In Vitro Evolution of Unmodified 16S rRNA for Simple Ribosome

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In vitro evolution of unmodified 16S rRNA for simple ribosome reconstitution Yoshiki Murase, Hiroki Nakanishi, Gakushi Tsuji, Takeshi Sunami, and Norikazu Ichihashi ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 23, 2017

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ACS Synthetic Biology

Title

In vitro evolution of unmodified 16S rRNA for simple ribosome reconstitution

Authors Yoshiki Murase[a], Hiroki Nakanishi[a], Gakushi Tsuji[b], Takeshi Sunami[b], Norikazu Ichihashi*[a, c]

Affiliations: [a] Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan [b] Institute for Academic Initiatives, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan [c] Graduate School of Frontier Biosciences, Osaka University University 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan

Contact Information Norikazu Ichihashi Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan Tel: +81-6-6879-4151 FAX: +81-6-6879-7433 E-mail: [email protected]

Key words Ribosome reconstitution, artificial evolution, 16S rRNA, rRNA modification, in vitro evolution, artificial cell

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Abstract One of the largest challenges in the synthesis of artificial cell that can reproduce is in

vitro assembly of ribosomes from in vitro synthesized rRNAs and proteins. In this study, to circumvent the posttranscriptional modification of 16S rRNA for reconstitution of fully active 30S subunit, we performed artificial evolution of 16S rRNA, which forms the functional 30S subunit without posttranscriptional modifications. We first established an in vitro selection scheme by combining the integrated synthesis, assembly, and translation (iSAT) system with the liposome sorting technique. After 15 rounds of selection cycles, we found one point mutation (U1495C) near the 3′ terminus that significantly enhanced the reconstitution activity of the functional 30S subunit from unmodified 16S rRNA to approximately 57% of that from native modified 16S rRNA. The effect of the mutation did not depend on the reconstitution scheme, anti-SD sequences, or the target genes to be translated. The mutation we found in this study enabled reconstitution of the active 30S subunit without rRNA modification, and thus would be a useful tool for simple construction of self-reproducing ribosomes.

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Artificial or minimal cell synthesis from isolated molecules is a challenge in the field of

in vitro synthetic biology(1-3). One of the central functions of the cell is translating the genetic information into proteins. The translation system of Escherichia coli has been reconstituted in vitro from purified molecules, such as ribosome, initiation proteins, and elongation proteins(4). To construct a self-reproducing system, some of the translational proteins, such as all the ribosomal proteins(5) and aminoacyl-tRNA synthetases, have been produced in the translation system(6). One of the remaining important challenges is reproduction of the ribosome from the components produced in the translation system.

Ribosome is a large complex consisting of 54 ribosomal proteins and three rRNAs. The 16S and 23S rRNA subunits are chemically modified after transcription in vivo. In vitro reconstitution studies revealed that rRNA modifications play an important role in ribosomal function. The reconstituted E. coli 50S subunit harboring the unmodified 23S rRNA is almost inactive for peptidyl transferase activity(7), whereas the reconstituted 30S subunit of E. coli containing the unmodified 16S rRNA shows tRNA binding activity similar to that of the native subunit when reconstituted under non-physiological conditions (at 50°C and high magnesium and salt concentrations)(8). In a crude E. coli extract, the reconstituted ribosome from unmodified rRNAs under physiological translation-integrated conditions shows an activity similar to that of the in

vivo-assembled ribosome(9). However, in a pure translation system, the reconstituted 30S subunit from unmodified 16S rRNA under physiological translation-integrated conditions shows significantly reduced activity compared to that from the modified 16S rRNA(10). Currently, several enzymes (seven pseudouridine synthases and 10 or 14 methyltransferases for 16S or 23S rRNAs, respectively) have been reported to participate in rRNA modifications(11). However, the modification system has not yet been reconstituted in vitro.

In this study, to circumvent the modification requirement, we attempted to obtain a 16S rRNA mutant that is functional without modification through artificial evolution. rRNA modifications are considered to play several roles in cells, such as stabilizing the

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ribosome structure as a molecular glue or acting as checkpoints to guarantee ribosome quality(11). We hypothesized that changes in the rRNA sequence could compensate for the role of rRNA modification as a molecular glue. If such a 16S rRNA is obtained, we would reconstitute a ribosome in a simple manner, which would be useful for in vitro ribosome reproduction in the future.

