A fully orthogonal system for protein synthesis in bacterial cells

Abstract

Disclosed are engineered polynucleotides, engineered ribosomes comprising the engineered polynucleotides, engineered cells and systems comprising the engineered polynucleotides and ribosomes, and methods of making and using the engineered polynucleotides, engineered ribosomes, engineered cells and systems. The engineered polynucleotides, engineered ribosomes, and engineered cells can be used to prepare sequence-defined polymers and to select mutant ribosomes that are capable of incorporating non-canonical amino acids into polymers.

Description

[Technical Field]

[0001] Statements relating to research or development funded by the federal government This invention was made with government assistance under MCB-1716766 and MCB-1615851, granted by the National Science Foundation. The government has certain rights to this invention. Cross-references to related patent applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 993,860, filed on March 24, 2020, under 35 U.S.SC § 119(e), the contents of which are incorporated herein by reference in their entirety.

[0003] This invention relates to engineered polynucleotides, engineered ribosomes containing engineered polynucleotides, engineered cells and systems containing engineered polynucleotides and ribosomes, and methods for producing and using engineered polynucleotides, engineered ribosomes, engineered cells and systems. Sequence-defined polymers can be prepared using engineered polynucleotides, engineered ribosomes, and engineered cells. [Background technology]

[0004] Ribosomes are the ribonucleoprotein machinery responsible for protein synthesis. In all biological kingdoms, ribosomes consist of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent yet cooperative functions of the subunits—for example, their ability to bind at initiation, rotate during elongation, and dissociate after protein release—form an established paradigm of protein synthesis. Furthermore, the bipartite nature of ribosomes is presumed to be essential for biogenesis, as dedicated assembly factors separate immature ribosomal subunits, preventing them from initiating translation [Karbstein 2013]. The free exchange of subunits limits the development of specialized orthogonal gene systems that can evolve toward new functions without interfering with native translation.

[0005] Ribosomes are highly complex mechanical devices. These large particles consist of two subunits, each composed of RNA as its primary structural and functional component, regulating distinct but complementary functions. The smaller subunit decodes mRNA, while the larger subunit catalyzes the formation of peptide bonds and creates the polypeptide exit tunnel. Subunit binding is strictly controlled throughout the translation cycle. First, during the maturation of the ribonucleoprotein, several assembly factors prevent the binding of the two subunits. Subsequently, the initiation of translation also involves initiation factors, mRNA, and fMet-tRNA. fMetThe process is strictly controlled, with the addition of subunits to the small subunit sequentially, forming a pre-initiation complex, after which the large subunit is recruited. During elongation, the subunits move slightly in one direction relative to each other at an angle of approximately 6 degrees (ratchet). Once translation is complete, the newly synthesized protein is released from the ribosome, and the subunits dissociate in an active process called ribosome recycling, preparing them for further translation rounds. Thus, the reason why ribosomes have been maintained as two subunits throughout evolution is likely explained by the need for programmed subunit binding and dissociation at specific stages of translation. While it has been suggested that initiation in leaderless mRNA occurs by 70S ribosomes with pre-bound subunits, there is no experimental evidence to show that the entire cycle of protein synthesis can be achieved by ribosomes with inseparable subunits.

[0006] Random exchange of ribosomal subunits during repeated protein biosynthesis is an obstacle to creating perfectly orthogonal ribosomes, a challenge of significant importance in both basic science and biotechnology. Previously, by placing an alternative Shine-Dalgarno (SD) sequence in the reporter mRNA and introducing complementary changes in the anti-SD region of 16S rRNA, it has been possible to redirect the subpopulation of small ribosomal subunits from translating commensal mRNA to specific mRNA [Hui 1987; Rackham 2005], enabling the selection of mutant 30S subunits with novel decoding characteristics [Wang 2007]. However, since the large subunit freely exchanges between the native small subunit and the orthogonal small subunit, it is impossible to create a fully orthogonal ribosome, which has limited the design of specific novel properties for the 50S subunit, including the peptidyltransferase center (PTC) and the nascent peptide exit tunnel.

[0007] The design of tethered ribosomes, in which subunits are linked to one another, has the potential to open up new avenues, such as creating orthogonal translation systems, evolving ribosomes for the uptake of non-natural amino acids in synthetic biology, and molecularly characterizing dominant lethal mutations. Previously, we and others have disclosed tethered ribosomes, as well as methods for their preparation and use (see International Publication No. 2015 / 184283, "Tethered Ribosomes and Methods of Making and Using Thereof," and Orelle et al., "Protein synthesis by ribosomes with tethered subunits," Nature, 6 August 2015, Vol. 524, page 119). Here, we disclose further improvements to systems and methods for incorporating ribosomes with tethered subunits. [Overview of the project]

[0008] Disclosed herein are modified polynucleotides, modified ribosomes, and modified cells and systems. Modified polynucleotides, modified ribosomes, and modified cells and systems can be used in methods for preparing sequence-controlled polymers. In some embodiments, the modified ribosome comprises a small subunit, a large subunit, and a linking portion containing a polynucleotide sequence, the linking portion ligating the small subunit to the large subunit, and the modified ribosome can assist in the translation of the sequence-controlled polymer.

[0009] In certain embodiments, the small subunit of the modified ribosome comprises rRNA and protein, the large subunit of the modified ribosome comprises rRNA and protein, and the ligation region ligates the rRNA of the small subunit to the rRNA of the large subunit. In certain embodiments, the large subunit comprises a permutated variant of 23S rRNA. In certain embodiments, the small subunit comprises a permutated variant of 16S rRNA. Thus, in certain embodiments, the modified ribosome comprises a modified polynucleotide comprising (a) 16S rRNA, its permutated variant, or fragments thereof, and (b) fusions of 23S rRNA, its permutated variant, or fragments thereof.

[0010] The rRNA of the small subunit of the modified ribosome of this disclosure may contain an anti-Shine-Dalgano (anti-SD) sequence. In some embodiments, the anti-SD sequence of the rRNA of the small subunit of the modified ribosome corresponds to or is identical to the native anti-SD sequence of the modified host cell containing the modified ribosome. In such embodiments, the anti-SD sequence of the rRNA of the small subunit of the modified ribosome exhibits reverse complementarity with the Shine-Dalgano (SD) sequence of the native mRNA of the modified host cell. In some embodiments of the modified ribosome of this disclosure, the rRNA of the small subunit of the modified ribosome of this disclosure may be ligated to the rRNA of the large subunit (submit) via a ligation region containing a polynucleotide sequence, in which case the modified ribosome may be described as containing the native anti-SD sequence of the modified host cell containing the modified ribosome, which has a ligated large subunit and small subunit and exhibits reverse complementarity with the SD sequence of the native mRNA of the modified host cell. Therefore, modified ribosomes with tethered large and small subunits can assist in translation using the native mRNA of the modified host cell.

[0011] In other embodiments, the anti-SD sequence of the rRNA of the small subunit of the modified ribosome is modified to include a base substitution with respect to the anti-SD sequence of the native mRNA of the modified host cell containing the modified ribosome (or with respect to the anti-SD sequence of the first modified ribosome). In such embodiments, the modified host cell can be designed to contain modified mRNA having the modified anti-SD sequence, which exhibits reverse complementarity with the modified anti-SD sequence of the rRNA of the small subunit of the modified ribosome, enabling translation of the modified mRNA by the modified ribosome having the modified anti-SD sequence.

[0012] The modified ribosomes of this disclosure can be combined for use in modified host cells. In some embodiments, the combination of ribosomes of this disclosure may include a first modified ribosome and a second modified ribosome. The first modified ribosome may include i) a small subunit comprising ribosomal RNA (rRNA) and protein, ii) a large subunit comprising ribosomal RNA (rRNA) and protein, and iii) a ligation portion comprising a polynucleotide sequence and ligating the rRNA of the small subunit to the rRNA of the large subunit. In some embodiments, the rRNA of the small subunit of the first modified ribosome includes an anti-SD sequence corresponding to the SD sequence of the native mRNA of the modified host cell, which enables translation of the native mRNA of the modified host cell and preferably does not allow translation of mRNA having a modified SD sequence (i.e., a modified SD sequence having one or more nucleotide substitutions relative to the anti-SD sequence of the native ribosome of the modified host cell). The second modified ribosome may comprise i) a small subunit containing rRNA and protein, and ii) a large subunit containing rRNA and protein, wherein the second modified ribosome lacks a junction between the large and small subunits. In some embodiments, the rRNA of the small subunit of the second modified ribosome comprises a modified anti-SD sequence having one or more nucleotide substitutions relative to the anti-SD sequence of the native ribosome of the modified host cell (and / or relative to the anti-SD of the first modified ribosome).The modified anti-SD sequence preferentially enables the translation of mRNA templates having an SD sequence that is complementary or congeneral to the SD sequence of the native cell mRNA and / or an anti-SD sequence that is different from the anti-SD sequence of the first modified ribosome (i.e., enables the translation of mRNA having an SD sequence that is complementary to the anti-SD of the rRNA of the small subunit of the second ribosome that enables the translation of mRNA having an SD sequence by the second ribosome, and preferably does not allow the translation of the native mRNA of the modified host cell by the second modified ribosome), and / or in that case (and / or where), the second modified ribosome contains one or more functionally transformative mutations in the large subunit and / or small subunit relative to the native ribosome of the modified host cell (or relative to the first modified ribosome), and such functionally transformative mutations are not present in the anti-SD sequence.

[0013] In a particular embodiment in which the large and small subunits of a modified ribosome are ligated together by a ligation region, the ligation region covalently bonds the helix of the large subunit to the helix of the small subunit. In a particular embodiment in which the large subunit contains 23S rRNA (or a permutation variant of 23S rRNA) and the small subunit contains 16S rRNA (or a permutation variant of 16S rRNA), the ligation region covalently bonds helix 10, helix 38, helix 42, helix 54, helix 58, helix 63, helix 78, or helix 101 of the 23S rRNA (or a permutation variant of 23S rRNA) to the helix of the 16S rRNA (or a permutation variant of 16S rRNA). In a particular embodiment in which the large subunit contains 23S rRNA (or a permutation variant of 23S rRNA) and the small subunit contains 16S rRNA (or a permutation variant of 16S rRNA), the ligation portion covalently binds helix 11, helix 26, helix 33, or helix 44 of the 16S rRNA (or a permutation variant of 16S rRNA) to the helix of the 23S rRNA (or a permutation variant of 23S rRNA).

[0014] In certain embodiments, the large subunit comprises an L1 polynucleotide domain, an L2 polynucleotide domain, and a C polynucleotide domain, where the C domain and L2 domain follow the L1 domain from 5' to 3' in order. In certain embodiments, the polynucleotide essentially consisting of the L1 domain followed by the L2 domain from 5' to 3' is substantially identical to 23S rRNA (e.g., 23S rRNA from E. coli). In certain embodiments, the polynucleotide essentially consisting of the L1 domain followed by the L2 domain from 5' to 3' is at least 95% identical to 23S rRNA. In certain embodiments, the C domain contains a polynucleotide having a length in the range of 1 to 200 nucleotides. In certain embodiments, the C domain contains a GAGA polynucleotide.

[0015] In certain embodiments, the small subunit comprises an S1 polynucleotide domain and an S2 polynucleotide domain, where the S2 domain is sequentially followed by the S1 domain from 5' to 3'. In certain embodiments, the polynucleotide essentially consisting of the S1 domain followed by the S2 domain from 5' to 3' is substantially identical to 16S rRNA (e.g., E. coli 16S rRNA). In certain embodiments, the polynucleotide essentially consisting of the S1 domain followed by the S2 domain from 5' to 3' is at least 95% identical to 16S rRNA.

[0016] In certain embodiments, the linking portion includes a T1 polynucleotide domain and a T2 polynucleotide domain. In certain embodiments, the T1 domain is linked to the S1 domain and the L1 domain, where the T1 domain and the L1 domain follow the S1 domain sequentially from 5' to 3'. In certain embodiments, the T1 domain contains a polynucleotide having a length in the range of 5 to 200 nucleotides. In certain embodiments, the T1 domain contains a polynucleotide having a length in the range of 7 to 40 nucleotides. In certain embodiments, the T1 domain contains a polyadenine polynucleotide. In certain embodiments, the T1 domain contains a polyadenine polynucleotide having a length in the range of 7 to 12 adenine nucleotides. In certain embodiments, the T2 domain is linked to the S2 domain and the L2 domain, where the T2 domain and the S2 domain follow the L2 domain sequentially from 5' to 3'. In certain embodiments, the T2 domain contains a polynucleotide having a length in the range of 5 to 200 nucleotides. In certain embodiments, the T2 domain contains a polynucleotide having a length in the range of 7 to 20 nucleotides. In certain embodiments, the T2 domain contains a polyadenine polynucleotide. In certain embodiments, the T2 domain contains a polyadenine polynucleotide having a length of 7 to 12 adenine nucleotides.

[0017] In certain embodiments, the modified ribosome includes an S1 domain from 5' to 3' followed by a T1 domain, an L1 domain, a C domain, an L2 domain, a T2 domain, and an S2 domain.

[0018] In certain embodiments, the modified ribosomes of this disclosure include mutations compared to wild-type host cells (e.g., compared to wild-type E. coli). In certain embodiments, the mutations are transformative mutations. In certain embodiments, the transformative mutations are gain-of-function mutations. In certain embodiments, the gain-of-function mutations are located at the peptidyltransferase center of the large subunit of the modified ribosome. In certain embodiments, the gain-of-function mutations are located at the A-site of the peptidyltransferase center of the large subunit of the modified ribosome. In certain embodiments, the gain-of-function mutations are located at the exit tunnel of the large subunit of the modified ribosome. In certain embodiments, the modified ribosomes include antibiotic resistance mutations located in the large and / or small subunits of the modified ribosome.

[0019] Furthermore disclosed herein are polynucleotides, which encode the rRNA of a modified ribosome. In certain embodiments, the polynucleotide is a vector. In certain embodiments, the polynucleotide further comprises a gene to be expressed by the modified ribosome. In certain embodiments, the gene is a reporter gene. In certain embodiments, the reporter gene is a green fluorescent protein gene. In certain embodiments, the modified ribosome comprises a modified anti-SD sequence, and its gene comprises a complementary modified SD sequence corresponding to the anti-SD sequence of the modified ribosome. In certain embodiments, the gene comprises a codon, and the codon encodes a non-natural amino acid. In some embodiments, the ribosome comprising the modified anti-SD sequence is an untethered ribosome.

[0020] Furthermore, disclosed herein are methods for preparing modified ribosomes, wherein a polynucleotide encoding the rRNA of the modified ribosome is expressed in a modified host cell, such as E. coli. In certain embodiments, the method further includes preparing modified ribosomes in a host cell, expressing a selection marker, and selecting modified ribosomes to express the selection marker in the modified host cell. In some embodiments, the selected modified ribosomes will contain one or more mutations compared to the modified ribosomes expressed in the modified host cell (and / or compared to the native ribosomes of the modified host cell). In certain embodiments, the selection step includes a negative selection step, a positive selection step, and a selection step that includes both negative and positive steps.

[0021] Furthermore disclosed herein are modified cells. A modified cell is (i) a polynucleotide encoding the rRNA of a modified ribosome, (ii) a modified ribosome, or a host cell such as an E. coli cell containing both (i) and (ii). In some embodiments, the modified host cell comprises a first modified ribosome having a tethered large subunit and a small subunit, the small subunit comprising rRNA having an anti-SD sequence corresponding to the SD sequence of the native mRNA of the modified host cell. In some embodiments, the modified host cell further comprises a second modified ribosome having an untethered large subunit and a small subunit, the small subunit comprising rRNA having an anti-SD sequence modified with respect to the SD sequence of the native mRNA of the modified host cell, and enabling translation of mRNA having a modified SD sequence corresponding to the modified anti-SD sequence of the rRNA of the small subunit of the second ribosome.

[0022] In some embodiments, the modified cell includes a first protein translation mechanism and a second protein translation mechanism. The first protein translation mechanism may include a first modified ribosome, in which the first modified ribosome includes a ligation region that tethers the first and second subunits. The second translation mechanism may include a second modified ribosome, in which the second modified ribosome lacks a ligation region between the large and small subunits. In some embodiments, the second modified ribosome includes a modified anti-SD sequence relative to the anti-SD sequence of the native ribosome (and / or relative to the anti-SD sequence of the first modified ribosome) that is complementary to the SD sequence of the native mRNA, and / or functionally transformative mutations other than the anti-SD sequence relative to the native ribosome of the modified cell (and / or relative to the first modified ribosome).

[0023] Furthermore, disclosed herein are methods for preparing sequence-controlled polymers, comprising (a) preparing modified ribosomes or modified cells containing one or more modified ribosomes, and (b) preparing an mRNA or DNA template encoding a sequence-controlled polymer, and preparing a sequence-controlled polymer using one or more modified ribosomes, wherein the modified cells contain one or more modified ribosomes and the mRNA or DNA template encodes a sequence-controlled polymer. Sequence-controlled polymers can be prepared in vitro and / or in vivo.

