same encapsulation concentration, the DFHBI-1T fluorescence intensity in 3-mol%-DOTAP-containing vesicles was the highest compared with that of the other two types of giant lipid vesicles (Fig. 4b,c, Fig. S4a,b), possibly because the transcription was activated by the positively charged lipids. The DFHBI-1T fluorescence intensities in all the giant lipid vesicles decreased gradually (Fig. 4). Regarding translational activity, sfCherry fluorescence was detected approximately after 1 h incubation. The sfCherry fluorescence in 3-mol%-DOTAP-containing vesicles was the highest compared with that of the other two types of giant lipid vesicles (Fig. 4b,c, Fig. S4c,d). The translational activity in the DOPC vesicle or negatively charged lipid vesicles did not change. Similarly, the transcriptional activity in the same vesicles did not change. There is no association between the vesicle areas and the amount of transcription and translation (Fig. S5a–c). The fluorescence intensity of sfCherry was dependent on the fluorescence intensity of DFHBI-1T, indicating that the translational activity depended on the activation of transcription. The results showed that transcriptional and translational activities in the 3-mol% DOTAP-containing vesicles were the highest among those in the DOPC- or DOPS-containing vesicles (Fig. S4a–d).

Moreover, we investigated changes in transcriptional and translational activities in the vesicles by changing DOTAP concentrations (1 mol% and 5 mol%) (Fig. 5a). When the DOTAP concentration increased from 3 to 5 mol%, the decrease in the amount of synthesized mRNA in the 5-mol%-DOTAP-containing vesicles was slower than that in the 3-mol%-DOTAP-containing vesicles. After incubating for 120 min, the synthesized amount of sfCherry in the 5-mol%-DOTAP-containing vesicles was higher than that in the 3-mol%-DOTAP-containing vesicles (Fig. 5b,c). Conversely, when the DOTAP concentration decreased from 3 to 1 mol%, the decrease in the amount of synthesized mRNA in the 1-mol%-DOTAP-containing vesicles was faster than that in the 3-mol%-DOTAP-containing vesicles. After incubating for 120 min, the synthesized amount of sfCherry in the 1-mol%-DOTAP-containing vesicles was lower than that in the 3-mol%-DOTAP-containing vesicles (Fig. 5b,c). Moreover, transcriptional and translational activities in the 1-mol%-DOTAP-containing vesicles were the highest among those in the DOPC or DOPS-containing vesicles (Figs. 4c and 5c). The amounts of mRNA synthesized at 30 min and sfCherry synthesized at 240 min in the vesicles containing 1 mol%, 3 mol%, or 5 mol% DOTAP were almost the same (Fig. 5c). Thus, the presence of DOTAP in the vesicles increased the transcriptional and translational activities and played a role in preventing a digestion of mRNA (especially 5-mol%-DOTAP-containing vesicles).

Figure 5
figure 5

Comparison of transcription and translation inside giant lipid vesicles changing the concentration of cationic lipids. (a) Typical confocal images of the fluorescence intensity change inside the lipid vesicles containing DOPC/DOTAP (99:1 molar ratio), DOPC/DOTAP (98:2 molar ratio), and DOPC/DOTAP (97:3 molar ratio) on both leaflets. (b) DFHBI-1T fluorescence inside GUVs with the 1-mol%-DOTAP, 3-mol%-DOTAP, or 5-mol%-DOTAP membrane in response to time changes is shown in the left (green). sfCherry fluorescence of GUVs with 1-mol%-DOTAP, 3-mol%-DOTAP, or 5-mol%-DOTAP membrane in response to time changes is shown in the right (red). 1-mol%-DOTAP vesicles; 30 min (n = 111 (111 vesicles), N = 3 (three independent experiments), 60 min (n = 108, N = 3), 120 min (n = 116, N = 3), and 240 min (n = 118, N = 3). 3-mol%-DOTAP vesicles; 30 min (n = 104, N = 5), 60 min (n = 102, N = 5), 120 min (n = 90, N = 5), and 240 min (n = 92, N = 5). 5-mol%-DOTAP vesicles; 30 min (n = 64, N = 3), 60 min (n = 57, N = 3), 120 min (n = 58, N = 3), and 240 min (n = 57, N = 3). (c) Time-dependent changes in the fluorescence intensity of DFHBI-1T bound to messenger RNAs in the lipid vesicles. Comparison of the median value of the 1-mol%-DOTAP-containing lipid vesicle (purple), 3-mol%-DOTAP-containing lipid vesicle (pink), and 5-mol%-DOTAP-containing lipid vesicle (green). Time-dependent changes in the fluorescence intensity of sfCherry in the lipid vesicles. Comparison of the median value of the 1-mol%-DOTAP-containing lipid vesicle (purple), 3-mol%-DOTAP-containing lipid vesicle (pink), and 5-mol%-DOTAP-containing lipid vesicle (green).