Two types of artificial evolution of 16S rRNA have been reported for obtaining orthogonal or drug-resistant ribosomes previously(12, 13). In those studies, ribosomes were constructed in the cell, and thus rRNA modification was inevitable. In this study, we first established an in vitro evolution method of unmodified 16S rRNA. After 15 rounds of selection cycles, starting from randomly mutagenized 16S rRNA gene library, we found that one of the mutations (U1495C) significantly improved the reconstitution activity of unmodified 16S rRNA to a level comparable to that of the native modified 16S rRNA.

Results Results Selection strategy To achieve ribosome reconstitution from unmodified 16S rRNA, we used the integrated synthesis, assembly, and translation (iSAT) system(10). In the iSAT system, 16S rRNA is transcribed from DNA and assembled from the total proteins of 30S subunit (TP30) to form the 30S subunit, which then assembles with the 50S subunit to translate a reporter gene in a single-pod reaction. Recently, iSAT reaction in liposome has been also reported(14). Although most of the previous iSAT reactions were performed using an E.

coli cell extract, we used the purified translation system of E. coli (PURE system)(4) in this study to avoid rRNA modification. As a selection method, we used the liposome sorting method, which we have developed previously(15). In this method, a fluorescent reaction system is encapsulated in liposomes, and highly fluorescent liposomes were collected using a fluorescence-activated cell sorter. Combining the iSAT and the liposome sorting methods, we attempted to establish in vitro evolution of 16S rRNA.

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The method of in vitro selection cycle is schematically illustrated in Figure 1. The 16S rRNA gene library was prepared using error-prone PCR, and was encapsulated into liposomes together with the reconstitution-translation reaction mixture, including the purified translation system: T7 RNA polymerase, all ribosomal proteins, 23S and 5S rRNAs, reporter gene (β-galactosidase), and a fluorogenic substrate. In the liposomes, the DNA library was transcribed to 16S rRNA, which assembled with TP30 to form 30S subunits. Similarly, the 50S subunit was also assembled from total ribosomal proteins of the 50S subunit (TP50) and a native modified 23S and 5S rRNAs. The reconstituted 30S and 50S subunits formed 70S ribosome to translate a reporter β-galactosidase, which produces green fluorescence by degrading the fluorogenic substrate. The liposomes that exhibited stronger green fluorescence were collected using a cell-sorter. The DNAs encoding 16S rRNA gene were extracted from the collected liposomes and used for the next round of the selection cycle after PCR amplification.

A technical hurdle in this selection scheme was the high background noise. For an effective selection, one copy of DNA encoding 16S rRNA should be present per liposome, which corresponds to less than 50 pM for the average liposome size used in this study. At such low concentrations, the number of liposomes showing sufficiently high green fluorescence was almost the same as that without the DNA (Figure 2A), indicating that reconstitution of 30S subunit could not be detected using this method because of the large background noise due to contamination of the 30S subunit. Therefore, we attempted to introduce an orthogonal SD and anti-SD pairs (A2) developed in a previous study as a possible solution for this issue(13). We introduced the orthogonal anti-SD into 16S rRNA gene and the corresponding orthogonal SD into the reporter β-galactosidase gene. When using these orthogonal pairs, we detected more liposomes with the DNA encoding 16S rRNA gene than without it (Figure 2B). The ratios of the liposomes in the region used for selection (indicated in red) were 0.5% with the 16S rRNA gene, which were 5-fold higher than those without the 16S rRNA gene (0.1%). Therefore, we used these orthogonal pairs in the selection cycle. In the following section, we used the terms, “n-16S rRNAs” and “o-16S rRNAs” for the 16S rRNAs that include the native and orthogonal anti-SDs, respectively.