[0024] In certain embodiments, the sequence-controlled polymer is prepared in vitro, and the method further comprises (c) preparing a ribosome-deficient cell extract or a purified translation system and preparing the sequence-controlled polymer using the ribosome-deficient cell extract or the purified translation system. In certain embodiments, the ribosome-deficient cell extract is a cell culture in the mid-to-late exponential growth phase or has an OD of at least about 2.0, 2.5, or 3.0 at harvesting. 600 It contains an S150 extract prepared from a culture containing [the specified compound].

[0025] In certain embodiments, sequence-controlled polymers are prepared in vivo. Sequence-controlled polymers can be prepared in modified cells comprising first and second translation systems comprising modified ribosomes, wherein the first translation system comprises tethered ribosomes having a wild-type anti-SD sequence (i.e., a native anti-SD sequence of the modified host cell's ribosome that is complementary to the SD sequence of the modified host cell's native mRNA), and wherein the second translation system comprises (a) modified anti-SD sequences (e.g., not complementary to the SD sequence of the host cell's native mRNA with respect to the native anti-SD sequence of the modified host cell's ribosome, or to the anti-SD of the tethered ribosome of the first translation system), and / or (b) non-tethered ribosomes having functionally transformative mutations other than anti-SD sequences, which are mutations with respect to the native ribosome of the modified host cell or to the tethered ribosome of the first translation system. In certain embodiments, the mRNA or DNA encoding the sequence-controlled polymer includes a modified SD sequence, and the non-tethered, modified ribosome of the second translation system includes a modified anti-SD sequence complementary to the modified SD sequence of the mRNA or DNA encoding the sequence-controlled polymer, enabling translation of the mRNA encoding the sequence-controlled polymer by the second translation system (and preferably by the first translation system).

[0026] In certain embodiments, the sequence-controlled polymer contains amino acids. In certain embodiments, the amino acids are native amino acids. In certain embodiments, the amino acids are unnatural or non-canonical amino acids, and the non-tethered, modified ribosome of the second translation system contains one or more mutations relative to the native ribosome (or relative to the tethered, modified ribosome of the first translation system), enabling the incorporation of the unnatural or non-canonical amino acids into the sequence-controlled polymer. [Brief explanation of the drawing]

[0027] [Figure 1]Figure 1 shows the OSYRIS setup. a) Structure of the rRNA gene and the dissociable 70S ribosome (left) and Ribo-T (right). The small and large subunits of Ribo-T are covalently joined by two RNA tethers that link a circularly-permutated 23S rRNA to a helix 44 loop of 16S rRNA13,16. b) In the first original orthogonal translation system based on Ribo-T13, wild-type dissociable ribosomes translate the cellular proteome, while orthogonal Ribo-T (oRibo-T) is involved in the translation of orthogonal reporter mRNA. c) In OSYRIS cells (Example 1), the proteome is synthesized by Ribo-T, while dissociable ribosomes function as a specialized orthogonal translation system. The tethered nature of Ribo-T restricts both dissociable ribosome subunits (the 30S and 50S subunits with modified ASDs) to translating only orthogonal mRNA.

[0028] [Figure 2]Figure 2 shows the performance of dissociable orthogonal ribosomes in OSYRIS cells. a) Agarose gel electrophoresis analysis of large rRNA species in OSYRIS cells compared with wild-type E. coli containing only dissociable 70S ribosomes (wild-type) and Ribo-T cells containing only tethered ribosomes (Ribo-T). b) Primer elongation analysis of ribosomes contained within OSYRIS cells. Top: Ribo-T and dissociable ribosomes can be distinguished by the A2058G mutation present in Ribo-T rRNA. Middle: Principle of primer elongation analysis. In the presence of ddCTP, reverse transcriptase elongates the primer by 4 nucleotides with a 23S rRNA template (containing A2058), but only by 3 nucleotides with a Ribo-T rRNA template (containing G2058). Bottom: Gel electrophoresis analysis of primer extension products purified with rRNA extracted from wild-type, Ribo-T, or OSYRIS cells. c) Expression of orthogonal GFP reporter in OSYRIS cells with dissociable ribosomes and orthogonal 30S subunits (oRbs) with wild-type 30S subunits (wt Rbs) or modified 16S rRNA ASD. Transcription of the reporter gene was induced by varying the concentration of the inducer homoserine lactone 19. Autofluorescence values ​​of cells lacking the reporter gene were subtracted from all values. The inset is an ultraviolet photograph of an agar plate with the labeled cells spotted and grown. d) Comparison of o-GFP reporter expression in OSYRIS cells (dark gray bars) and expression in BL21 cells transformed with o-pAM552 or poRibo-T expressing wild-type ribosomes (light gray bars) (see Figure 1 for expanded data). The moderate copy number plasmids used to introduce o-ribosomes or oRibo-T into BL21 cells were based on the pBR322 origin (322), while the low copy number plasmids expressing OSYRIS o-ribosomes were based on the pSC101 origin (101). Error bars indicate the standard deviation (sd) of n=3 repeats. *** indicates p<0.0005 by Student's t-test.

[0029] [Figure 3] Figure 3 shows the orthogonality of the small and large subunits of the dissociable o-ribosome in OSYRIS cells. a) The sensitivity of orthogonal GFP reporter expression in OSYRIS cells to erythromycin (left, dark gray bars) indicates that its translation is primarily carried out by dissociable o-ribosomes, and not by EryR Ribo-T or the Ribo-T / 30S hybrid (right illustration (cartoon)). Consistently, translation of the wild-type GFP gene driven by EryR Ribo-T is not inhibited by the antibiotic (light gray bars). Error bars indicate the standard deviation of n=3 repeats. b) Top: OSYRIS cells transformed with the poRbs plasmid containing the lethal mutation A2602U in the 23S rRNA gene were able to colonize, demonstrating that the large subunit of the o-ribosome is not involved in the translation of the cellular proteome. The dominant lethality of the A2602U mutation in non-orthogonal translation systems is demonstrated by the absence of colonies when OSYRIS cells are transformed with the same plasmid (pRbs) containing the unmodified (wild-type) ASD (see also Figures 12b and c). Below: Primer extension analysis showing that OSYRIS cells stably maintain macroribosomal subunits with the 23S rRNA mutation, which can be dominantly lethal in wild-type E. coli cells. cDNA bands are shown, resulting from extension of primers annealed proximal to the relevant mutation site of the mutant 23S rRNA (up arrow) or the unmutated Ribo-T rRNA (down arrow). Primer elongation analysis around the rRNA residue at position 2058 further confirmed the coexistence of Ribo-T (containing G2058) and dissociable ribosomes with a lethal 23S rRNA mutation (but with wild-type adenine at position 2058) (Figure 12d). Right: Illustration illustrating the conclusions from these experiments, which support the finding that the dissociable 50S subunit is largely separated from the translation of the cellular proteome, whose expression is Ribo-T dependent.

[0030] [Figure 4]Figure 4 shows the selection of gain-of-function mutations from a PTC mutant library in OSYRIS cells. a) Adding a TnaC-encoding sequence to the terminus of GFP is expected to reduce reporter expression due to the inhibitory effect of TnaC on translation termination when translation occurs at high concentrations of L-tryptophan28. The presence of the W12R mutation in TnaC is known to partially mitigate the termination problem28 and should lead to higher levels of reporter expression. b) Expression of the GFP-TnaC fusion in OSYRIS cells is suppressed by 94% in the presence of the L-tryptophan analog 1-methyltryptophan (1m-Trp), while expression of the GFP-TnaC(W12R) mutant is reduced by only 48%. Error bars indicate the standard deviation of n=3 repeats. ** indicates p<0.005 by Student's t-test. c) Cross-cut of a 50S ribosomal subunit, showing the location of mutated 23S rRNA nucleotides (arrows) in the PTC mutant library. P-site and A-site tRNAs are shown. d) Mutant 23S rRNA residues in the PTC library. Left and center: In the PTC library, most mutated 23S rRNA residues were found within a 10 Å radius (inner shell) of the PTC active site, and most residues within a 25 Å radius (second shell). The aminoacylated acceptor ends of the P-site and A-site tRNAs are shown in pink and green, respectively. Right: Location of mutated nucleotides shown in the secondary structure of the central loop of the 23S rRNA domain V. The associated 23S rRNA hairpins are shown. e) Staling bypass score of translational activity of PTC library mutants expressing an orthogonal GFP-TnaC reporter in OSYRIS cells. Dots corresponding to mutants that show efficient termination of the TnaC peptide while maintaining high translation efficiency (>60% of wild-type controls) (increased bypass score) are enclosed in squares and are darker. The dotted line indicates the background level of expression of the orthogonal GFP-TnaC(W12R) mutant in Ribo-T cells lacking orthogonal ribosomes.Black dots (arrows) indicate reporter translation by o-ribosomes containing wild-type 23S rRNA. f) Isolated ribosomes with specific gain-of-function mutations identified in OSYRIS cells were tested in a cell-free translation system. In in vitro tests, 50S subunits of ribosomes with lethal mutations (U2500G, A2060C, A2450U) were isolated from OSYRIS cells and conjugated with wild-type 30S subunits. Ribosomes with non-lethal mutations were isolated from SQ171 cells. Towprinting assays (Figure 16c) were used to assess the degree of translational arrest at the tnaC gene stop codon due to insufficient TnaC release. Error bars represent the standard deviation from three independent experiments. The statistical significance of the difference from the wild-type value is determined by Student's t-test and is indicated by * (p < 0.05), ** (p < 0.005), or *** (p < 0.0005). g) Arrangement of the 23S rRNA residue (blue) relative to TnaC-tRNA (green) and RF2 (orange) in the structure 30 of the TnaC stalled ribosome, where the mutation results in gain-of-function (dark dot in panel E).

[0031] [Figure 5]Figure 5 shows key plasmids of OSYRI. a) Map of the pRibo-Tt plasmid. The pRibo-T gene, encoding 16S-23S rRNA hybrid and 5S rRNA, is expressed under the control of the lambda-PL promoter. In the 16S-23S rRNA hybrid, a circular permutation 23S rRNA opened in the loop at helix 101 is inserted into the loop at helix 44 of the 16S rRNA by two RNA tethers whose sequences were redesigned in Ribo-T v.2.0 compared to the original Ribo-T version3,4. The 23S rRNA segment has the A2058G mutation, which gives Ribo-T erythromycin resistance. A cluster of 10tRNA genes, which are not found in host cells due to a deletion in the chromosomal rRNA operon, is under the control of the Ptac promoter. This plasmid contains the pBR322 origin of replication and the ampicillin resistance gene. b) The poRBS plasmid derived from pAM5524 has an E. coli rrnB operon in which the ASD sequence GUGGUU is modified in the 16S rRNA gene.3 This plasmid has the pSC101 origin of replication and the kanamycin resistance gene. The control plasmid pRbs (not shown) is identical to poRbs except that the 16S rRNA gene contains wild-type ASD. c) The reporter plasmid poGFP has either the gene for the superfolder green fluorescent protein (sf-GFP) (poGFP) or the same gene plus a gene for red fluorescent protein (poRFP / oGFP). Prior to the reporter coding sequence is a modified (orthogonal) SD sequence, AACCAC3, which is complementary to the ASD sequence of the 16S rRNA encoded by the poRBS plasmid shown in panel b. The transcription of the orthogonal GFP gene in poGFP is regulated by an inducible PLux promoter, which is regulated by the binding of N-(β-ketocaproyl)-L-homoserine lactone (HSL) to the LuxR repressor. Two copies of the luxR gene are present in the plasmid. The TpoGFP plasmid has a pA15 origin of replication and an Spc resistance gene. d) The reporter plasmid poRFP / oGFP contains genes for green (sfGFP) and red (RFP) fluorescent proteins, respectively, under the control of the Plpp5 and PT5 promoters.Both genes are preceded by the orthogonal SD sequence AACCAC. This plasmid contains the pA15 origin of replication and the Spc resistance gene. e) The poLuc plasmid is similar to the poGFP plasmid (panel c), but the sf-gfp gene is replaced by the luc gene, which encodes firefly luciferase. The luc gene is preceded by the orthogonal SD sequence AACCAC. The complete annotated sequences of the plasmids shown in this figure can be found in Appendix I.

[0032] [Figure 6] Figure 6 shows the assembly of OSYRIS in E. coli cells. a. Plasmid composition of OSYRIS cells. Ribo-T, which translates the cellular proteome, is expressed from the pRibo-Tt plasmid. mRNA transcribed from the orthogonal reporter gene of the poGFP (or poRFP / oGFP) plasmid is translated by the o-ribosome, whose rRNA is encoded by the poRbs plasmid. b. A series of steps for constructing OSYRIS cells. After assembly step III, the cell genome was fully sequenced (see panel c). In the next two steps, the cells were subsequently transformed with the reporter plasmid (poGFP in the illustrated example) and then with poRbs (or plasmids from the PTC mutant library described in Figure 4). Antibiotic resistance of the cells resulting from each step is shown. c. Genome of OSYRIS cells. The starting SQ171 FG strain was derived from Escherichia coli MG1655 cells.26 Five spontaneous mutations (arrows) were obtained during the assembly and proliferation of OSYRIS cells, and the exact location of the mutations and the function of the affected genes are shown in the table in Figure 20. The numbers outside the circles indicate the nucleotide numbers of the genome.d. Gel electrophoresis analysis of intracellularly contained plasmids at different steps of OSYRIS assembly (shown in panel b). Plasmid preparations were digested with a mixture of restriction enzymes KpnI, BamHI, and HindIII. Restriction enzyme digestion of individual plasmids is shown for reference.

[0033] [Figure 7]Figure 7 shows that oRbs is stably expressed in OSYRIS cells. a. Agarose gel electrophoresis analysis of total RNA maintained in OSYRIS cells after dilution of overnight cultures. Two independent colonies of OSYRIS cells (A and B) containing the poGFP plasmid were cultured overnight and diluted 1:50 in two tubes in LB medium supplemented with 50 μg / ml ampicillin, 25 μg / ml kanamycin, and 15 μg / ml spectinomycin. Total RNA was isolated after the indicated time intervals. Two technical repeats from each culture were processed independently and run on separate lanes of the gel. b. Time-course primer elongation analysis of the expression (representation) of oRbs (with wild-type A2058) against Ribo-T (with A2058G mutation) in OSYRIS cells. The principle of primer elongation analysis is illustrated on the sequencing gel. Total RNA prepared from OSYRIS cells (see panel a) was used as a template for primer elongation. RNA samples prepared from wild-type E. coli cells ("A2058") and cells expressing only Ribo-T ("A2058G") were used as controls. Lanes marked "Pr" contain DNA primers labeled with [32P]. The relative expression of the two ribosome species was evaluated by quantifying the relative intensities of Ribo-T and oRbs-specific bands.

[0034] [Figure 8]Figure 8 shows the efficient translation of orthogonal reporters in OSYRIS cells. a. Growth curve of OSYRIS cells containing o-ribosomes (solid line) or wild-type ribosomes (dashed line) (top) and expression of orthogonal GFP reporter within them (right). b. Growth curve of OSYRIS cells expressing o-ribosomes (solid line) or wild-type ribosomes (dashed line) (left) and expression of orthogonal GFP (center) and RFP reporter (right). The highest fluorescence reading (relative fluorescence units, RLU) in each experiment was set to 100%. c. Expression of orthogonal luciferase reporter in OSYRIS cells containing either o-ribosomes (dark gray bars) or wild-type ribosomes (light gray bars). The highest fluorescence reading (relative fluorescence units, RLU) was set to 100%. Error bars indicate the standard deviation of n=3 replicates, *** indicates p<0.0005, and ns indicates no statistical significance by Student's t-test.

[0035] [Figure 9] Figure 9 shows the expression of orthogonal GFP reporters in OSYRIS cells and E. coli BL21. (a) Growth curves, (b) GFP fluorescence, and (c) GFP fluorescence normalized by cell density of OSYRIS cells and BL21 cells cultured in 96-well plates. Both cell types express either wild-type ribosomes (dashed line) or o-ribosomes (solid line). Note that normalized orthogonal GFP fluorescence (or oGFP fluorescence per cell) is higher in OSYRIS cells than in BL21 cells. The data represent the results of three independent biological replicates, and error bars indicate the standard deviation. In (b), the highest fluorescence reading (relative fluorescence units) (for BL21 cells) is set to 100%. In (c), the highest normalized fluorescence reading (relative fluorescence units relative to A600) (for OSYRIS cells) is set to 100%.