Next, we investigated the activations of the transcription and translation in pDNA-encapsulated giant lipid vesicles, with pDNA concentration changing from 20 to 35 ng/μL. The encapsulated pDNA varied among the three types of giant lipid vesicles when prepared with 35 ng/μL of pDNA (Fig. S3). The highest pDNA encapsulation concentration was in DOTAP-containing vesicles, followed by DOPC- and DOPS-containing vesicles. The DFHBI-1T fluorescence intensities in all types of giant lipid vesicles prepared with 35 ng/μL (approximately 9 nM) of pDNA were higher than those in the giant lipid vesicles prepared with 20 ng/μL (approximately 5 nM) of pDNA (Figs. 4b,c, 6a,b; Fig. S6a–c). Similarly, the fluorescence intensities of sfCherry in all types of giant lipid vesicles prepared with 35 ng/μL of pDNA were higher than those in the giant lipid vesicles prepared with 20 ng/μL of pDNA (Figs. 4b,c, 6a,b; Fig. S6d–f). In a previous study, the amounts of the synthesized mRNA and protein in the giant lipid vesicles containing pDNA concentrations of 1.75 nM and 3.5 nM were significantly different30. Thus, herein, changes in transcriptional and translational activities observed in the range of 5–9 nM of pDNA concentrations were reasonable. Among giant lipid vesicles prepared with 35 ng/μL pDNA, the DFHBI-1T fluorescence and sfCherry amount in DOTAP-containing vesicles were difficult to compare with the other two types of vesicles because the pDNA encapsulation concentration in DOTAP-containing vesicles was markedly high compared with that of other two types of vesicles (Fig. 2).

Figure 6
figure 6

Comparison of transcriptional and translational activity under high plasmid DNA (pDNA) concentration condition (35 ng/μL of pDNA). (a) DFHBI-1T fluorescence inside GUVs with DOPC, 25-mol%-DOPS or 3-mol%-DOTAP membrane in response to time changing shown in left (green). SfCherry fluorescence of GUVs with DOPC, 25-mol%-DOPS or 3-mol%-DOTAP membrane in response to time changing shown in right (red). DOPC vesicles; 30 min (n = 92 (92 vesicles), N = 3 (three independent experiments)), 60 min (n = 83, N = 3), 120 min (n = 89, N = 3), and 240 min (n = 78, N = 3). 25-mol%-DOPS vesicles; 30 min (n = 66, N = 3), 60 min (n = 64, N = 3), 120 min (n = 58, N = 3), and 240 min (n = 60, N = 3). 3-mol%-DOTAP vesicles; 30 min (n = 61, N = 3), 60 min (n = 52, N = 3), 120 min (n = 57, N = 3), and 240 min (n = 47, N = 3). (b) Time-dependent changes in fluorescence intensity of DFHBI-1T bound to messenger RNA in the lipid vesicles. Comparison of the median value of DOPC-containing lipid vesicle (blue), 25-mol%-DOPS-containing lipid vesicle (orange), and 3-mol%-DOTAP-containing lipid vesicle (pink). Time-dependent changes in fluorescence intensity of sfCherry in the lipid vesicles. Comparison of the median value of DOPC-containing lipid vesicle (blue), 25-mol%-DOPS-containing lipid vesicle (orange), and 3-mol%-DOTAP-containing lipid vesicle (pink).

Additionally, to investigate the effect of transcriptional and translational activities attributed to the membrane surface charge, the zeta potential values of each vesicle composition were measured at a lipid vesicle concentration of approximately 1 mM, which was estimated by the molar concentration of the phospholipid in the inner leaflet of the lipid vesicle with a diameter of 10 μm. The zeta potential values of the vesicle membrane surface comprising DOPC, DOPC/DOPS [3:1 molar ratio], and DOPC/DOTAP [99:1 molar ratio, 97:3 molar ratio, and 95:5 molar ratio] hydrated by 10 mM HEPES/75 mM NaCl (pH 7.4) were − 3.18 ± 0.03 mV, − 42.0 ± 0.35 mV, − 2.49 ± 0.08 mV, − 0.71 ± 0.08 mV, and + 1.14 ± 0.39 mV, respectively (Fig. S7). Each zeta potential value of the lipid vesicles had the significant difference.

These results suggested that changes in transcriptional and translational activities depended on the lipid compositions of the giant lipid vesicles. The presence of positively charged lipids in high concentrations inhibited the transcriptional and translational activities when the protein was synthesized by the CFPS system outside the lipid vesicles23,28. In contrast, in this study, transcriptional and translational activities in the vesicles containing a low concentration (1–5 mol%) of positively charged lipids increased and 5-mol%-DOTAP-containing vesicles especially inhibited the digestion of mRNA. Although pDNA normally interacts with the membrane surface of vesicles containing positively charged lipids owing to electrostatic interactions, the present study showed that the pDNA did not interact with the positively charged vesicles containing the CFPS solution with the S30 extract (Fig. 2a, Fig. S1a). Electrostatic interactions did not occur between pDNA and the positively charged membrane owing to the presence of various biomolecules in the CFPS solution, implying that CFPS biomolecules were present in the lipid vesicles. Consequently, the transcriptional activity in the vesicles containing the positively charged lipid increased. Regarding the translation step, the ribosomal protein may be present in the inner phase of the vesicles containing positively charged lipids although the ribosomal protein with the positive charge and hydrophobicity easily interacted with the lipid membrane in the absence of positively charged lipids23. Consequently, the translational activity in the positively charged vesicles increased. The role of DOTAP (positively charged lipids) in protein synthesis using the CFPS system differed according to CFPS system conditions such as vesicle concentration in the reaction solution, charged lipid concentration in the vesicles, and CFPS types.