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Selection cycle We started the selection cycle from a randomly mutagenized o-16S rRNA gene library, and repeated the cycle for 15 rounds. In each round, we measured the average fluorescence of the reacted liposomes after the reconstitution-translation step, and plotted it as a relative value to that of the non-mutagenized original o-16S rRNA gene (Figure 3). We also plotted the ratio of collected liposomes to the total number of liposomes in the same size range (Figure S2). Both results showed similar increasing trajectories with some fluctuations at rounds 8 and 14. The DNA extracted from the liposomes were stored for longer periods (more than two weeks) prior to these two rounds, which probably caused these temporal reductions.

Cloning assay After round 15, we ligated the DNA mixture encoding the o-16S rRNA gene with a plasmid and transformed the ligated products into an E. coli strain. We randomly selected six colonies and extracted the plasmids. The genes encoding six o-16S rRNA were amplified from the plasmids and assayed for reconstitution-translation activities. This assay was the same as the reaction occurring in the liposomes during the selection cycle, except for performing under bulk conditions and using 10 nM of the DNA encoding o-16S rRNA gene. After reaction at 37°C for 6 h, the green fluorescence was measured. Five of the six clones showed higher fluorescence than the original o-16S rRNA (Figure 4A), indicating that these clones had higher reconstitution-translation activities.

The sequences of these clones are shown in Figure 4B. These clones had 3–8 mutations, and two of them (U1495C and C206U) were common in four clones. Since common mutations are expected to be responsible for the increased activity, we isolated these mutations and assayed the reconstitution-translation activity. The o-16S rRNA encoding C206U mutation alone showed the same level of fluorescence as the original one, while those encoding U1495C alone and both C206U and U1495C showed higher

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activities, comparable to that of clone 1 (Figure 4C). These results indicate that the increased activity of the round 15 clones was sufficiently explained by the effect of a single mutation, U1495C.

Effect on the n-16S rRNA, rRNA, including the native antianti-SD The mutation, U1495C, was located close to the anti-SD region (Figure 5A, 3-D structure is shown in Figure S3), and thus the effect of the mutation possibly depended on the orthogonal anti-SD. To examine this possibility, we changed the orthogonal anti-SD sequence back to the native SD sequence to make n-16S rRNA and measured the reconstitution-translation activities. The fluorescence of the original n-16S rRNA gene was slightly higher than that without the n-16S rRNA gene (NC), while the U1495C mutant showed significantly higher fluorescence (Figure 5B). This result indicates that the effect of U1495C mutation is independent of the anti-SD sequence.

Translation of other proteins Next, we investigated the effect of the mutation on the translation of two other proteins, firefly luciferase and GFP. We performed the reconstitution-translation assay using these proteins as reporters, instead of β-galactosidase, and measured luminescence or fluorescence, respectively. In both cases, the U1495C mutant showed significantly higher signal than that of the original o-16S rRNA gene (Figure 6), demonstrating that U1495C improves the expression of proteins other than β-galactosidase as well.

Reconstitution with 50S subunit instead of TP50 and 23S rRNA In the previous reconstitution-translation reactions, the 50S subunit was also reconstituted from TP50 and 23S and 5S rRNAs in the same reaction mixture. To examine the possibility that the effect of U1495C mutation depends on the 50S reconstitution, we performed the reconstitution-translation assay in the presence of isolated 50S subunits instead of TP50 and 23S and 5S rRNAs (Figure 7A). The U1495C mutant showed significantly higher reporter activity even under this condition (Figure 7B), similar to the previous experimental condition, indicating that the effect of U1495C mutation does not depend on 50S reconstitution.

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ReconstitutionReconstitution-translation translation assay from rRNA In