[0036] [Figure 10]Figure 10 shows that oRbs is superior to oRibo-T in the expression of the orthogonal luciferase reporter. Expression of o-luc in BL21 or OSYRIS cells advanced by dissociable oRbs or oRibo-T. BL21 cells carrying the reporter plasmid poLuc were transformed with a moderate copy number (pBR322 ori) plasmid o-pAM552 or poRibo-T, expressing either oRbs or oRibo-T, respectively. OSYRIS cells express oRbs from the low copy number plasmid poRbs. Control cells were transformed with the same plasmid except that they contained rRNA with wild-type ASD. Relative reporter expression was recorded as described in the experimental procedure. Error bars indicate the standard deviation of n=3 repeats. *** indicates p<0.0005, and ns indicates no statistical significance by Student's t-test.

[0037] [Figure 11]Figure 11 shows that resistance to erythromycin (Ery) in OSYRIS cells is due to the functional separation of orthogonal, dissociable ribosomes. a: Ribosome composition of OSYRIS cells expressing wild-type (top two cells and left side of the bottom cell) or orthogonal (right side of the bottom cell) ribosomes. The tethered ribosomes have the A2058G mutation, which confers resistance to Ery, but the dissociable ribosomes are sensitive to Ery. b: Optical density of OSYRIS cell cultures expressing wild-type or orthogonal ribosomes after 24 hours of growth in a 96-well plate in the presence of the indicated concentrations of Ery. It has been shown that expressing wild-type dissociable EryS ribosomes along with EryR Ribo-T makes cells sensitive to erythromycin (Ribo-T = + wt Rbs, the third bar on each X axis), and that protein synthesis and cell proliferation are inhibited if the free 50S subunit is involved in translation (in this case, due to its interaction with the wild-type 30S subunit). In contrast, cells expressing oRbs remain EryR, and it has been shown that the 50S subunit of the orthogonal dissociable ribosomes in OSYRIS cells is functionally separated and not involved in the translation of the cellular proteome. In Figure 11b, the first bar of each X-axis data value is Ribo-T only, the second bar is Ribo-T + wild-type Rbs, and the third bar is Ribo-T + oRbs.

[0038] [Figure 12]Figure 12 shows that the viability of OSYRIS cells expressing a lethal mutation in the rRNA of the 50S subunit of orthogonal ribosomes exhibits functional isolation of the two orthogonal translation systems. a. Positions of 23S rRNA nucleotides G2553, A2602, and A2451 (orange) in the PTC active site (PDB 1VY4)22. Mutations in these nucleotides are dominantly lethal in wild-type E. coli cells27. A-site tRNA is green and P-site tRNA is blue. b. Transformation of OSYRIS cells produces viable colonies when the mutant 23S rRNA with the lethal mutation is co-expressed with orthogonal 16S rRNA (poRbs), but not when co-expressed with wild-type 16S rRNA (pRbs). c. Transformation of POP2136 strain 2, in which the expression of rRNAs operons from pRbs and poRbs plasmids is suppressed, does not produce colonies. d. (Top) Ribosome composition of OSYRIS cells expressing Ribo-T with the A2058G mutation and dissociable orthogonal ribosomes with a lethal mutation in the 50S subunit (right). (Bottom) Primer extension analysis of the rRNA region proximal to nucleotide 2058, which shows stable maintenance in OSYRIS cells of the 50S subunit with the lethal mutation (without the A2058G mutation) together with Ribo-T (with the A2058G mutation). Lanes 1-3: Control primer extension in the preparation of wild-type 23S rRNA (lane 1), 23S rRNA with the A2058G mutation (lane 2), or RNA extracted from OSYRIS cells expressing only Ribo-T (lane 3). Lane 4: rRNA derived from OSYRIS cells transformed with pRbs and expressing wild-type dissociable ribosomes. Lanes 5-8: rRNA derived from cells expressing orthogonal ribosomes with no mutation in 23S rRNA (lane 4) or with the lethal mutation indicated in 23S rRNA. The numbers below the lanes on the gel indicate the estimated 23S rRNA content (%), calculated as the ratio of the sum of the intensities of the cDNA band representing 23S rRNA (the two lower arrows) and the 23S rRNA and Ribo-T specific bands (the two lower arrows and the upper arrow, respectively). Representative gels from three independent biological repeats are shown.

[0039] [Figure 13] Figure 13 shows TnaC-mediated inhibition of in vitro translation of the reporter protein. Translation of GFP-TnaC or GFP-TnaC(W12R) reporters was performed in the presence of low (50 μM) or high (5 mM) concentrations of L-tryptophan in a PURExpress cell-free system. The TnaC mutant W12R is known to reduce the inhibition of TnaC-mediated protein release at the stop codon when L-tryptophan concentrations are high.28 The data represent the results of three independent experiments, and the error bars indicate experimental error. The DNA template sequences can be found in Appendix I. The highest fluorescence reading (relative fluorescence units) in each experiment was set to 100%.

[0040] [Figure 14] Figure 14 shows the translational activity of PTC library mutants in OSYRIS cells. The translational activity of individual mutants was estimated by comparing the expression of the o-GFP-TnaC(W12R) reporter (showing partial stall relief) (see Figures 4a-b) in OSYRIS cells with mutant o-ribosomes with the expression of the same reporter (100%) in OSYRIS cells with o-ribosomes containing wild-type 23S rRNA. Each wild-type 23S rRNA residue is shown, indicating the identity of the evaluated mutant. High translational activity was defined as reporter expression of 60% or higher. Gain-of-function mutants with both high bypass scores and high translational activity are shown with darker bars (see Figures 4e and 13). The data represent the results of two independent biological replications, and error bars indicate experimental error. Numerical data can be seen in Figure 17. The normalized fluorescence reading (relative fluorescence units on A600) of OSYRIS cells containing o-ribosomes with wild-type 23S rRNA was set to 100%.

[0041] [Figure 15]Figure 15 shows the termination stalling bypass score for individual PTC mutants. The TnaC stalling bypass score was calculated as the ratio of GFP fluorescence (normalized by cell density) in OSYRIS cells expressing GFP-TnaC to fluorescence in cells with the GFP-TnaC(W12R) reporter. The bypass score for cells with o-ribosomes containing wild-type 23S rRNA is 0.17. High threshold bypass scores (≥0.3, red dashed line) are those resulting from U2609C mutations 29 and 30, which have been reported to reduce translation arrest at the tnaC stop codon. Each wild-type 23S rRNA residue is shown, and the identity of the evaluated mutant is indicated. Gain-of-function mutants exhibiting both high bypass scores and high translational activity (see Figures 4e and 14) are shown in blue. The data represent the results of two independent biological replicates, and error bars indicate experimental error. Student's t-tests comparing the values ​​of each mutant to wild-type ribosomes showed that ns indicated no statistically significant difference, * indicated p<0.05, ** indicated p<0.005, and ** indicated p<0.0005. Numerical data can be seen in Figure 17.

[0042] [Figure 16]Figure 16 shows the testing of gain-of-function mutants in a cell-free translation system. a. Sucrose gradient fractionation of ribosome material from OSYRIS cells under subunit dissociation conditions. 30S and 50S subunits (arrows) prepared from dissociated wild-type ribosomes were used as markers. The gray shading indicates the 50S subunit fraction collected and used in the cell-free translation experiment. b. Analysis of the purity of 50S material (isolated as described in A) by agarose gel electrophoresis of rRNA. Wild-type 16S and 23S and purified Ribo-T rRNA were used as migration markers. c. Principle (top) and results (bottom) of the in vitro towprinting experiment. The tnaC template was translated using ribosomes assembled from isolated mutant 50S subunits and wild-type 30S subunits. Translation reactions were performed for each mutant under three different conditions. (I) In the presence of L-PSA21, a prolyl-tRNA synthetase inhibitor that stalls ribosomes with the tnaC Pro24 codon at the A site. The intensity of the corresponding toe-printing band (indicated by a white (open) arrowhead) reflects the translational activity of the mutant ribosome. (H) In the presence of high concentrations of L-tryptophan (5 mM) leading to translation arrest at the stop codon (indicated by a green arrowhead) if the rRNA mutation does not alleviate the stalling. (L) When no stalling at the stop codon was detected, or limited stalling was detected, with low concentrations of L-tryptophan (5 μM). The termination stalling efficiency (Figure 4f) was calculated as the ratio of the intensity of the stop codon toe-printing band in sample H to the intensity of the Pro24 codon band in sample I.

[0043] [Figure 17] Figure 17 provides a table showing the translational activity and termination / stalling bypass score of the PTC library variant described in Example 1.

[0044] [Figure 18] Figure 18 provides a table showing the genotypes of the E. coli strains used in Example 1.

[0045] [Figure 19]Figure 19 provides a table showing the primers used in Example 1.

[0046] [Figure 20] Figure 20 provides a table showing the genotypes of the OSYRIS cells used in Example 1.

[0047] [Figure 21] Figure 21 provides a table showing the primer and nucleotide combinations used in the primer extension analysis of Example 1.

[0048] [Figure 22] Figure 22 shows: A) Secondary structures of the large and small subunit rRNAs; B) Genes encoding the large and small subunit rRNAs.

[0049] [Figure 23] Figure 23 shows A) a tethered ribosome with a large subunit, a small subunit, and a junction. B) the gene encoding the tethered ribosome in Figure 23A.

[0050] [Figure 24] Figure 24 shows Per mutations in ribosomal rRNA.

[0051] [Figure 25] Figure 25 shows A) a plasmid containing a gene encoding rRNA, and B) a plasmid containing a gene encoding rRNA for tethered ribosomes. [Modes for carrying out the invention]

[0052] A tethered and therefore inseparable subunit ribosome ("Ribo-T") capable of successful protein synthesis is disclosed. Ribo-T can be prepared by genetic engineering a ribosome comprising a small subunit, a large subunit, and a ligation region that tethers the small subunit to the large subunit. The modified ribosome may contain a hybrid rRNA comprising a small subunit rRNA sequence, a large subunit rRNA sequence, and an RNA linker that can covalently link the small subunit rRNA sequence and the large subunit rRNA sequence into a single entity. The modified ribosome can be prepared by expressing a polynucleotide encoding the rRNA of the modified ribosome. The modified ribosome can also be evolved by positive or negative selection of mutations. Notably, Ribo-T is not only functional in vitro but can also assist cell proliferation even in the absence of wild-type ("wt") ribosomes. As a result, Ribo-T has a variety of applications. For example, Ribo-T can be used to prepare sequence-controlled polymers such as naturally derived proteins or non-naturally derived amino acid polymers, to create fully orthogonal ribosome-mRNA systems in vitro or in vivo, to investigate unknown functions of ribosomes, and to design ribosomes with novel functions.

[0053] Tethered ribosome

[0054] U.S. Patent Application Publication No. 2017 / 0073381, which discloses tethered ribosomes and methods for constructing and using tethered ribosomes, is referenced and incorporated herein by reference in its entirety. The modified ribosome comprises a small subunit, a large subunit, and a linking portion, the linking portion tethering the small subunit to the large subunit. The modified ribosome can assist in the translation of sequence-controlled polymers.

[0055] In contrast to naturally occurring ribosomes, modified ribosomes have inseparable large and small subunits. Figure 22 shows a portion of a wild-type ribosome with separable small and large subunits. Figure 22A shows the secondary structure of the large subunit rRNA101 and small subunit rRNA102 that together form a functional portion of the ribosome. Figure 22B shows the rRNA gene 200, which includes the operon encoding the large subunit rRNA202 and the operon encoding the small subunit rRNA201. In wild-type rRNA, the rRNAs of the large and small subunits are excised from the primary transcript and processed to mature the individual subunits.

[0056] One embodiment of a modified tethered ribosome is shown in Figure 23. Figure 23A shows the secondary structure of a portion of the rRNA of the modified ribosome 300. The modified ribosome includes a large subunit 301, a small subunit 302, and a ligation portion 303 that ligates the small subunit 302 to the large subunit 301. In this example, the ligation portion 303 ligates the rRNA of the small subunit 302 to the rRNA of the large subunit 301. The modified ribosome may include a connector 304 that closes the end of the native large subunit rRNA. Figure 23B shows an example of an rRNA gene 400 and the operon encoding to the modified ribosome 300.

[0057] Large subunit

[0058] The large subunit 301 contains subunits that can bind amino acids to form a polypeptide chain. The large subunit 301 may include a first large subunit domain ("L1 polynucleotide domain" or "L1 domain"), a second large subunit domain ("L2 polynucleotide domain" or "L2 domain"), and a connector domain ("C polynucleotide domain" or "C domain") 304, with the C domain and L2 domain following the L1 domain in order from 5' to 3'.

[0059] Figure 23B shows an example of an rRNA gene 400 encoding a modified ribosome 300 and provides an alternative representation for understanding the modified ribosome. The encoding polynucleotide 400 may contain different sequences encoding various domains of the modified ribosome 300. As shown in Figure 23B, the polynucleotide encoding the large subunit rRNA 301 includes a polynucleotide encoding the L1 domain 402, a polynucleotide encoding the C domain 406, and a polynucleotide encoding the L2 domain 403.

[0060] The large subunit rRNA301 can be a permutation substitution variant. In certain embodiments, the permutation substitution variant is a circular permutation substitution variant of the separable large subunit rRNA. The separable large subunit can be any functional large subunit. In certain embodiments, the separable large subunit can be 23S rRNA. In certain embodiments, the separable large subunit is wild-type large subunit rRNA. In specific embodiments, the separable large subunit is wild-type 23S rRNA.

[0061] If the large subunit 301 is a permutation substitution variant of the large subunit rRNA, then the polynucleotide consisting of the L1 domain from 5' to 3' essentially from the following L2 domain can be substantially identical to the large subunit rRNA. In certain embodiments, the polynucleotide consisting of the L1 domain from 5' to 3' essentially from the following L2 domain is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the large subunit rRNA.

[0062] In certain embodiments, where the large subunit 301 is a permutational variant of a separable large subunit rRNA, the large subunit 301 may further include a C domain 304 ligating the native 5' and 3' ends of the separable large subunit rRNA. The C domain may include polynucleotides having lengths ranging from 1 to 200 nucleotides. In certain embodiments, the C domain 304 may include polynucleotides having lengths ranging from 1 to 150 nucleotides, 1 to 100 nucleotides, 1 to 90 nucleotides, 1 to 80 nucleotides, 1 to 70 nucleotides, 1 to 60 nucleotides, 1 to 50 nucleotides, 1 to 40 nucleotides, 1 to 30 nucleotides, 1 to 20 nucleotides, 1 to 10 nucleotides, 1 to 9 nucleotides, 1 to 8 nucleotides, 1 to 7 nucleotides, 1 to 6 nucleotides, 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, or 1 to 2 nucleotides. In certain embodiments, the C domain may include a GAGA polynucleotide.

[0063] Small sub-unit

[0064] The small subunit 302 can bind to mRNA. The small subunit 302 comprises a first small subunit domain ("S1 polynucleotide domain" or "S1 domain") and a second small subunit domain ("S2 polynucleotide domain" or "S2 domain"), with the S2 domain following the S1 domain sequentially from 5' to 3'. Referring again to Figure 23B, the polynucleotide encoding the small subunit rRNA 302 includes a polynucleotide encoding the S1 domain 401 and a polynucleotide encoding the S2 domain 404.

[0065] The small subunit rRNA302 can be a permutational variant of the separable small subunit rRNA. In certain embodiments, the permutational variant is a circular permutational variant of the separable small subunit rRNA. The separable small subunit can be a small subunit. In certain embodiments, the separable small subunit can be 16S rRNA. In certain embodiments, the separable small subunit is wild-type small subunit rRNA. In specific embodiments, the separable small subunit is wild-type 23S rRNA.

[0066] If small subunit 302 is a permutational substitution variant of small subunit rRNA, the polynucleotide consisting essentially of the S2 domain followed by the S1 domain from 5' to 3' can be substantially identical to the small subunit rRNA. In certain embodiments, the polynucleotide consisting essentially of the S2 domain followed by the S1 domain from 5' to 3' is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the small subunit rRNA.

[0067] The small subunit may further contain a modified anti-Shine-Dalgarno sequence. The modified anti-Shine-Dalgarno sequence enables the translation of templates that have a complementary Shine-Dalgarno sequence different from that of endogenous cellular mRNA.

[0068] connecting part

[0069] Referring again to Figure 23B, the connecting portion 303 connects the small subunit 302 to the large subunit 301. In a particular embodiment, the connecting portion covalently connects the helix of the large subunit 301 to the helix of the small subunit 302.

[0070] The ligation portion may also include a first tether domain ("T1 polynucleotide domain" or "T1 domain") and a second tether domain ("T2 polynucleotide domain" or "T2 domain"). Referring again to Figure 23B, the polynucleotide encoding ligation portion 303 includes a polynucleotide encoding T1 domain 405 and a polynucleotide encoding T2 domain 407.

[0071] The T1 domain ligates its S1 and L1 domains, with the T1 and L1 domains following the S1 domain in order from 5' to 3'. The T1 domain may contain polynucleotides having lengths in the range of 5-200 nucleotides, 5-150 nucleotides, 5-100 nucleotides, 5-90 nucleotides, 5-80 nucleotides, 5-70 nucleotides, 5-60 nucleotides, 5-50 nucleotides, 5-40 nucleotides, 5-30 nucleotides, or 5-20 nucleotides, for example, polynucleotides having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In certain embodiments, T1 contains polyadenine. In certain embodiments, T1 contains polyuridine. In certain embodiments, T1 contains unstructured polynucleotides. In certain embodiments, T1 includes a nucleotide that forms a base pair with the T2 domain.

[0072] The T2 domain ligates its L2 and S2 domains, with the L2 domain followed by the T2 and S2 domains in order from 5' to 3'. The T2 domain may include polynucleotides having lengths in the range of 5-200 nucleotides, 5-150 nucleotides, 5-100 nucleotides, 5-90 nucleotides, 5-80 nucleotides, 5-70 nucleotides, 5-60 nucleotides, 5-50 nucleotides, 5-40 nucleotides, 5-30 nucleotides, or 5-20 nucleotides, for example, polynucleotides having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In certain embodiments, T1 contains polyadenine. In certain embodiments, T2 contains polyuridine. In certain embodiments, T2 contains unstructured polynucleotides. In certain embodiments, T2 contains nucleotides that form base pairs with the T1 domain.

[0073] In embodiments having T1 and T2 domains, the T1 and T2 domains may have the same number of polynucleotides. In other embodiments, the T1 and T2 domains may have different numbers of polynucleotides.

[0074] In certain embodiments, the modified ribosome may include an S1 domain that is followed in order from 5' to 3' by a T1 domain, an L1 domain, a C domain, an L2 domain, a T2 domain, and an S2 domain. In specific embodiments, the modified ribosome may essentially consist of an S1 domain that is followed in order from 5' to 3' by a T1 domain, an L1 domain, a C domain, an L2 domain, a T2 domain, and an S2 domain.

[0075] mutation

[0076] In certain embodiments, the modified ribosome may contain one or more mutations. In specific embodiments, the mutations are function-converting mutations. Function-converting mutations may be gain-of-function mutations or loss-of-function mutations. Gain-of-function mutations may be any mutation that confers a new function. Loss-of-function mutations may be any mutation that results in the loss of a function that the parent had.

[0077] In certain embodiments, the functional transformation mutation can be located at the peptidyltransferase center of the ribosome. In specific embodiments, the functional transformation mutation can be located at the A site of the peptidyltransferase center. In other embodiments, the functional transformation mutation can be located at the exit tunnel of the modified ribosome.

[0078] In certain embodiments, the functional transformation mutation may be an antibiotic resistance mutation. The antibiotic resistance mutation may be located in either the large or small subunit. In certain embodiments, the antibiotic resistance mutation can confer resistance to the modified ribosome to aminoglycosides, tetracyclines, patamycin, streptomycin, edein, or any other antibiotic that targets the small ribosomal subunit. In certain embodiments, the antibiotic resistance mutation can confer resistance to macrolides, chloramphenicol, lincosamides, oxazolidinones, pleuromutilin, streptogramin, or any other antibiotic that targets the large ribosomal subunit.

[0079] Design of tethered ribosomes

[0080] For a chimeric construct linking a large and small subunit to be successful, it must have a linker that is i) appropriately interacts with ribosomal proteins and biogenesis factors for functional ribosome assembly, ii) avoids ribonuclease degradation, and iii) is short enough to ensure cis-association of the subunits, but long enough to minimize subunit movement required for translation initiation, elongation, and peptide release. Considering the design constraints outlined above, the native ends of the large and small subunits are unsuitable. For example, in native prokaryotic ribosomes, the 5' and 3' ends of 16S and 23S rRNAs are too far apart (>170 Å) to be linked by a nuclease-resistant RNA linker. Consequently, alternative designs are needed to achieve functional modified ribosomes.

[0081] One method for designing tethered ribosomes is to create new 5' and 3' ends by permuting the large subunit. In certain embodiments, the native ends of the large subunit are proximal to each other, so circular permutation (CP) is employed. Circular permutation can be shown in the following scheme. [ka]

[0082] Therefore, in circular permutation mutations of polynucleotides, the sequence of the polynucleotide is maintained in each permutation, but each nucleotide is located at the end of the individual permutation. Permutation mutations are used to replace the ends of polynucleotides at different positions while maintaining the secondary structure of the polynucleotide.

[0083] The CP method was first developed in vitro by Polacek and his colleagues (coworkers) [Erlacher 2005], and subsequent pilot studies showed that three 23S rRNA circular permutation variants can be assembled into a functional subunit in vivo [Kitahara 2009]. The approach is shown in Figure 24. In Figure 24, the native large subunit ribosome 510 contains a second large subunit domain (L2 domain) 513 from 5' to 3', followed by the first large subunit domain (L1 domain). The native end of the large subunit ribosome 510 (a simplified representation of large subunit rRNA 101 shown in Figure 22A) is joined through a connector domain (C domain) 511, and a new end is created at 512. The circular permutation subunit produced by this method contains a first large subunit domain (L1 domain), followed by a connector domain (C domain) and a second large subunit domain (L2 domain) from 5' to 3'. Figure 24 also illustrates a portion of gene 500 encoding a new circular permutation large subunit containing a small subunit 501 and an L1 domain 502, followed by a C domain 506 and an L2 domain 503 from 5' to 3'.

[0084] Continuing with the approach outlined above, as shown in Figures 23A and 23B, the new ends of the small subunits need to be fabricated so that they can be connected to the new ends of the large subunits by a connecting portion.

[0085] Using the approach outlined above, a collection of circular permutation mutants with new ends can be generated. The new ends can be created at any position on the native subunit. While the results of new ends in some permutation mutants may not be viable, the methods disclosed herein allow for the generation and testing of collections of circular permutation mutants.

[0086] In some embodiments, the location of the new terminal of a small or large subunit can be selected based on the secondary structure of the subunit, its proximity to other subunits, the viability of the ribosome, or a combination thereof.

[0087] The location of the new end can be determined using the secondary structures of either or both of the large and small subunits. In certain embodiments, the new end is fabricated on the helix of the native subunit. In some specific embodiments, the new end is fabricated on the hairpin of the native subunit.

[0088] The location of the new end can be selected on either the large subunit, the small subunit, or both, based on its proximity to other subunits. In certain embodiments, the new end is located on the solvent side of the native subunit. In some other embodiments, the new end is located near the interface rim. In certain specific embodiments, the new end is located on the solvent side of the subunit and near the interface rim.

[0089] By utilizing the viability of ribosomes, it is possible to select a new terminal position for either the large subunit, the small subunit, or both. For example, a new terminal position can be selected by utilizing polynucleotide sequences or secondary structures present in either the large subunit, the small subunit, or both that are not highly conserved within a population.

[0090] In certain embodiments where the modified ribosome is a 23S construct, the ligation region can covalently bond to helix 10, helix 38, helix 42, helix 54, helix 58, helix 63, helix 78, or helix 101 of a permutation variant of 23S rRNA. In certain embodiments where the modified ribosome is a 16S rRNA construct, the ligation region can covalently bond to helix 11, helix 26, helix 33, or helix 44 of a permutation variant of 16S rRNA. In certain other embodiments where the modified ribosome is a 16S construct, the ligation region can covalently bond near the E-site of a permutation variant of 16S rRNA. In a specific embodiment where the modified ribosome is a 16S-23S construct, the ligation portion covalently connects helix 44 of the permutation substitution variant 16S rRNA with helix 101 of the permutation substitution variant 23S rRNA, the ligation portion covalently connects helix 26 of the permutation substitution variant 16S rRNA with helix 10 of the permutation substitution variant 23S rRNA, the ligation portion covalently connects helix 33 of the permutation substitution variant 16S rRNA with helix 38 of the permutation substitution variant 23S rRNA, the ligation portion covalently connects helix 11 of the permutation substitution variant 16S rRNA with helix 58 of the permutation substitution variant 23S rRNA, the ligation portion covalently connects helix 44 of the permutation substitution variant 16S rRNA with helix 58 of the permutation substitution variant 23S rRNA, and the ligation portion is the permutation substitution variant 16S The ligation site is covalently linked between helix 26 of rRNA and helix 54 of permutation substitution variant 23S rRNA, or between helix 11 of permutation substitution variant 16S rRNA and helix 63 of permutation substitution variant 23S rRNA, or between helix 44 of permutation substitution variant 16S rRNA and helix 63 of permutation substitution variant 23S rRNA.

[0091] As explained above, the ligation region must be short enough to prevent degradation and ensure the cis bond of the subunits, while being long enough to minimize the movement of the subunits required for translation initiation, elongation, and peptide release. As a result, the ligation region must span several tens of angstroms between the new ends of the large and short subunits.

[0092] Polynucleotides encoding tethered riposomes

[0093] Furthermore, polynucleotides encoding tethered ribosomes are also disclosed. These polynucleotides can be any polynucleotide that can be expressed to produce tethered ribosome rRNA. Figure 23B shows a polynucleotide for preparing tethered ribosome rRNA. Polynucleotide 400 includes, from 5' to 3', a sequence encoding rRNA for T1 linker 405, a sequence encoding rRNA for L1 domain 402, a sequence encoding rRNA for C domain 406, a sequence encoding rRNA for L2 domain 403, a sequence encoding rRNA for T2 linker 407, and a sequence encoding rRNA for S2 domain 404, followed in that order by a sequence encoding rRNA for S1 domain 401.

[0094] A polynucleotide encoding a tethered ribosome may further contain genes encoding other rRNA subunits or ribosomal proteins of the ribosome. For example, a polynucleotide encoding a modified ribosome containing a permutation-substituted 23S rRNA tethered to a permutation-substituted 16S rRNA may further contain a gene encoding a 5S rRNA.

[0095] In certain embodiments, polynucleotides are vectors that can introduce foreign genetic material into host cells. The vector can be a plasmid, a viral vector, a cosmid, or an artificial chromosome.

[0096] Figures 25A and 25B show examples of plasmids encoding prokaryotic ribosomes with separable subunits (Figure 25A) and polynucleotides encoding tethered ribosomes (Figure 25B). In Figure 25A, plasmid 600 includes a promoter 612, a gene 601 encoding a 16S subunit with representation of a processing stem indicated by a smaller rectangle, a tRNA gene 613, a gene 602 encoding a 23S subunit with representation of a processing stem indicated by a smaller rectangle, a gene 611 encoding a 5S subunit, a gene 614 encoding antibiotic resistance, and an origin of replication gene 615. In some embodiments, the 16S subunit 601 includes a modified antishine-Dalgano sequence. The modified antishine-Dalgano sequence can be located in either the small subunit domain, i.e., S1 or S2.

[0097] By elective selection, plasmids encoding prokaryotic ribosomes with separable subunits contain one or more additional genes. These additional genes (one or more) may include modified Shine-Dalgano sequences that are complementary to the modified anti-Shine-Dalgano sequences of the small subunit of the non-tethered ribosome.

[0098] In contrast to plasmids encoding ribosomes with separable subunits, plasmids encoding tethered ribosome 700 have chimeric genes encoding a large subunit, a small subunit, and ligation regions 701-707 that connect the large subunit to the small subunit. The plasmid contains genes for the expression of tethered ribosome 720. Optionally, the plasmid may further contain one or more additional genes 740.

[0099] The gene encoding the tether subunit contains, from 5' to 3', a sequence encoding the rRNA of the S1 domain, followed by a sequence encoding the rRNA of the T1 linker (705), the L1 domain (702), the C domain (706), the L2 domain (703), the T2 linker (707), and the S2 domain (704). The processing sequences of the small subunit adjacent to the chimeric gene, indicated by the small rectangle, can be retained for proper maturation of the small subunit ends, while the processing sequence of the large subunit (716) can be moved to another location on the plasmid or removed entirely to prevent the large subunit from being cleaved from the hybrid.

[0100] In a particular embodiment, the plasmid encoding the tether subunit further comprises a gene 711 encoding the 5S subunit, a gene 714 encoding antibiotic resistance, and a replication origin gene 715.

[0101] Optionally, the plasmid encoding the tether subunit may contain a modified anti-shine-Dalgano sequence 708(round). Although the modified anti-SD sequence is shown in Figure 25B to be located within the sequence encoding the S2 domain, the modified anti-shine-Dalgano sequence may be located in either of the small subunit domains, i.e., S1 or S2. In some embodiments, the plasmid containing the tether subunit also contains the wild-type anti-shine-Dalgano sequence.

[0102] Optionally, the plasmid encoding the tether subunit contains one or more additional genes 740. These additional genes may include modified Shine-Dalgano sequences complementary to the modified anti-Shine-Dalgano sequence of the tether ribosome. In certain embodiments, the additional gene may be a reporter gene. In specific embodiments, the reporter gene is a green fluorescent protein. In some embodiments, the additional gene contains a wild-type anti-Shine-Dalgano sequence.

[0103] Preparation of polynucleotides

[0104] Furthermore, methods for preparing polynucleotides are also disclosed herein. These methods include preparing a plasmid encoding a permutation-subunit rRNA construct, identifying viable permutation-subunit rRNA constructs, and preparing polynucleotides encoding a modified ribosome comprising a large subunit, a small subunit, and a ligation region connecting the small subunit to the large subunit.

[0105] The preparation of plasmids encoding permutation-substituted subunit rRNA constructs can be achieved by circular permutation, which involves ligating the native ends of subunits and preparing new ends (Figure 24). Plasmid preparation can include the steps of template preparation, plasmid backbone preparation, and assembly. The template preparation step can be achieved by plasmid digestion and ligation. For example, the CP23S template can be prepared from the pCP23S-EagI plasmid by EagI digestion and ligation. Each CP23S variant is generated by PCR using a unique primer pair with a circularized 23S rRNA gene as a template and a sequence that overlaps with the target plasmid backbone. The plasmid backbone preparation step can be achieved by restriction enzyme digestion of the plasmid to linearize the backbone with a subunit that processes the stem site. For example, the plasmid backbone is prepared by digesting pAM552-23S-AflII with the AflII restriction enzyme to linearize the backbone at the 23S processing stem site. In the assembly step, the template is incorporated into the plasmid backbone, and a plasmid encoding a permutation substitution subunit rRNA is prepared. The assembly step can be achieved by Gibson assembly.

[0106] To identify viable constructs of permutation subunit rRNA, plasmids encoding permutation subunit rRNA are introduced into host cell lines, and transformants are identified using a screening mechanism. Host cells containing the plasmid and the plasmid encoding the wild-type rRNA operon can be spotted onto an agar plate along with antibiotics. The selection mechanism includes identifying antibiotic-resistant transformants. For example, the plasmid is transformed into the Δ7 rrn SQ171 strain containing the pCSacB plasmid with the wild-type rRNA operon, and transformants resistant to ampicillin, erythromycin, and sucrose are selected. To confirm that the wild-type rRNA operon is completely replaced by the plasmid encoding the permutation subunit rRNA, a three-primer diagnostic PCR check can be performed on the whole plasmid extract.

[0107] Preparing a polynucleotide encoding a modified ribosome comprising a large subunit, a small subunit, and a ligation site that ligates the small subunit to the large subunit involves grafting a permutation-substituted subunit rRNA construct and the ligation site to the other subunits. In certain embodiments, the preparation step may also include preparing a plasmid containing a polynucleotide encoding a modified ribosome comprising a large subunit, a small subunit, and a ligation site that ligates the small subunit to the large subunit. In other embodiments, the preparation step may also include preparing a plasmid containing a polynucleotide encoding a modified ribosome comprising a large subunit, a small subunit, and a ligation site that ligates the small subunit to the large subunit, as well as a polynucleotide encoding an additional gene.

[0108] Preparation of tethered ribosomes

[0109] Further disclosed are methods for preparing tethered ribosomes. Tethered ribosomes can be prepared by expressing a polynucleotide encoding a modified ribosome. In certain embodiments, the preparation of tethered ribosomes further includes preparing a polynucleotide encoding a modified ribosome. In other embodiments, the preparation of tethered ribosomes further includes transforming cells with a polynucleotide encoding a modified ribosome. In some specific embodiments, the preparation of tethered ribosomes further includes preparing a polynucleotide and transforming cells with the polynucleotide.

[0110] Evolution of tethered ribosomes

[0111] Further disclosed are methods for evolving tethered ribosomes. These methods for tethered ribosome evolution include expressing a polynucleotide encoding a modified ribosome and selecting mutants. The selection steps may include a negative selection step, a positive selection step, or both negative and positive selection steps. The selected mutants may include tethered ribosomes having function-converting mutations. These function-converting mutations may be gain-of-function mutations or loss-of-function mutations.

[0112] Uses and applications of tethered ribosomes

[0113] Some uses and applications of tethered ribosomes are listed below.