all

of

the

previous

experiments,

we

performed

reconstitution

under

transcription-coupled conditions, in which transcription of 16S rRNA and reconstitution of 30S subunit occurred at the same time. Next, we examined the effect of U1495C mutation on the reconstitution under a transcription-uncoupled condition, in which 30S subunit was reconstituted from 16S rRNA prepared in advance. We prepared unmodified original and U1495C n-16S rRNAs by in vitro transcription, and added into the reconstitution-translation reaction mixture instead of the DNAs encoding 16S rRNA genes (Figure 8A). For comparison, we also prepared native modified n-16S rRNA from the 30S subunit purified from E. coli. Similar to the transcription-coupled condition, the U1495C mutant (IVT U1495C) showed a significantly higher luciferase activity than the original n-16S rRNA (IVT Original), and it showed approximately 57% of the activity of the native modified n-16S rRNA (Navive, Figure 8B). This result indicated that the effect of U1495C is independent of transcription, and the mutation enhanced the reconstitution activity of the unmodified n-16S rRNA up to approximately half of that of the modified native n-16S rRNA. For comparison, we also showed the results of the native 30S subunit purified from E. coli (30S subunit). The activity was approximately three times higher than that of native n-16S rRNA, indicating that even the modified rRNA was not fully reconstituted in this system.

The effect of U1495C in vivo The in vivo effect of U1495C has been reported to be deleterious(16, 17). To confirm this result, we used E. coli strain KT101(18), in which all the rRNA genes were removed from the genome and complemented by a plasmid, pRB10 (ampicillin resistant). We also used other plasmids, which encoded another drug-resistant gene (zeocin resistant), and the original n-16S rRNA genes or U1495C mutant (pRB103-16SrRNA and pRB103-16SrRNA_U1495C). We introduced these new plasmids into the strain KT101, and selected the transformants using zeocin. The surviving colonies were smaller with the U1495C mutant (1.45 ± 0.07 mm) than those observed with the original n-16S rRNA (2.0 ± 0.06 mm), suggesting that this mutation inhibits cell growth. We found that the

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colonies contained both the original and newly introduced plasmids because they were resistant to both ampicillin and zeocin. To remove the original plasmid, we cultured four colonies after four transfers, until they became ampicillin sensitive. We then sequenced the remaining plasmids, and found that the U1495C mutations in the newly introduced plasmids were reverted to the original sequence for all four lineages. These results indicate that U1495C mutation is significantly toxic in vivo, consistent with previous reports(16, 17).

Discussion In this study, we established an artificial evolutionary method for unmodified 16S rRNA

in vitro by combining the iSAT system in PURE system and the liposome sorting technique. After 15 rounds of the selection, we found one effective mutation, U1495C, which significantly enhanced the reconstitution-translation activity of 16S rRNA, independent of the anti-SD types (Figure 5), reporter genes (Figure 6), and reconstitution

schemes

(Figures

7

and

8).

The

mutation

enhanced

the

reconstitution-translation activity of unmodified 16S rRNA up to approximately half the level of that of the native modified 16S rRNA (Figure 8). The mutation we found in this study partially circumvented the requirement of posttranscriptional modification of 16S rRNA and enabled reconstitution of the 30S subunit in vitro under pure and physiological conditions by using a simple scheme.

The nucleotide U1495 is located in a highly conserved region (helix 44) near the 3’ terminus, called the decoding center, where the mRNA and tRNA interact(19). The helix has been reported to form a bridge (B2a bridge) to interact with the 50S subunit, and this inter-subunit interaction is important for translation initiation and translational processivity(20). The mutations in U1495 change the conformation of the helix and significantly decrease association with the 50S subunit, indicating the importance of U1495 for interaction with the 50S subunit(16). This role of U1495 is consistent with the toxicity of U1495C mutations observed in this study. However, the question remains as to why the mutation did not negatively affect reconstitution and translation in vitro. We can speculate two possibilities to answer this question. First, the toxic effect of

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U1495C might depend on the posttranscriptional modifications of the 16S rRNA. There are several modifications around U1495, such as 1402 m4Cm, 1407 m5C, and 1498 m3U (indicated with arrowheads in Figure 5A)(21). They are all close vicinity of the U1495 in 3-D structure (Figure S3). These modifications are required for proper 16S rRNA folding in the decoding center(22-25). U1495 mutations were also reported to change rRNA folding in vivo(16). Taken together, the U1495C mutation might compensate for the absence of modification by changing 16S rRNA conformation, and both modification and U1495C mutation might change the folding to an extent that is not tolerated by the cell. The second possibility is that the U1495C mutation might impair one of the in

vivo-specific essential functions of 16S rRNA, such as ribosome quality control(21). Studies suggested that ribosome assembly in vivo is monitored by modifying rRNAs at several steps to maintain the quality of the ribosome(26). Perturbations in the quality control might be toxic in vivo but may have negligible effects in vitro. Further studies focusing on the different effects of the mutation between in vivo and in vitro conditions would provide useful information regarding the role of the 16S rRNA modifications inside the cell.

In this study, we used a highly-purified translation system, all components of which showed

almost

single

bands

in

sodium

dodecyl

sulfate-polyacrylamide

gel

electrophoresis (SDS-PAGE) analysis(27). However, we cannot deny that a small amount of modifying enzyme contamination may have affected the results.

We performed in vitro evolution of unmodified 16S rRNA in this study. One of the remaining challenges is in vitro evolution of unmodified 23S rRNA. In contrast to the 30S subunit, the modification of 23S rRNA is essential for the function of 50S subunit of

E. coli(10). Therefore, we may have to introduce some of the modification enzymes in the reconstitution system for the in vitro evolution. Another possible strategy is to use 50S subunits of other bacteria. It has been reported that functional 50S subunits of

Thermus aquaticus and Geobacillus stearothermophilus (previously known as Bacillus stearothermophilus) were reconstituted from unmodified 23S rRNAs(28, 29). Additionally, we found that the 50S subunit in the purified E. coli translation system

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was interchangeable with those of other species with a modest decrease in the translation activity(27). Thus, in vitro evolution of unmodified 23S rRNA could be achieved using the 50S subunits of these bacteria.

Methods Plasmids The plasmid encoding n-16S rRNA of E. coli (pUC-16SrRNA) was constructed by introducing the 16S rRNA genes of E. coli into the pUC19 plasmid. The fragment was prepared by reverse-transcription with PrimeScript RTase (Takara, Japan) and primer 1 at 42°C for 30 min followed by PCR amplification using primers 1 and 2. The amplified fragment of the target size was selected by agarose-gel extraction and ligated using the In-Fusion cloning kit (Takara) to another fragment, which was PCR-amplified using pUC19 as a template and primers 3 and 4. The amplified rRNA sequence was a variation of rrnB and was the same as the registered sequence in GenBank (GenBank: J01859.1). The resultant plasmid contained GGG nucleotides at the 5′-terminus of the 16S rRNA gene and substitution at the second nucleotide for transcription with T7 RNA polymerase(30). To prepare an orthogonal o-16S rRNA, we constructed another plasmid (pUC-16SrRNA-A2) by introducing gu722-723cg and ccuccu1535-1540cugugg(13) into pUC-16SrRNA. The whole sequence of the orthogonal o-16S rRNA gene in pUC-16SrRNA-A2 is shown in Figure S1. For preparation of mutant o- and n-16S rRNAs, each mutation was introduced into pUC-16SrRNA-A2 by PCR with mutagenized primers, followed by self-ligation. To prepare the plasmid encoding lacZ genes under orthogonal SD, the SD sequence of pET-lacZ(31) was exchanged with that of the orthogonal SD by PCR amplification with primers 12 and 13, followed by self-ligation with the In-Fusion cloning kit (Takara, Japan). The plasmids for other reporter genes, luciferase and gfp, was prepared in the same method, except for using pEX-T7-Fluc and pETG5tag(32) as templates. pEX-T7-Fluc was constructed using the chemically synthesized firefly luciferase gene(33) and pEX vector (Eurofin genomics). The

plasmids

used

for

in

vivo

analysis,

pBR103-16SrRNA

and

pBR103-16SrRNA-U1495C, were constructed by ligating each vector fragment and

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insert fragment with the In-Fusion cloning kit (Takara, Japan). The vector fragment was prepared by PCR using pBR103(18) as a template and primers 14 and 15. The insert fragments were prepared by PCR using primers 16 and 17 and pUC-16SrRNA-A2 or the U1495C derivative as templates. All primer sequences are shown in Table S1.