[0114] artificial cells

[0115] Artificial cells are disclosed. The artificial cells may contain a polynucleotide encoding a modified ribosome, the modified ribosome comprising a small subunit, a large subunit, and a ligation region, the ligation region ligating the small subunit to the large subunit. Artificial cells containing a polynucleotide encoding a modified ribosome may be able to express the polynucleotide to prepare the modified ribosome. In other embodiments, the artificial cells include modified ribosomes. In some specific embodiments, the artificial cells include a polynucleotide encoding a modified ribosome and the modified ribosome itself.

[0116] The artificial cell may have one or more translation mechanisms. In the first embodiment, the artificial cell has one translation mechanism including a modified ribosome, the modified ribosome including a small subunit, a large subunit, and a ligator, the ligator ligating the small subunit to the large subunit.

[0117] In another embodiment, the artificial cell may have two translation mechanisms. The first translation mechanism may include a ribosome, the ribosome lacking a ligation region between the large and small subunits. The second translation mechanism may include a modified ribosome, the modified ribosome including a small subunit, a large subunit, and a ligation region, the ligation region tethering the small subunit to the large subunit. In some embodiments, the first or second translation mechanism is an orthogonal translation mechanism. In some embodiments, the first and second translation mechanisms are orthogonal translation mechanisms. The orthogonal translation mechanism can be prepared by modifying the anti-Shine-Dalgano sequence of the ribosome to enable translation of a template having a Shine-Dalgano sequence that is complementary to endogenous cellular mRNA and different from that of endogenous cellular mRNA.

[0118] In another embodiment, a cell comprising a first and a second mechanism for protein translation is disclosed. The first mechanism comprises a tethered ribosome having a wild-type anti-shine Dalgano sequence, and mRNA is translated by the ribosome according to the innate genetic code (i.e., the triplet code inherent in the cell). The second mechanism comprises an artificial mechanism derived from a non-tethered ribosome that functions to enable the expression of a heterologous gene. In some embodiments, the second mechanism comprises a ribosome having a modified anti-shine Dalgano sequence.

[0119] Preparation of sequence-defined polymers

[0120] Furthermore, methods for preparing sequence-controlled polymers are also provided. In certain embodiments, the method for preparing a sequence-controlled polymer includes preparing a modified ribosome and preparing an mRNA or DNA template encoding the sequence-controlled polymer. In some embodiments, the modified ribosome includes a small subunit, a large subunit, and a ligation region, the ligation region ligating the small subunit to the large subunit, and therein the modified ribosome includes a modified anti-Shine-Dalgano sequence. In some embodiments, the modified ribosome includes a small subunit, a large subunit, lacks a ligation region, and includes a modified Shine-Dalgano sequence. In one aspect of the method, any one of the steps includes adding at least one exogenous DNA template encoding the mRNA of the sequence-controlled polymer.

[0121] In one aspect of the method of the present invention, the sequence-controlled polymer is a natural biopolymer. In another aspect of the method of the present invention, the sequence-controlled polymer is a non-natural biopolymer. In certain embodiments, the sequence-controlled polymer comprises amino acids. In certain embodiments, the amino acids may be natural amino acids. As used herein, natural amino acids are proteinogenic amino acids directly encoded by codons of the universal genetic code. In certain embodiments, the amino acids may be non-natural amino acids. As used herein, non-natural amino acids are non-proteinogenic amino acids. Examples of non-natural amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcpβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, and p-amino-L-phenylalanine. Amino acids, isopropyl-L-phenylalanine, unnatural analogs of tyrosine amino acids; unnatural analogs of glutamine amino acids; unnatural analogs of phenylalanine amino acids; unnatural analogs of serine amino acids; unnatural analogs of threonine amino acids; unnatural analogs of methionine amino acids; unnatural analogs of leucine amino acids; unnatural analogs of isoleucine amino acids; alkyl, aryl, acyl, azide, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thio acid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino-substituted amino acids, or combinations thereof; photoactivatable crosslinked amino acids; spin-labeled amino acids; fluorescent amino acids;Examples include, but are not limited to, metal-linked amino acids; metal-containing amino acids; radioactive amino acids; photo-caged amino acids and / or photoisomerizable amino acids; biotin or biotin-analog-containing amino acids; keto-containing amino acids; amino acids containing polyethylene glycol or polyether; heavy atom-substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids with extended side chains; amino acids containing toxic groups; sugar-substituted amino acids; carbon-linked sugar-containing amino acids; redox-active amino acids; α-hydroxy acids; aminothioacids; α,α-disubstituted amino acids; β-amino acids; γ-amino acids; cyclic amino acids other than proline or histidine; and aromatic amino acids other than phenylalanine, tyrosine, or tryptophan. In certain embodiments, the sequence-defined polymer is a polypeptide or protein.

[0122] In one aspect of the present invention, the arrangement of the tether subunit includes a ligation region between 23S and 16S rRNA. In one aspect of this aspect, the ligation region covalently bonds helix 101 of 23S rRNA to helix 44 of 16S rRNA. In another aspect of this aspect, the ligation region includes a polynucleotide having a length ranging from 5 to 200 nucleotides. The ligated ribosome may further include a modified 16S rRNA having a modified anti-shine-Dalgano sequence to enable in vitro translation of a translation template having a complementary SD sequence different from that of endogenous cellular mRNA. Thus, selective in vitro translation of mRNA is possible, which efficiently generates sequence-directed biopolymers.

[0123] In one aspect of the present invention, the modified ribosome is not tethered and contains a modified anti-shine-dalgano (SD)16S sequence to enable in vitro or in vivo translation of a translation template having a complementary SD sequence different from that of endogenous cellular mRNA. Thus, selective translation of mRNA is possible, which efficiently generates sequence-defined biopolymers.

[0124] In one aspect of the present invention, the mRNA or DNA template encodes a modified Shine-Dalgano sequence. In a particular embodiment, the modified ribosome includes an anti-Shine-Dalgano sequence complementary to the Shine-Dalgano sequence encoded by the mRNA or DNA template.

[0125] In some embodiments, the mRNA or DNA template is supplied to a modified cell (e.g., a cell containing two different protein translation mechanisms), an extract from such a cell, or a purified translation system from such a cell.

[0126] Sequence-controlled polymers can be prepared in vitro. In some embodiments, the method for preparing sequence-controlled polymers in vitro further includes preparing a ribosome-deficient cell extract or a purified translation system. In certain embodiments, the ribosome-deficient cell extract includes an S150 extract prepared from cell cultures in the mid-to-late exponential growth phase or cultures having an OD600 of ~3.0 at harvesting. In one aspect of the present invention, the ribosome-deficient extract is prepared with one or more polyamines, such as spermine, spermidine, and putrescine, or a combination thereof. In one aspect of the present invention, the ribosome-deficient extract is prepared at a salt concentration of about 50 mM to about 300 mM.

[0127] Methods for preparing ribosome-deficient cell extracts and using them to aid in the in vitro translation of sequence-controlled polymers are disclosed in international application PCT / US14 / 35376, filed April 24, 2014, by Michael Jewett et al., the contents of which are incorporated herein by reference in their entirety.

[0128] In one aspect of this method, the mRNA encodes a modified Shine-Dalgano sequence different from the endogenous cellular mRNA present in ribosome-deficient cell extracts. In one aspect of this method, the modified ribosomes contain modified 16S rRNA having a modified anti-Shine-Dalgano sequence complementary to the modified Shine-Dalgano sequence, enabling in vitro translation of the mRNA and preparation of a sequence-directed biopolymer in vitro.

[0129] In one embodiment, the method is configured for a flow batch operation or a continuous operation. In another embodiment of the method, at least one substrate is replenished during the operation.

[0130] In one embodiment of this method, at least one step includes a DNA-dependent RNA polymerase. In one embodiment of this method, at least one high molecular weight crowding agent is included in one of the steps. In one embodiment of this method, at least one reducing agent (e.g., dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride, etc.) is included in one of the steps.

[0131] Sequence-controlled polymers can be prepared in vivo. Methods for preparing sequence-controlled polymers in vivo can be carried out using the artificial cells disclosed above. The artificial cells may have a translation mechanism including modified ribosomes, where the modified ribosomes include a small subunit, a large subunit, and a ligation region, the ligation region tethering the small subunit to the large subunit. In certain embodiments, the artificial cells have one translation mechanism. In other embodiments, the cells have two translation mechanisms. In some embodiments, the cells have two protein translation mechanisms, where the first protein translation mechanism includes ribosomes, where the ribosomes lack a ligation region between the large and small subunits, and the second protein translation mechanism includes ribosomes, where the ribosomes include a ligation region linking the large and small subunits. In some embodiments, the ribosomes of the first translation system include a modified anti-shine-dalgano sequence, and the ribosomes of the second translation system include a wild-type (unmodified) anti-shine-dalgano sequence.

[0132] term

[0133] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. It should be understood that all definitions defined and used herein take precedence over dictionary definitions, definitions in documents referenced by reference, and / or the ordinary meanings of the defined terms.

[0134] As used herein and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless otherwise indicated.

[0135] The range includes individual members. Therefore, for example, a group consisting of 1 to 3 members means a group that has 1, 2, or 3 members.

[0136] Furthermore, unless otherwise explicitly stated, it should be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which they are described.

[0137] The modal verb "may" signifies a preferred use or selection of one or more options or choices among several described embodiments or features contained herein. If no options or choices relating to a particular embodiment or feature are disclosed, the modal verb "may" signifies an affirmative act relating to how to construct or use an aspect of a described embodiment or feature, or a definitive decision relating to a specific skill relating to a described embodiment or feature. In this latter context, the modal verb "may" has the same meaning and connotation as the modal verb "can".

[0138] In both the claims and the specification, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and similar phrases are understood to be open-ended, meaning that “including” is not limited. Only the transitional phrases “consisting of” and “consisting essentially of” are considered closed or semi-closed transitional phrases, respectively, as provided in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures.

[0139] As used herein, the terms “nucleic acid” and “oligonucleotide” mean polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base. There is no intended length distinction between the terms “nucleic acid,” “oligonucleotide,” and “polynucleotide,” and these terms are to be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms encompass double-stranded and single-stranded DNA as well as double-stranded and single-stranded RNA. As used in the present invention, oligonucleotides may also include analogues with modified base, sugar, or phosphate skeletons, and non-purine or non-pyrimidine nucleotide analogues.

[0140] A "fragment" of a polynucleotide is a portion of a polynucleotide sequence that is identical in sequence to a reference sequence but shorter in length. A fragment may include the full-length reference sequence minus at least one nucleotide. For example, a fragment may contain 5 to 1000 consecutive nucleotides of the reference polynucleotide. In some embodiments, a fragment may contain at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 consecutive nucleotides of the reference polynucleotide. Fragments can be preferentially selected from a specific region of the molecule. The term "at least one fragment" encompasses the full-length polynucleotide. A "variant," "mutant," or "derivative" of a reference polynucleotide sequence may include a fragment of the reference polynucleotide sequence.

[0141] With respect to polynucleotide sequences, the identity percentage may be measured over the entire length of the defined polynucleotide sequence, for example, as defined by a particular sequence number, or over shorter lengths, for example, over fragments taken from a larger defined sequence, such as over the length of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 consecutive nucleotide fragments. Such lengths are merely illustrative, and it is understood that the lengths over which the identity percentage can be measured may be described using the lengths of any fragments supported by sequences shown in tables, figures, or sequence listings herein.

[0142] With respect to polynucleotide sequences, a “variant,” “mutant,” or “derivative” can be defined as a nucleic acid sequence that has at least 50% sequence identity with a particular nucleic acid sequence over a certain length, using the blastn and “BLAST 2 Sequences” tools available on the National Center for Biotechnology Information website (see Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences - a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). Such pairs of nucleic acids can exhibit sequence identity of, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more over a certain defined length.

[0143] "Recombinant nucleic acids" are sequences that do not exist in nature, or sequences created by artificially combining two or more normally separate sequence segments. This artificial combination is often achieved by chemical synthesis, or more generally by the artificial manipulation of isolated nucleic acid segments, for example, by genetic engineering techniques known in the art. The term recombinant includes nucleic acids modified only by the addition, substitution, or deletion of some nucleic acids. Often, recombinant nucleic acids may include nucleic acid sequences operably linked to a promoter sequence. Such recombinant nucleic acids may, for example, be part of a vector used to transform cells.

[0144] The nucleic acids disclosed herein may be “substantially isolated or purified.” The term “substantially isolated or purified” means nucleic acids that have been taken out of their natural environment and which contain at least 60%, preferably at least 75%, more preferably at least 90%, and even more preferably at least 95% of other components to which they are naturally bound.

[0145] Oligonucleotides can be prepared by any preferred method, including direct chemical synthesis by methods such as the phosphotriester method (Narang et al., 1979, Meth. Enzymol. 68:90-99), the phosphodiester method (Brown et al., 1979, Meth. Enzymol. 68:109-151), the diethylphosphoramidite method (Beaucage et al., 1981, Tetrahedron Letters 22:1859-1862), and the solid support method (U.S. Patent No. 4,458,066). These references are incorporated herein by reference. A review of methods for synthesizing oligonucleotides and modified nucleotide conjugates is provided in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187, which is incorporated herein by reference.

[0146] As used herein, the term "primer" refers to an oligonucleotide that can act as a starting point for DNA synthesis under suitable conditions. Such conditions include inducing the synthesis of a primer extension product complementary to the nucleic acid chain in a suitable buffer and temperature in the presence of four different nucleoside triphosphates and an extension agent (e.g., DNA polymerase or reverse transcriptase).

[0147] Preferably, the primer is single-stranded DNA. The appropriate length of the primer depends on the intended use of the primer, but is typically in the range of about 6 to about 225 nucleotides, including intermediate ranges such as 15 to 35 nucleotides, 18 to 75 nucleotides, and 25 to 150 nucleotides. Shorter primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template. The primer does not need to precisely reflect the sequence of the template nucleic acid, but it must be sufficiently complementary to the template to hybridize. The design of primers suitable for amplification of a given target sequence is well known in the art and is described in the literature cited herein.

[0148] Primers can incorporate additional features that enable detection or immobilization of the primer, but their fundamental property as a starting point for DNA synthesis remains unchanged. For example, a primer may contain additional nucleic acid sequences at its 5' end that do not hybridize to the target nucleic acid but facilitate the cloning or detection of the amplification product, or enable RNA transcription (e.g., by including a promoter) or protein translation (e.g., by including 5'-UTR elements such as an internal ribosome entry site (IRES) or poly(A)n sequences (n ranging from approximately 20 to approximately 200)). The region of the primer that is sufficiently complementary to and hybridizes with the template is referred to herein as the hybridizing region.

[0149] The term "promoter" refers to a cis-acting DNA sequence that initiates RNA transcription from a DNA template containing a cis-acting DNA sequence by guiding RNA polymerase and other trans-acting transcription factors.

[0150] As used herein, the terms “target,” “target sequence,” “target region,” and “target nucleic acid” are synonymous and mean a region or sequence of nucleic acid to be amplified, sequenced, or detected.

[0151] As used herein, the term “hybridization” means that two single-stranded nucleic acids form a double-stranded structure through complementary base pairing. Hybridization can occur between perfectly complementary nucleic acid strands or between “substantially complementary” nucleic acid strands containing small regions of mismatch. Conditions under which hybridization of perfectly complementary nucleic acid strands is strongly preferred are called “stringent hybridization conditions” or “sequence-specific hybridization conditions.” Stable double helix of substantially complementary sequences can be achieved under lower stringent hybridization conditions, and the acceptable degree of mismatch can be controlled by suitable adjustment of the hybridization conditions. Those skilled in nucleic acid technology can empirically determine double-strand stability by considering numerous variables, including, for example, the length and base pair composition of oligonucleotides, ionic strength, and the rate of mismatched base pairs, in accordance with the guidelines provided by the art (see, for example, Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3 / 4):227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353, which are incorporated herein by reference).

[0152] The term "amplification reaction" refers to a chemical reaction, including enzymatic reactions, that results in an increase in the copy number of a template nucleic acid sequence or in the transcription of a template nucleic acid. Examples of amplification reactions include reverse transcription, polymerase chain reaction (PCR) including real-time PCR (see U.S. Patent Nos. 4,683,195 and 4,683,202, and PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), and ligase chain reaction (LCR) (see Barany et al., U.S. Patent No. 5,494,810). Exemplary "amplification reaction conditions" or "amplification conditions" typically include either a two-step or three-step cycle. A two-step cycle has a high-temperature denaturation step followed by a hybridization / extension (or ligation) step. A three-step cycle includes a denaturation step followed by a hybridization step followed by an extension step.

[0153] As used herein, “polymerase” means an enzyme that catalyzes the polymerization of nucleotides. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase I, Escherichia coli DNA polymerase I, T7 DNA polymerase, and Thermus aquaticus (Taq) DNA polymerase. “RNA polymerase” catalyzes the polymerization of ribonucleotides. The aforementioned examples of DNA polymerases are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases also fall within the range of DNA polymerases. Reverse transcriptases, including viral polymerases encoded by retroviruses, are an example of RNA-dependent DNA polymerases. Known examples of RNA polymerases ("RNAP") include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, and Escherichia coli RNA polymerase. The RNA polymerases mentioned above are also known as DNA-dependent RNA polymerases. The polymerase activity of any of the above enzymes can be measured by means well known in the art.