DNA and RNA fragments for reconstitution The

DNA

fragments

encoding

n-

or

o-16S

rRNA

gene

used

for

the

reconstitution-translation assay were prepared by PCR using pUC-16SrRNA-A2 as a template and each primer sets: primers 5 and 6 for the orthogonal o-16S rRNA gene used in Figure 2, 4, 6, and 7, and primers 5 and 7 for the n-16S rRNA gene including native anti-SD used in Figure 5. The in vitro-transcribed n-16S rRNA used for results shown in Figure 8 was prepared by in vitro transcription with the T7 RNA polymerase (Takara, Japan) using the DNA fragments prepared with primers 5 and 7, according to the manufacturer’s instructions. The RNA was purified using PureLink RNA purification kit (Life Science technologies), followed by DNase I treatment (Takara, Japan), according to the manufacturer’s instructions and further purification using the same kit. The o-16S rRNA gene library was prepared using the GeneMorph II Random Mutagenesis Kit (Agilent Technologies) to introduce 0–4.5 mutations/kb, according to the manufacturer’s instructions. The DNA fragments encoding reporter genes with the orthogonal SD, lacZ, firefly luciferase, and gfp, were prepared using pET-lacZ_A2SD, pEX-Fluc_A2SD, and pET-gfp_A2SD as templates and primers 8 and 9, 10 and 11, 8 and 9, respectively. All DNA fragments were purified using the PureLink DNA purification kit (Life Science Technologies) before use.

Ribosomal subunits, subunits, ribosomal proteins, and rRNAs rRNAs The 70S ribosome was prepared according to method from a previous study(34). Ribosomal

subunits

were

prepared

by

fractionation

after

sucrose-gradient

centrifugation, according to the previous study(10). The total proteins of ribosomal subunit (TP30 and TP50) and rRNAs (native n-16S, 23S, and 5S rRNAs) were prepared from each subunit, according to previous study(35).

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Translation system We used a reconstituted E. coli translation system (PURE SYSTEM)(4) minus the ribosome. All translation-related proteins were purified additionally by gel-filtration chromatography after affinity chromatography to reduce contamination(34). The composition and SDS-PAGE profile of each component were shown in our previous study(27).

Liposome Liposomes were prepared by modifying the phase-transition method(36, 37). Palmitoyl-oleoyl-phosphatidylcholine (POPC, Avanti polar lipid) and cholesterol (Avanti polar lipid) were separately dissolved in chloroform at 100 mg/ml, and then liquid paraffin was added at a final concentration of 5 mg/ml. After incubation at 80°C for 30 min, POPC solution (360 µl) was vigorously mixed with the cholesterol solution (40 µl), followed by incubation at 80°C for 30 min. After cooling to 20–25°C, the internal solution described below was dispersed in the lipid mixture by vigorously mixing. The emulsion was kept on ice for 10 min and moved onto 180 µl of the outer solution described below. After keeping on ice another 10 min, the solution was centrifuged at 18 k×g for 30 min at 4°C. The liposome pellet and a part of the supernatant was collected from bottom of the tube by making a hole with an injection needle (φ = 0.8 mm). The liposome was centrifuged at 18 k×g for 5 min at 4°C. The pellet was suspended with 50 µl

of the

dilution solution and incubated at 37°C for 19–20 h for the

reconstitution-translation reaction. The internal solution includes the purified translation system of E. coli without ribosome(27), 0.2 M sucrose, 2.3 µM Alexa Fluor™ 647 Conjugate (TA647, Thermo Fisher Scientific), 1 U/µl RNase inhibitor (RNaseIn Plus, Promega), 0.1 mM 5-chloromethylfluorescein di-β-D-galactopyranoside (CMFDG, Thermo Fisher Scientific), 0.7 U /µl T7 RNA polymerase (Takara, Japan), 0.3–0.5 µM TP30 and TP50, 0.1 µM 23S and 5S rRNAs, 3.5 nM lacZ DNA, and 50 pM DNA fragments encoding 16S rRNA gene. The outer solution included 74 mM glucose, 0.74 mM magnesium acetate, and the translation system, except all proteins and tRNAs. The dilution solution included 50 mM Hepes-KOH (pH 7.8), 300 mM glutamate potassium (pH 8.0), 12.6 mM magnesium acetate, and 150 mM glucose.