[0154] As used herein, the term “sequence-controlled polymer” means a polymer having a specific primary sequence. A sequence-controlled polymer may be equivalent to a genetically-encoded polymer if the gene codes for a polymer having a specific primary sequence.

[0155] As used herein, a primer is "specific" to a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer primarily hybridizes to the target nucleic acid. Typically, a primer is specific to a target sequence if the stability of the primer-target sequence double helix is ​​greater than the stability of the double helix formed between the primer and other sequences in the sample. Those skilled in the art will recognize that various factors, such as salt conditions, as well as the primer's base composition and mismatch location, affect the specificity of the primer, and that primer specificity often needs to be routinely confirmed experimentally. Hybridization conditions can be selected that allow the primer to form a stable double helix only with the target sequence. Therefore, by using target-specific primers under suitably stringent amplification conditions, selective amplification of target sequences containing target primer binding sites becomes possible.

[0156] As used herein, “expression template” means a nucleic acid that serves as a substrate for transcribing at least one RNA that can be translated into a polypeptide or protein. An expression template includes a nucleic acid composed of DNA or RNA. Suitable sources of DNA for use as nucleic acid for expression templates include genomic DNA, plasmid DNA, cDNA, and RNA that can be converted to cDNA. Genomic DNA, cDNA, and RNA may originate from any biological source, in particular, such as tissue samples, biopsies, swabs, sputum, blood samples, fecal samples, urine samples, or scraping specimens. Genomic DNA, cDNA, and RNA may be derived from host cells or viruses, and may originate from any species, including extant and extinct organisms. As used herein, “expression template” and “transcription template” have the same meaning and are interchangeable.

[0157] As used herein, “tethered,” “conjoined,” “linked,” “connected,” “coupled,” and “covalently bonded” have the same meaning as modifiers.

[0158] As used herein, "tethered ribosome" and "Ribo-T" are to be used interchangeably.

[0159] As used herein, the term “modified ribosome” means a modified ribosome. Exemplary modifications include, but are not limited to, tethering one or more subunits, altering or modifying subunits, and altering one or more rRNA sequences. Exemplary, non-limiting modifications include, one or more modifications such as the introduction of 16S rRNA, 23S rRNA, anti-shine-dalgano sequences, peptidyltransferase centers, nascent exit tunnels, ribosome coding centers, elongation factor interaction sites, tRNA binding sites, chaperone binding sites, nascent chain-modifying enzyme binding sites, GTPase centers, and antibiotic resistance sequences.

[0160] As used herein, the terms “wild-type,” “native,” or “endogenous” mean substances or conditions typically found in a given organism.

[0161] As used herein, the terms “mutant,” “exogenous,” “orthogonal,” and “non-nonnative” mean a substance or state not typically found in a given organism.

[0162] Where used herein, "CP" means circular permutation subunit. Where used herein, when CP is followed by "23S", it means circular permutation 23S rRNA. Where used herein, when CP is followed by a number, depending on the context, it may refer to the position of the new 5' end in the secondary structure (e.g., CP101 means the new 5' end is at helix 101 of the 23S rRNA) or to the position of the new 5' nucleotide (e.g., CP2861 means the new 5' nucleotide is nucleotide 2861 of the 23 rRNA).

[0163] As used herein, “translation template” means the RNA product of transcription from an expression template that can be used by ribosomes to synthesize polypeptides or proteins.

[0164] As used herein, “ribosome binding site” or “RBS” refers to a sequence of nucleotides upstream of the start codon of an mRNA transcript that plays a role in recruiting ribosomes during the initiation of protein translation. RBS may include the Shine-Dalgano sequence. The Shine-Dalgano (SD) sequence is the ribosome binding site of prokaryotic messenger RNA and is generally located about 8 nucleotides upstream of the start codon AUG. The SD sequence helps recruit ribosomes to messenger RNA (mRNA) and initiate protein synthesis by aligning them with the start codon. The 6-nucleotide consensus sequence is AGGAGG, and in E. coli it is AGGAGGU.

[0165] others

[0166] All methods described herein can be performed in any preferred order, unless otherwise specifically indicated herein or unless the context clearly contradicts it. The use of all examples or preferred (exemplary) language provided herein (e.g., “such as”) is intended solely to better illustrate the invention and, unless otherwise claimed, does not imply any limitation to the scope of the invention. Nothing in this specification should be construed as indicating that any unclaimed element is essential to the carrying out of the invention.

[0167] Preferred embodiments of the present invention, including the best embodiments known to the inventors of the present invention for carrying out the invention, are described herein. Variations of these preferred embodiments will be apparent to those skilled in the art by reading the foregoing description. The inventors of the present invention anticipate that those skilled in the art will appropriately adopt such variations, and they intend that the present invention will be carried out in ways other than those specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter described in the appended claims as permitted by applicable law. Furthermore, unless otherwise specifically indicated herein, or unless it is clearly inconsistent with the context, any combination of the above elements in all possible variations of those elements is encompassed by the present invention.

[0168] Exemplary Embodiments The following embodiments are illustrative and should be construed to limit the scope of the claimed subject matter.

[0169] Embodiment 1. A modified ribosome comprising a small subunit, a large subunit, and a connecting portion, wherein a. the connecting portion ties the small subunit to the large subunit, and b. the connecting portion can assist in the translation of a sequence-defined polymer.

[0170] Embodiment 2. A modified ribosome of Embodiment 1, wherein the small subunit contains rRNA and protein, the large subunit contains rRNA and protein, and the linking portion ligates the rRNA of the small subunit to the rRNA of the large subunit.

[0171] Embodiment 3. A modified ribosome of Embodiment 1 or 2, wherein the large subunit comprises a permutation substitution variant of 23S rRNA (e.g., a circular permutation substitution variant of 23 rRNA).

[0172] Embodiment 4. A modified ribosome of Embodiments 1-3, wherein the small subunit contains a permutation substitution variant of 16S rRNA (e.g., a circular permutation substitution variant of 23 rRNA).

[0173] Embodiment 5. A modified ribosome of any of Embodiments 1 to 4, wherein the small subunit includes a modified anti-Shine Dalgano sequence that enables translation of a template having a Shine Dalgano sequence that is different from and complementary to the endogenous cellular mRNA (for example, the modified anti-Shine Dalgano sequence of the small subunit is complementary to a Shine Dalgano sequence that is different from the endogenous cellular mRNA).

[0174] Embodiment 6. A modified ribosome of any of Embodiments 1 to 5, wherein the connecting portion covalently bonds the helix of the large subunit to the helix of the small subunit.

[0175] Embodiment 7. A modified ribosome of any of Embodiments 3 to 6, wherein the linking portion is covalently linked to a permutation substitution variant helix 10, helix 38, helix 42, helix 54, helix 58, helix 63, helix 78, or helix 101 of 23S rRNA.

[0176] Embodiment 8. A modified ribosome of any of Embodiments 4 to 7, wherein the ligation portion covalently binds helix 11, helix 26, helix 33, or helix 44 of a permutation substitution variant of 16S rRNA.

[0177] Embodiment 9. A modified ribosome of any of Embodiments 1 to 8, wherein the large subunit comprises or essentially consists of an L1 polynucleotide domain (e.g., a fragment of 23S rRNA), an L2 polynucleotide domain (e.g., a fragment of 23S rRNA), and a C polynucleotide domain, wherein the C domain and the L2 domain are sequentially followed behind the L1 domain from 5' to 3'.

[0178] Embodiment 10. A modified ribosome of Embodiment 9, wherein a polynucleotide comprising, or essentially derived therefrom, an L2 domain following an L1 domain from 5' to 3' is substantially identical to 23S rRNA or a fragment of 23S rRNA.

[0179] Embodiment 11. A modified ribosome of Embodiment 9 or 10, wherein a polynucleotide comprising, or essentially derived therefrom, an L2 domain following an L1 domain from 5' to 3' is at least 95% identical to 23S rRNA or a fragment of 23S rRNA (or at least 96%, 97%, 98%, or 99% identical to 23S rRNA or a fragment of 23S rRNA).

[0180] Embodiment 12. A modified ribosome of any of Embodiments 9 to 11, wherein the C domain comprises a polynucleotide having a length in the range of 1 to 200 nucleotides.

[0181] Embodiment 13. A modified ribosome of any of Embodiments 9 to 12, wherein the C domain contains a GAGA polynucleotide.

[0182] Embodiment 14. A modified ribosome of any of Embodiments 1 to 13, wherein the small subunit comprises or is essentially composed of an S1 polynucleotide domain (e.g., a fragment of 16S rRNA) and an S2 polynucleotide domain (e.g., a fragment of 16S rRNA), with the S2 domain sequentially following the S1 domain from 5' to 3'.

[0183] Embodiment 15. A modified ribosome of Embodiment 14 in which a polynucleotide comprising, or essentially derived therefrom, an S1 domain followed by an S2 domain from 5' to 3' is substantially identical to 16S rRNA (or a fragment of 16S rRNA).

[0184] Embodiment 16. A modified ribosome of Embodiment 14 or 15, wherein a polynucleotide comprising an S1 domain followed by an S2 domain from 5' to 3', or essentially derived therefrom, is at least 95% identical to 16S rRNA (or at least 96%, 97%, 98%, or 99% identical to 23S rRNA or a fragment of 23S rRNA).

[0185] Embodiment 17. A modified ribosome of any of Embodiments 1 to 16, wherein the linking portion includes a T1 polynucleotide domain and a T2 polynucleotide domain.

[0186] Embodiment 18. A modified ribosome of Embodiment 17, wherein the T1 domain ligates the S1 domain and the L1 domain, and thereafter, the T1 domain and the L1 domain are sequentially followed from 5' to 3' after the S1 domain.

[0187] Embodiment 19. A modified ribosome of Embodiment 17 or 18, wherein the T1 domain comprises a polynucleotide having a length in the range of 5 to 200 nucleotides.

[0188] Embodiment 20. A modified ribosome of Embodiment 19 in which the T1 domain comprises a polynucleotide having a length in the range of 7 to 20 nucleotides.

[0189] Embodiment 21. A modified ribosome of any of Embodiments 17-20, wherein the T1 domain contains a polyadenine polynucleotide.

[0190] Embodiment 22. A modified ribosome of any of Embodiments 17-20, wherein the T1 domain comprises a polyadenine polynucleotide having a length of 7-12 adenine nucleotides.

[0191] Embodiment 23. A modified ribosome of any of Embodiments 17 to 22, wherein the T2 domain ligates the S2 domain and the L2 domain, and thereafter, the T2 domain and the S2 domain are sequentially followed from 5' to 3' after the L2 domain.

[0192] Embodiment 24. A modified ribosome of any of Embodiments 17 to 24, wherein the T2 domain comprises a polynucleotide having a length in the range of 5 to 200 nucleotides.

[0193] Embodiment 25. A modified ribosome of Embodiments 17, 23, or 24, wherein the T2 domain comprises a polynucleotide having a length in the range of 7 to 20 nucleotides.

[0194] Embodiment 26. A modified ribosome of any of Embodiments 17-25, wherein the T2 domain contains a polyadenine polynucleotide.

[0195] Embodiment 27. A modified ribosome of any of Embodiments 17 to 26, wherein the T2 domain comprises a polyadenine polynucleotide having a length of 7 to 12 adenine nucleotides.

[0196] Embodiment 28. A modified ribosome of any of Embodiments 17 to 27, wherein the ribosome includes an S1 domain in which a T1 domain, L1 domain, C domain, L2 domain, T2 domain, and S2 domain are sequentially followed from 5' to 3'.

[0197] Embodiment 29. A modified ribosome of any of Embodiments 17 to 28, comprising a polynucleotide essentially consisting of an S1 domain followed by a T1 domain, an L1 domain, a C domain, an L2 domain, a T2 domain, and an S2 domain in sequence from 5' to 3'.

[0198] Embodiment 30. A modified ribosome from any of Embodiments 1 to 29, wherein the modified ribosome contains a mutation.

[0199] Embodiment 31. A modified ribosome of Embodiment 30 in which the mutation is a functional transformation mutation.

[0200] Embodiment 32. A modified ribosome of Embodiment 31 in which a functionally transformative mutation is located at the peptidyltransferase center.

[0201] Embodiment 33. A modified ribosome of Embodiment 31 in which the functionally transformative mutation is at the A site of the peptidyltransferase center.

[0202] Embodiment 34. A modified ribosome of Embodiment 31 in which a functionally transformative mutation is located in one or more of the exit tunnels, translocon interaction sites, or translation-facilitating accessory protein interaction sites of the modified ribosome.

[0203] Embodiment 35. A modified ribosome according to any of Embodiments 1 to 35, wherein the modified ribosome has an antibiotic resistance mutation.

[0204] Embodiment 36. A polynucleotide that encodes the rRNA of a modified ribosome according to any of Embodiments 1 to 35.

[0205] Embodiment 37. The polynucleotide of Embodiment 36, wherein the polynucleotide is a vector.

[0206] Embodiment 38. The polynucleotide of Embodiment 36 or 37, further comprising a gene expressed by a modified ribosome.

[0207] Embodiment 39. The polynucleotide of Embodiment 38, wherein the gene is a reporter gene.

[0208] Embodiment 40. The polynucleotide of Embodiment 39, wherein the reporter gene is the green fluorescent protein gene.

[0209] Embodiment 41. A polynucleotide of any of Embodiments 36 to 40, wherein the modified ribosome contains a modified anti-Shine-Dalgano sequence, and the gene contains a Shine-Dalgano sequence complementary to the modified ribosome.

[0210] Embodiment 42. A polynucleotide of any of Embodiments 36 to 41, wherein the gene contains a codon and the codon encodes a non-natural amino acid.

[0211] Embodiment 43. A method for preparing a modified ribosome, comprising expressing any of the polynucleotides of Embodiments 36 to 42.

[0212] Embodiment 44. The method of Embodiment 43, further comprising selecting a variant.

[0213] Embodiment 45. The method of Embodiment 44, wherein the selection step includes a negative selection step, a positive selection step, or both negative and positive selection steps.

[0214] Embodiment 46. Modified cells comprising (i) a polynucleotide of any of Embodiments 36 to 42, (ii) a modified ribosome of any of Embodiments 1 to 35, or both (i) and (ii).

[0215] Embodiment 47. A modified cell comprising a first protein translation mechanism and a second protein translation mechanism, wherein a. the first protein translation mechanism comprises a ribosome, wherein the ribosome lacks a junction between a large subunit and a small subunit, and b. the second protein translation mechanism comprises a modified ribosome according to any of Embodiments 1 to 35.

[0216] Embodiment 48. A method for preparing a sequence-controlled polymer, comprising (a) preparing a modified ribosome according to any of Embodiments 1 to 35, and (b) preparing an mRNA or DNA template encoding a sequence-controlled polymer.

[0217] Embodiment 49. The method of Embodiment 48 for preparing a sequence-defined polymer in vitro.

[0218] Embodiment 50. The method of Embodiment 49, further comprising preparing a ribosome-deficient cell extract or a purified translation system.

[0219] Embodiment 51. The method of Embodiment 50, comprising an S150 extract, in which the ribosome-deficient cell extract is used to prepare cell cultures in the mid-to-late exponential growth phase or cultures having an OD of 600-3.0 at the time of harvesting.

[0220] Embodiment 52. The method of Embodiment 48 for preparing a sequence-defined polymer in vivo.

[0221] Embodiment 53. The method of Embodiment 48 or 52, wherein the sequence-controlled polymer is prepared in the cells of either Embodiment 46 or 47.

[0222] Embodiment 54. Any method of Embodiments 48 to 53, wherein mRNA or DNA encodes a modified Shine-Dalgano sequence, and the modified ribosome includes an anti-Shine-Dalgano sequence complementary to the modified Shine-Dalgano sequence.

[0223] Embodiment 55. Any method of Embodiments 48 to 54, wherein the sequence-defined polymer contains amino acids.

[0224] Embodiment 56. The method of Embodiment 55, wherein the amino acid is a natural amino acid.

[0225] Embodiment 57. The method of Embodiment 55, wherein the amino acid is a non-natural amino acid.

[0226] Embodiment 58. A modified cell of Embodiment 47, wherein the ribosomes of the first protein translation system contain a modified anti-shine-dalgano sequence, and the ribosomes of the second protein translation system contain an unmodified (e.g., wild-type) anti-shine-dalgano sequence.

[0227] Embodiment 59. Any one of Embodiments 48 to 53 further comprising a non-tethered ribosome containing a modified anti-shine-dalgano sequence.

[0228] Embodiment 60. The method of Embodiment 59, wherein mRNA or DNA encodes a modified Shine-Dalgano sequence, and the untethered ribosome contains an anti-Shine-Dalgano sequence complementary to the modified Shine-Dalgano sequence.