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Selection cycle The liposome solution was incubated at 37°C for 19–20 h for ribosome reconstitution and translation, and subjected to flow cytometry (FACS AriaIII, Becton Dickinson). Ten thousand liposomes that exhibited 1000–3000 units of red fluorescence of TA647 (volume marker) and more than 1000 units of green fluorescence (reaction marker) were collected from unilamellar population determeind from forward and side scatterings(38). The volume of liposome was estimate from the red fluorescence of TA647 included in the internal solution, according to a previous study(15). The DNA was extracted from the collected liposomes using the Purelink PCR Micro Kit (Thermo Fisher Scientific) and PCR-amplified with primers 5 and 6. The PCR fragment was purified using the same kit and used for the next round of selection.

ReconstitutionReconstitution-translation assay in test tubes The composition of the reaction mixture was the same as that of the internal solution of the liposomes described above, except for 6 nM of the DNA fragment encoding each reporter gene and 10 nM DNA fragment encoding each 16S rRNA. When using lacZ or gfp genes as reporters, the reaction mixture was incubated at 37°C for 6 h, and the final fluorescence was measured with Mx3005P (Agilent technology). When using luciferase gene, the reaction mixture was incubated at 37°C for 4 h and then assayed luciferases activity using Luciferase Assay System (Promega). When using luciferase or gfp genes, CMFDG was omitted from the reaction mixture. For reconstitution using 50S subunit, as shown in Figure 7, 1 µM 50S subunit was used instead of TP50 and 23S rRNA. For reconstitution from rRNAs, as shown in Figure 8, 0.15 µM rRNAs and 30S subunit were used instead of the DNA fragments encoding 16S rRNA genes in absence of sucrose and TA647.

In vivo experiment We introduced plasmids pRB103-16SrRNA and pRB103-16SrRNA_U1495C, which encode the n-16S rRNAs with or without U1495C mutation, into E. coli strain KT101(18), and selected colonies on Luria Bertani (LB) agar medium containing 100

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µg/ml zeocin (Thermo Fisher Scientific) after overnight incubation at 37°C. To determine whether the colonies harbored the original pRB101 plasmid, we tested their ampicillin sensitivity by inoculating colonies on LB agar medium containing 50 µg/ml ampicillin and incubated them overnight at 37°C. To remove the original plasmid, we transferred four colonies on new LB agar plates containing 100 µg/ml zeocin, and incubated them overnight at 37°C. All colonies showed ampicillin sensitivity after four transfers. Next, we extracted and sequenced the plasmids.

Acknowledgements This work was supported by JSPS KAKENHI Grant Number, 15H01534.

Supporting Information Table S1 Primer list, Figure S1 Sequence of the orthogonal o-16S rRNA gene used in this study, Figure S2 Average ratio of the collected liposomes during selection cycle. Figure S3 S3 3-D structure around the mutational cite.

Author contributions YM, HN, GT, and NI performed experiments. NI wrote the paper. TS designed and experiments.

The authors declare no competing financial interest.

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Figure 1 Scheme of in vitro evolution of 16S rRNA. rRNA. 16S

rRNA

gene

library

was

encapsulated

in

liposomes

with

the

reconstitution-translation reaction mixture, which included total ribosomal proteins (TP30 + TP50), 23S and 5S rRNAs, reporter gene, fluorogenic substrate, T7 RNA polymerase, and the reconstituted E. coli translation system. In the liposomes, ribosomes were reconstituted from the 16S rRNA library and the reporter gene was translated, which produced green fluorescence. The liposomes were subjected to flow cytometry, and those showing high fluorescence intensities were collected. The DNA encoding 16S rRNA gene was extracted from the collected liposome and subjected to the next round of selection cycle. 19



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Figure 2 Flow cytometry of liposomes after the reconstitutionreconstitution-translation reaction. reaction. The reconstitution-translation reaction mixture was encapsulated in liposomes with or without the DNAs encoding 16S rRNA gene containing wild-type (A) or orthogonal anti-SD sequences (B) and incubated at 37°C for 20 h. The liposomes (100000) were analyzed using a flow cytometer. Red fluorescence, which reflected the volume of liposome, and green fluorescence, which reflected the translation level of the reporter gene (β-galactosidase), were analyzed. The regions where unreacted liposomes appeared are shadowed. The liposomes collected in the selection experiment (1000–3000 red fluorescence, corresponding to approximately 10–30 fL, and more than 1000 green fluorescence) were colored in red. The ratios of the liposomes in this region to all liposomes in the same volume range are indicated on each panel.