[0229] Embodiment 61. The method of Embodiment 60, wherein the sequence-defined polymer comprises natural or non-natural amino acids.

[0230] Embodiment 62. A modified cell comprising two or more protein translation mechanisms, wherein (a) the first mechanism is a natural translation mechanism in which mRNA is translated by tethered or stapled ribosomes according to the natural genetic code, and (b) the second mechanism is an artificial mechanism derived from dissociable ribosomes that regulates the host metabolic load or in which orthogonal mRNA containing orthogonal codons is translated by these orthogonal ribosomes.

[0231] Embodiment 63. A modified cell comprising two or more protein translation mechanisms, wherein (a) the first mechanism is a natural translation mechanism in which mRNA that sustains the cell's life is translated by tethered or stapled ribosomes, and (b) the second mechanism is an artificial mechanism derived from dissociable ribosomes that perform an orthogonal function.

[0232] Embodiment 64. A modified cell comprising two or more protein translation mechanisms, wherein orthogonal dissociable ribosomes are superior to orthogonal tethered ribosomes in terms of protein expression.

[0233] Embodiment 65. Modified cells in which not only o-30S but also the free 50S subunit is manipulated to achieve a new function.

[0234] Embodiment 66. Modified cells in which not only o-30S but also the free 50S subunit is manipulated to achieve new functions without interfering with the expression of the cellular proteome.

[0235] Embodiment 67. Modified cells in which not only the o-30S but also the free 50S subunit is manipulated to achieve gain-of-function ribosome mutations.

[0236] Embodiment 68. Modified cells in which not only the o-30S but also the free 50S subunit is manipulated to realize gain-of-function ribosome mutations, wherein these mutations specifically overcome the translation of the problematic polymer sequence.

[0237] Embodiment 69. A modified cell comprising a first protein translation mechanism and a second protein translation mechanism, wherein the first protein translation mechanism comprises a first modified ribosome, the first modified ribosome comprising i) a small subunit comprising ribosomal RNA (rRNA) and protein, ii) a large subunit comprising ribosomal RNA (rRNA) and protein, and iii) a ligation portion, the ligation portion comprising a polynucleotide sequence and ligating the rRNA of the small subunit to the rRNA of the large subunit, and the second protein translation mechanism comprises a second modified ribosome, the second modified ribosome comprising i) rRNA and protein A modified cell comprising: ii) a small subunit containing protein; ii) a large subunit containing rRNA and protein; and iii) wherein the second modified ribosome lacks a ligation region between the large subunit and the small subunit, and wherein the small subunit of the second modified ribosome contains a modified anti-Shine-Dalgano sequence, enabling translation of a template having a complementary and / or congenital Shine-Dalgano sequence different from that of endogenous cellular mRNA; and / or the second modified ribosome contains one or more functionally transformative mutations, the functionally transformative mutations being absent in the anti-Shine-Dalgano sequence.

[0238] Embodiment 70. A modified cell of Embodiment 69 in which the first and second protein translation mechanisms can assist in the translation of a sequence-defined polymer.

[0239] Embodiment 71. A modified cell of any one of Embodiments 69-70 in which the first protein translation mechanism can assist in the translation of native endogenous RNA.

[0240] Embodiment 72. A modified cell according to any one of Embodiments 69 to 71, in which the second protein translation mechanism can assist in the translation of non-native exogenous RNA.

[0241] Embodiment 73. A modified cell according to any one of Embodiments 69 to 72, in which the small subunit of the second modified ribosome contains a modified anti-Shine-Dalgarno sequence selected from the group consisting of 3'-GGUGUU-5', 3'-UGGUGU-5', 3'-GGUGUC-5', 3'-GUUUAG-5', 3'-UGGAAU-5', 3'-GGAUCU-5', 3'-UGGAUC-5', 3'-UGGUAA-5', and 3'-UGGAUC-5.

[0242] Embodiment 74. A modified cell according to any one of Embodiments 69 to 74, in which the second modified ribosome contains a functional conversion type mutation at one or more of: a) peptidyl transferase center (PTC), b) nascent peptide exit tunnel (NPET), c) interaction site with elongation factor, d) tRNA binding site, e) chaperone binding site, f) binding site for nascent chain modifying enzyme, g) GTPase center.

[0243] Embodiment 75. A modified cell according to any one of Embodiments 69 to 74, in which the large subunit of the second modified ribosome contains a functional conversion type mutation at one or more of the following residues of 23S rRNA: G2061, C2452, U2585, G2251, G2252, A2057, A2058, C2611, A2062, A2503, U2609, G2454, and G2455.

[0244] Embodiment 76. A modified cell according to any one of Embodiments 69 to 75, in which the first, second, or both the first and second modified ribosomes contain an antibiotic resistance mutation.

[0245] Embodiment 77. A modified cell of any one of Embodiments 69 to 76, wherein the large subunit of the first modified ribosome contains a permutation substitution variant or mutant of 23S rRNA, and / or the small subunit contains a permutation substitution variant or mutant of 16S rRNA.

[0246] Embodiment 78. A modified cell of any one of Embodiments 69 to 77, wherein the connecting portion is covalently bonded to a helix of a large subunit selected from the group consisting of helix 10, helix 38, helix 42, helix 54, helix 58, helix 63, helix 78, and helix 101, to a helix of a small subunit selected from the group consisting of helix 11, helix 26, helix 33, and helix 44.

[0247] Embodiment 79. A method for preparing a sequence-controlled amino acid polymer, comprising: (a) preparing one or more: (i) any one cell from Embodiments 69 to 78; (ii) a cell extract derived from any one cell from Embodiments 69 to 78; (iii) a purified translation system derived from any one cell from Embodiments 69 to 78; and (b) providing mRNA encoding a sequence-controlled polymer to the cell or cell extract.

[0248] Embodiment 80. The method of Embodiment 79 for preparing a sequence-defined amino acid polymer in vivo.

[0249] Embodiment 81. The method of Embodiment 79 for preparing a sequence-defined amino acid polymer in vitro.

[0250] Embodiment 82. Any one of the methods of Embodiments 79 to 81, wherein the sequence-defined amino acid polymer comprises one or more unnatural amino acids. [Examples]

[0251] The following examples are illustrative and should not be construed as limiting the claimed subject matter.

[0252] Example 1. Development and testing of a fully orthogonal system for protein synthesis in bacterial cells.

[0253] A. Abstract

[0254] Ribosomes synthesize genetically encoded polypeptides from proteogenic amino acids. Ribosome engineering has become a powerful method for expanding the catalytic activity of the protein synthesis machinery and elucidating its origin, evolution, and function. Because the properties of modified ribosomes may be detrimental to normal protein synthesis, genetically engineered (designer) ribosomes need to be functionally isolated from the translation mechanism that synthesizes cellular proteins. The first solution to this problem is provided by Ribo-T, a modified ribosome with a tethered subunit that can translate the desired protein while being excluded from translation of the cellular proteome. Here, we present a conceptually different design of a modified cell with two orthogonal translation systems, where cellular proteins are translated by Ribo-T, while the native ribosome acts as an isolated protein synthesis machinery in which both subunits are involved in the translation of specific types of mRNA. The inventors demonstrate that both subunits of a specialized ribosome can maintain autonomy from Ribo-T, decouple from cellular proteome translation, and thus be recombined for new functions. The inventors demonstrate the usefulness of this system by creating a comprehensive collection of mutants with variations in all rRNA nucleotides of the peptidyltransferase center and by isolating gain-of-function mutations that enable the ribosome to overcome the blockage of translation termination by arrest peptides.

[0255] B. Introduction

[0256] Ribosomes perform a distinctive, complex, and highly coordinated function during protein synthesis. Ribosomes consist of two subunits: a small subunit and a large subunit, which in bacteria are 30S and 50S, respectively (Figure 1a). The 30S subunit initiates translation using the complementarity between the Shine-Dalgarno sequence (SD) near the mRNA start codon and the anti-Shine-Dalgarno sequence (ASD) at the 3' end of the 16S rRNA. 1 The 30S subunit performs decoding function during the elongation phase of protein synthesis by maintaining codon-anticodon interactions, while facilitating the recognition of the stop codon by release factors during termination. The 50S subunit provides the site (hosts) for the peptidyltransferase center (PTC), where polymerization of amino acids into polypeptides takes place, and peptide release is catalyzed during the termination phase. The elongated amino acid chain then proceeds from the PTC to the nascent peptide exit tunnel (NPET) and exits the ribosome. 2、3 .

[0257] Ribosomes have evolved to operate using natural substrates (mRNA, tRNA, and proteogenic amino acids) to synthesize genetically encoded proteins. Nevertheless, molecular engineering could extend their synthetic capabilities, potentially enabling the use of alternative genetic codes, polymerization of a wider variety of amino acids, or even the programmable synthesis of non-proteinoid polymers. 4 Ribosome engineering could also be used to elucidate the origin, evolution, and function of the protein synthesis machinery. However, all such attempts require altering a fundamental property of the ribosome itself. 5 This inevitably reduces, or even eliminates, the ability of ribosomes to synthesize cellular proteins. 6、7 An interesting solution to this problem may be provided by cell-free translation systems. 8 However, due to issues of efficiency and scalability, there are currently limitations to their application.

[0258] The predicament that ribosome engineering is struggling with can be overcome by creating an orthogonal protein synthesis apparatus within the cell that is not involved in the production of the cellular proteome and is specialized solely for the translation of one or a few specific mRNAs 9 By mutating the ASD of 16S rRNA and introducing a complementary SD sequence into the mRNA, a part of the small subunit can be directed to translate only cognate mRNA 10、11 This is a strategy that has been utilized to expand the decoding ability of ribosomes 12 However, the orthogonality of this setup is limited to only the small subunit. The reason is that due to the probabilistic nature of the binding of the large and small ribosomal subunits in multiple translations, both the wild-type (wt) and the orthogonal 30S subunit share the same pool of 50S subunits. Since an orthogonal 50S subunit cannot be created, efforts to modify the PTC and NPET, which are the most important sites for designing a translation apparatus with altered or expanded catalytic ability, have been limited. Recombinant engineering of the first fully orthogonal translation system became possible with the emergence of the ribosome Ribo-T 13 with tethered subunits. In Ribo-T and subsequent similar designs 14-16 , a circularly permuted 23S rRNA is embedded in the 16S rRNA, and a ribosome is obtained in which the subunits are ligated with two RNA linkers (Figure 1a). In the orthogonal Ribo-T (oRibo-T) with a modified ASD, the small and large subunits of Ribo-T cannot be separated, so both subunits are exclusively involved in translating cognate mRNA, and oRibo-T thus functions independently of the wild-type ribosome that translates cellular proteins (Figure 1b). With the help of oRibo-T, specific PTC mutations that facilitate the polymerization of amino acid sequences problematic for wild-type ribosomes could be selected 13、15However, while achieving perfect orthogonality, Ribo-T's unusual design limits its function. Ribo-T translates proteins only at half the rate of dissociable ribosomes. 13 Furthermore, compared to wild-type ribosomes, they detach from the start codon more slowly. 17 Furthermore, even the biogenesis of "wild-type" ribosomes is quite slow and inefficient. 17 Furthermore, if the functional center of the ribosome undergoes further changes, assembly problems could worsen. 7 The inventors anticipate similar challenges with "stapled" ribosomes, although they have not characterized them in detail. In summary, all of these factors make it difficult to directly use Ribo-T, or any tethered ribosome, in further engineering endeavors.

[0259] C. Development and testing of "flipped" orthogonal systems for protein synthesis in bacterial cells

[0260] To overcome the shortcomings of the original oRibo-T-based method for designing cells with two functionally independent translation mechanisms, we have now created a conceptually novel in vivo system design utilizing dissociable and even completely isolated ribosomes specifically for the translation of particular mRNAs. By "flipping" the roles of Ribo-T and dissociable ribosomes, we have designed a bacterial cell in which proteome translation is performed by Ribo-T, while ribosomes composed of dissociable orthogonal 30S (o-30S) subunits and wild-type 50S subunits function as a fully orthogonal translation machine (Figure 1c). In the resulting setup, which the inventors have named OSYRIS (Orthogonal translation SYstem based on Ribosomes with Isolated Subunits), complete orthogonality is achieved because Ribo-T, due to its tethered nature, cannot bind to either the dissociable o-30S or 50S ribosomes. Therefore, in OSYRIS cells, the physically unbound o-30S and 50S ribosome subunits are forced to interact with each other and function as fully orthogonal ribosomes (o-ribosomes), despite not being bound together. As a result, not only the o-30S but also the free 50S subunit can be designed to realize new functions without interfering with the expression of the cellular proteome (Figure 1c).

[0261] The components of the system (Figure 5) are the chromosomal rrn alleles. 18 (Figure 6) was assembled in an E. coli strain lacking this trait. In the resulting OSYRIS cells, improved 16S-23S tethers were produced from the optimized pRibo-Tt plasmid. 16Ribo-T rRNA is expressed. Another plasmid, poRbs, has a dissociable o-ribosome rRNA gene, and its 16S rRNA gene has a modified ASD (Figure 5). In cells transformed with these two plasmids, o-ribosomes account for approximately 15% of the total ribosome population (Figures 2a, b and 7). A specialized reporter gene (gfp, rfp, or luc) with an SD of the same family as the o-ribosomal ASD reporter gene is introduced into a third plasmid (poGFP, poRFP / oGFP, or poLuc) (we will refer to these orthogonal reporters as o-reporters) (Figure 5).

[0262] In OSYRIS cells, o-reporter expression is dependent on o-ribosomes; in their absence, reporter proteins encoded by o-mRNA (GFP, RFP, or luciferase) are produced at low levels, while the presence of dissociable o-ribosomes greatly stimulates o-reporter expression (Figures 2c, d, and 8). Therefore, previous studies 11、16 The o-30S subunit, which has been confirmed to be decoupled from cellular mRNA translation, efficiently promotes the translation of OSYRIS o-mRNA. Notably, dissociable o-ribosomes performed better than o-Ribo-T in o-reporter expression when introduced into the same host (E. coli, BL21) with equivalent vectors. Furthermore, the relative expression of the o-GFP reporter in OSYRIS cells expressing o-ribosomes from low-copy-number plasmids is higher compared to cells expressing o-Ribo-T from high-copy-number plasmids (Figure 2d, Figure 8, dark bars).

[0263] To investigate whether only the small subunit of the dissociable o-ribosome in OSYRIS cells, or both the small and large subunits, remain functionally separated from Ribo-T, we investigated the A2058G mutation present in Ribo-T that confers resistance to the antibiotic erythromycin (Ery). 13We utilized the following: If the free 50S subunit, which is Ery-sensitive, can somehow cooperate with the small subunit of Ribo-T in translating the proteome, then Ery would inhibit overall protein synthesis and hinder cell proliferation. However, OSYRIS cells continued to proliferate even at the highest antibiotic concentration tested (1 mg / ml), demonstrating the functional autonomy of the dissociable 50S subunit and Ribo-T (Figure 11, set of second bars for each group). In contrast, o-GFP reporter expression gradually decreased with increasing Ery concentration in the culture medium (Figure 3a). This result indicates that o-reporter translation is primarily driven by ribosomes composed of dissociable o-30S and 50S subunits, in contrast to the o-30S / Ribo-T hybrid (Figure 3a). Therefore, neither the o-30S subunit nor the 50S subunit interacts with Ribo-T, and despite the lack of physical connection between them, the two subunits remain functionally dedicated to each other.

[0264] A more rigorous demonstration of the orthogonality of the dissociable 50S subunit in OSYRIS cells was obtained by introducing a mutation into its 23S rRNA, which is known to be dominantly lethal in wild-type E. coli cells. 20、21 Two of these mutations, A2451C and A2602U, alter key nucleotides in the PTC active site, while the mutation G2553C disrupts the essential rRNA-tRNA interaction necessary for the proper placement of A-site aminoacyl-tRNA for peptide bond formation. 22、23(Figure 12a). If the dissociable 50S subunit of the variant mainly interacts with the o-30S subunit, the o-ribosome is excluded from normal translation, so the survival of OSYRIS cells should not be impaired. Conversely, if the free 50S subunit binds to Ribo-T and is involved in proteome translation, the dominant lethal 23S rRNA mutation will prevent or significantly impair the growth of OSYRIS cells. Attempts to express the variant 50S subunit in cells lacking o-30S (by transforming Ribo-T cells with the pRbs plasmid encoding the variant 23S rRNA together with the wild-type 16S rRNA) did not yield transformants, confirming the dominant lethality of the 23S rRNA mutation (Figures 3b and 12b, c). In contrast, when the variant 23S rRNA gene was introduced into a plasmid with orthogonal 16S rRNA in OSYRIS cells, many transformants appeared (Figures 3b and 12b, c). Analysis of rRNA isolated from cultures of transformed cells revealed a rather high expression level of free 50S subunits containing the variant 23S rRNA (Figure 3b, Figure 12d). Overall, these results clearly show that the dissociable large ribosome subunit remains functionally separated from Ribo-T. These results clearly show that the dissociable large ribosome subunit remains functionally separated from Ribo-T.