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cycle.. Figure 3 Average fluorescence of the liposomes during selection cycle The average green fluorescence of the reacted liposomes was calculated in every round and normalized by that of the original o-16S rRNA gene. The reacted liposomes were defined as liposomes that have higher green fluorescence than those of unreacted liposomes (the liposomes in unshaded regions in Figure 2).

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Figure 4 o-16S rRNA clones obtained through the selection cycle. cycle. A) The reconstitution-translation assay of six clones obtained after 15 rounds of the selection cycle. The reaction scheme was the same as that in the liposome in the selection cycle, except for 10 nM of the DNA encoding o-16S rRNA. The fluorescence after 6 h incubation at 37°C was plotted. As control experiments, the original o-16S rRNA before selection (Original) and no 16S rRNA experiment (NC) were also performed. The error bars represent standard errors (n = 3). B) Sequence analysis of the six

clones.

Common

mutations

among

the

clones

are

shaded.

C)

The

reconstitution-translation assay of the mutant genes that have isolated common mutations. For comparison, the results from clone 1 are shown. As control experiments, the original o-16S rRNA before selection (Original) and no o-16S rRNA experiment (NC) were also performed. The error bars represent standard errors (n = 3).

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Figure 5 ReconstitutionReconstitution-translation from n-16S rRNA containing native antianti-SD. SD. A) The secondary structure of rRNA around U1495C mutation site (indicated with the arrow). The structure and base number were according to a previous study (39). Known modification sites are indicated with arrowheads(21). B) The reconstitution-translation assay using n-16S rRNA gene containing native anti-SD. As a control, no n-16S rRNA experiment (NC) was also performed. The error bars represent standard errors (n = 3).

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genes.. Figure 6 The effect of the mutation on the translation of other genes A) The reconstitution-translation assay using firefly luciferase gene as a reporter. After 4 h incubation at 37°C, the luciferase activity was measured, as described in the Methods section. B) The reconstitution-translation assay used gfp gene as a reporter. After 6 h incubation at 37°C, GFP fluorescence was measured. DNAs encoding orthogonal o-16S rRNAs were used in these experiments. Control experiments without o-16S rRNA gene were also performed (NC). The error bars represent standard errors (n = 3).

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Figure 7 Effect of the mutation on the reconstitution with 50S subunit. subunit. A) Reaction scheme. In this experiment, 50S subunit was used instead of TP50 and 23S rRNA. B) The reconstitution-translation assay using luciferase gene as a reporter. After 4 h incubation at 37°C, luciferase activity was measured, as described in the Methods section. DNAs encoding orthogonal o-16S rRNAs were used in this experiment. Control experiments without o-16S rRNA gene were also performed (NC). The error bars represent standard errors (n = 3).

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Figure 8 Reconstitution from rRNAs. rRNAs. A) Reaction scheme. In this experiment, 16S rRNAs were used instead of the DNAs encoding 16S rRNA genes. B) The reconstitution-translation assay using luciferase gene as a reporter. After 4 h incubation at 37°C, luciferase activity was measured, as described in the Methods section. In vitro-transcribed n-16S rRNAs (IVT original and IVT U1495C), the native modified n-16S rRNA purified from native 30S subunit (Native), or native 30S subunit purified form E. coli (30S subunit) were used at a concentration of 0.15 µM. Control experiment without n-16S rRNA was also performed (NC). The error bars represent standard errors (n = 3).

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275x397mm (300 x 300 DPI)


In Vitro Evolution of Unmodified 16S rRNA for Simple Ribosome

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