[0265] In summary, the results of o-reporter expression and resistance to dominant lethal mutations indicate that in OSYRIS cells, the dissociable o-ribosome translates o-mRNA but does not significantly contribute to proteome translation. Therefore, both subunits of the dissociable o-ribosome in OSYRIS cells are suitable for biomolecular engineering.

[0266] Having established the orthogonality of dissociable ribosomes in OSYRIS cells, the inventors conducted proof-of-principle experiments to test the potential of a system for selecting mutations in the large subunit rRNA that would enable ribosomes to perform problematic tasks. Specifically, the inventors aimed to design ribosomes capable of efficiently releasing difficult-to-terminate proteins. Generally, the release of fully synthesized polypeptides is a very nuanced reaction catalyzed by PTCs assisted by class 1 release factors. 24、25 Most proteins are efficiently released at stop codons, but the termination of others can be more problematic. 26、27 An extreme example of inefficient termination in E. coli is shown in programmed translation arrest at the stop codon of mRNA encoding the regulatory protein TnaC. 28-30 In high concentrations of tryptophan, the release of fully translated TnaC is inhibited, and the resulting ribosome stalling at the tnaC stop codon leads to the activation of downstream gene expression of the tna operon. 31 Termination arrest at the tnaC stop codon is mediated by undesirable interactions between nascent TnaC and rRNA nucleotides NPET and PTC. 30、31 TnaC-mediated termination arrest represents an inefficient paradigm of protein release and highlights one of the problems that can arise, for example, from the suppression of the expression of bioengineered modified polypeptides containing non-canonical amino acids.

[0267] To identify mutations that may mitigate the inefficient termination of TnaC, we constructed a reporter in which the TnaC-encoding sequence was appended to the end of the GFP gene (Figure 4a). As expected, the expression of the GFP-TnaC chimera was inhibited in vivo and in vitro at high concentrations of tryptophan (Figure 4b, Figure 13). The W12R mutation is known to mitigate termination arrest. 28 When introduced into the segment encoding TnaC, reporter expression was significantly stimulated in the presence of tryptophan (Figure 4b, Figure 13).

[0268] Next, the inventors created a comprehensive library of 120 single-nucleotide 23S rRNA variants of the poRbs plasmid containing the following modifications (Figure 5, Table in Figure 17): i) all nine rRNA residues in the PTC active site located within a 10 Å radius of the attacking amine of the receptor amino acid involved in the peptidyl transfer reaction; ii) 41 nucleotides of the second shell (located within a 25 Å radius of the PTC); and iii) six residues of the P-loop and A-loop of the 23S rRNA involved in the repositioning of the receptor ends of P-site tRNA and A-site tRNA (Figure 4c, d). Notably, the majority of the individual mutations contained in the library from OSYRIS cells are harmful or lethal in wild-type E. coli cells. 20、21 This has been reported, and therefore it could be easily tested based solely on the orthogonality of the dissociable ribosomes in OSYRIS cells.

[0269] The inventors characterized the ability of individual variants to successfully terminate the GFP-TnaC polypeptide by estimating a stalling bypass (SB) score. The SB score indicates the efficient termination of the GFP-TnaC(W12R) variant. 28This reflects the relative expression of the less terminated GFP-TnaC reporter compared to the wild-type ribosome. Furthermore, the effect of PTC mutations on the normal translational activity of mutant ribosomes was evaluated using the expression level of the GFP-TnaC(W12R) construct. Notably, many mutants with changes in PTC rRNA residues showed significantly higher bypass scores than OSYRIS cells with wild-type 50S subunits (Figure 4e and Figures 14 and 15). Of these, 19 mutants exhibited both high translational activity (>60% of wild-type) and significantly increased SB scores compared to wild-type controls (>0.3 vs. 0.17 of wild-type controls) (Figure 4e, Table in Figure 17). The identified mutations were located in the PTC active site (G2061, C2452, U2585), the P-loop (G2251, G2252), and 23S rRNA residues located in the second PTC shell, as well as residues at the NPET entry point (A2057, A2058, C2611, A2062, A2503, U2609) and two residues (G2454 and G2455) via the A2453 stack on C2452 of the PTC (Figure 4g). Two of the non-lethal mutations in this list (U2609C and A2058U) have been previously reported. 29、31 These mutations served as internal controls, confirming that the newly isolated mutants indeed revealed PTC residues involved in TnaC-mediated termination arrest and that the identified mutations help overcome ribosome stalling at the TnaC stop codon.

[0270] A unique opportunity offered by OSYRIS cells is that the dissociable 30S or 50S subunits can be separated from Ribo-T by sucrose gradient centrifugation. 13Therefore, it is possible to isolate individual ribosome subunits even if they have lethal mutations (Figure 16a, b). Using the characteristics of this system, the inventors prepared macroribosome subunits with lethal mutations U2500G, A2060C, and A2450U exhibiting bypass scores >0.37, recombined them with wild-type (non-orthogonal) 30S subunits, and investigated in a cell-free translation system whether these mutations alleviated ribosome stalling at the stop codon of the tnaC ORF (Figure 16c). The inventors further tested several non-lethal mutants (A2503G, A2062G, C2611G, C2611U) with SB scores of 0.35 to 0.55. Consistent with in vivo data, all tested mutant ribosomes showed reduced stalling at the tnaC stop codon compared to wild-type ribosomes during cell-free translation (Figure 4g and Figure 16), revealing their ability to more efficiently terminate TnaC peptide translation. The identified termination-arrest-release mutation locations suggest that either alterations in peptidyl-tRNA placement or less strict repositioning of the P-site substrate and / or release factor in the PTC of mutant ribosomes promote TnaC release. Almost certainly, mutations that mitigate TnaC-mediated termination arrests are not found in previous oRibo-T based methods. 13、15 It may be possible to isolate them using this method. However, compared to the dissociable o-ribosomes of OSYRIS (Figure 2d), the reduced reporter expression level induced by oRibo-T is likely to limit the number of mutants that exceed the minimum efficiency threshold imposed in our screen, so some of the mutations identified in OSYRIS are likely to be missed.

[0271] Our proof-of-principle experiments have shown that the OSYRIS design, based on Ribo-T's ability to maintain cell proliferation while forcing the dissociable subunits of O-ribosomes to interact with each other, presents a viable and conceptually novel method for creating a fully orthogonal cell translation system. Ribo-T is sufficiently active to translate the entire intracellular proteome. 13、17Therefore, the design of OSYRIS was possible. However, translation driven by Ribo-T is slow, and Ribo-T assembly is inefficient, which is likely one of the factors contributing to the slow proliferation rate of OSYRIS cells (τ ~ 300 minutes in a 96-well plate compared to a doubling time τ ~ 45 minutes for the BL21 strain) (Figure 9a). Therefore, optimizing the function and assembly of Ribo-T could improve the proliferation rate of OSYRIS cells and further expand the versatility of the orthogonal system. The setup of three plasmids (Figure 5) makes OSYRIS highly modular and thus easily adaptable to various applications. In principle, OSYRIS could be made even simpler by introducing the Ribo-T rRNA gene into the chromosome and combining it with an orthogonal rRNA gene and a reporter gene on the same plasmid. Reducing the number of plasmids could further promote the proliferation of OSYRIS cells. Increasing the proportion of o-ribosomes in OSYRIS cells by adjusting either plasmid copy number or promoter strength may be another way to improve the system's performance and tailor it to specific needs. Therefore, while our experiments showed that o-ribosomes were involved in the expression of only a single reporter gene, a properly balanced proportion of o-ribosomes could potentially lead to the simultaneous translation of several genes with modified SDs, opening up the possibility of, for example, orthogonal expression of multi-subunit protein complexes.

[0272] One obvious application of OSYRIS is the recognition of polypeptides (backbond-modified D-amino acids or β-amino acids) by ribosomes. 32This involves designing ribosomes that can incorporate non-canonical amino acids (such as ribosomes). Extending ribosome synthesis capacity requires many elements, from specialized aminoacylation systems to genetically engineered modified genetic codes, but fully orthogonal, dissociable ribosomes working in OSYRIS cells can accelerate the achievement of this goal. Importantly, OSYRIS enables many other attempts, from employing ribosome retro-engineering to elucidate the origins of the translation machinery to evolving novel catalytic functions for the programmable synthesis of non-proteinaceous polymers.

[0273] References 1 Shine, J. & Dalgarno, L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71, 1342-1346 (1974). 2 Jenni, S. & Ban, N. The chemistry of protein synthesis and voyage through the ribosomal tunnel. Curr. Opin. Struct. Biol. 13, 212-219 (2003). 3 Rodnina, MV Translation in Prokaryotes. Cold Spring Harbor Perspect. Biol. 10, pii: a032664 (2018). 4 Des Soye, BJ, Patel, JR, Isaacs, FJ & Jewett, MC Repurposing the translation apparatus for synthetic biology. Curr. Opin. Chem. Biol. 28, 83-90 (2015). 5 Davis, L. & Chin, J. W. Designer proteins: applications of genetic code expansion in cell biology. Nat. Rev. Mol. Cell Biol. 13, 168-182 (2012). 6 Dedkova, L. M., Fahmi, N. E., Golovine, S. Y. & Hecht, S. M. Construction of Modified Ribosomes for Incorporation of d-Amino Acids into Proteins. Biochemistry 45, 15541-15551 (2006). 7 Ward, F. R., Watson, Z. L., Ad, O., Schepartz, A. & Cate, J. H. D. Defects in the assembly of ribosomes selected for beta-amino acid incorporation. Biochemistry, 58, 4494-4504 (2019). 8 Silverman, A. D., Karim, A. S. & Jewett, M. C. Cell-free gene expression: an expanded repertoire of applications. Nat. Rev. Genet., doi:10.1038 / s41576-019-0186-3 (2019). 9 Arranz-Gibert, P., Vanderschuren, K. & Isaacs, F. J. The role of orthogonality in genetic code expansion. Life 9, 58 (2019). 10 Hui, A. & de Boer, H. A. Specialized ribosome system: preferential translation of a single mRNA species by a subpopulation of mutated ribosomes in Escherichia coli. Proc. Natl. Acad. Sci. USA 84, 4762-4766 (1987). 11 Rackham, O. & Chin, J. W. A network of orthogonal ribosome x mRNA pairs. Nat. Chem. Biol. 1, 159-166 (2005). 12 Wang, K., Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nat. Biotechnol. 25, 770-777 (2007). 13 Orelle, C. et al. Protein synthesis by ribosomes with tethered subunits. Nature 524, 119-124 (2015). 14 Fried, S. D., Schmied, W. H., Uttamapinant, C. & Chin, J. W. Ribosome subunit stapling for orthogonal translation in E. coli. Angew. Chem. Int. Edit. 54, 12791-12794 (2015). 15 Schmied, W. H. et al. Controlling orthogonal ribosome subunit interactions enables evolution of new function. Nature 564, 444-448 (2018). 16 Carlson, E. D. et al. Engineered ribosomes with tethered subunits for expanding biological function. Nat. Commun. 10, 3920 (2019). 17 Aleksashin, N. A. et al. Assembly and functionality of the ribosome with tethered subunits. Nat. Commun. 10, 930 (2019). 18 Quan, S., Skovgaard, O., McLaughlin, R. E., Buurman, E. T. & Squires, C. L. Markerless Escherichia coli rrn deletion strains for genetic determination of ribosomal binding sites. G3 5, 2555-2557 (2015). 19 Davis, J. H. et al. Modular assembly of the bacterial large ribosomal subunit. Cell 167, 1610-1622 (2016). 20 Sato, N. S., Hirabayashi, N., Agmon, I., Yonath, A. & Suzuki, T. Comprehensive genetic selection revealed essential bases in the peptidyl-transferase center. Proc. Natl. Acad. Sci. USA 103, 15386-15391 (2006). 21 Cochella, L. & Green, R. Isolation of antibiotic resistance mutations in the rRNA by using an in vitro selection system. Proc. Natl. Acad. Sci. USA 101, 3786-3791 (2004). 22 Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920-930 (2000). 23 Polikanov, Y. S., Steitz, T. A. & Innis, C. A. A proton wire to couple aminoacyl-tRNA accommodation and peptide-bond formation on the ribosome. Nat. Struct. Molec. Biol. 21, 787-793 (2014). 24 Korostelev, A. A. Structural aspects of translation termination on the ribosome. RNA 17, 1409-1421 (2011). 25 Tate, W. P., Cridge, A. G. & Brown, C. M. ’Stop’ in protein synthesis is modulated with exquisite subtlety by an extended RNA translation signal. Biochem. Soc. Trans. 46, 1615-1625 (2018). 26 Li, G. W., Burkhardt, D., Gross, C. & Weissman, J. S. Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell 157, 624-635 (2014). 27 Baggett, N. E., Zhang, Y. & Gross, C. A. Global analysis of translation termination in E. coli. PLoS Genet. 13, e1006676 (2017). 28 Gong, F. & Yanofsky, C. Instruction of translating ribosome by nascent peptide. Science 297, 1864-1867 (2002). 29 Martinez, A. K. et al. Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan. Nucleic Acids Res. 42, 1245-1256 (2014). 30 Bischoff, L., Berninghausen, O. & Beckmann, R. Molecular basis for the ribosome functioning as an L-tryptophan sensor. Cell Rep. 9, 469-475 (2014). 31 Cruz-Vera, LR, Rajagopal, S., Squires, C. & Yanofsky, C. Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression. Mol. Cell 19, 333-343 (2005). 32 Lee, J. et al. Expanding the limits of the second genetic code with ribozymes. Nat. Commun. 10, 5097, doi:10.1038 / s41467-019-12916-w (2019).

[0274] D. Materials and Methods

[0275] 1. Assembly of OSYRIS cells

[0276] a. Plasmid construction

[0277] Figure 5 shows the plasmids used to create and optimize the OSYRIS setup. The nucleotide sequences and characteristics of key plasmids are shown in the source data file.

[0278] All plasmids are Gibson Assembly 1The plasmid backbones were constructed using [material name], prepared by inverse PCR or restriction nuclease digestion, and cloned inserts were PCR-amplified from their respective templates or chemically synthesized by Integrated DNA Technologies. PCR reactions were performed using Q5 High-Fidelity DNA polymerase (New England Biolab), and the PCR products were purified using the DNA Clean and Concentrator Kit (Zymo Research). Gibson assembly reactions of rRNA-encoding plasmids were electroporated into E. coli POP2136 cells (all bacterial species are listed in the table in Figure 18), and transformants were collected on LB plates supplemented with appropriate antibiotics. The plates were incubated at 30°C and LambdaP [unclear]. L The expression of rRNA genes controlled by promoters was prevented. 2 All other plasmids were transformed and amplified in E. coli JM109 strain cultured in LB medium supplemented with 100 μg / ml ampicillin (Amp), 50 μg / ml kanamycin (Kan), or 50 μg / ml spectinomycin (Spc) as needed. Plasmids were isolated using the High Pure Plasmid Isolation Kit (Roche), confirmed by PCR and capillary sequencing, and used for modifying OSYRIS cells. The construction of the major plasmids is outlined in the following sections.

[0279] i) pRibo-Tt plasmid

[0280] pRibo-T v2.0 plasmid with erythromycin resistance mutation A2058G 3 The backbone was linearized using SgsI restriction enzyme and then purified. The transcription of this was P tac A group of deletion tRNA genes (tRNA) controlled by promoters and T1 terminators Glu tRNA Ala tRNA Ile tRNA Trpand tRNA Asp The DNA (encoding) was synthesized as gBlock (Integrated DNA Technology) and PCR-amplified using primers NA1 and NA2 (all primers are listed in the table in Figure 19). The PCR reaction was catalyzed using Q5 High-Fidelity DNA polymerase (New England Biolab) under the following conditions according to the manufacturer's protocol: 98°C for 30 seconds, followed by 30 cycles (98°C for 10 seconds; 64°C for 30 seconds; 72°C for 20 seconds), followed by a final incubation at 72°C for 2 minutes. The purified PCR product (20 ng) was linearized using SgsI to form a pRibo-T v.2.0 backbone (80 ng), and then subjected to a Gibson assembly reaction (1.7% PEG-800, 3.1 mM DTT, 0.31 mM β-nicotinamide adenine dinucleotide, 62.5 μM each dNTP, 3.1 mM MgCl2, 31.3 mM Tris / HCl, pH 7.5, 0.004 U / μl T5 exonuclease (Epicentre), 4 U / μl Taq DNA ligase (New England Biolab), 0.025 U / μl Phusion High Fidelity). The reaction mixture was mixed in DNA polymerase (New England Biolab). After incubation at 50°C for 1 hour, 3 μl of the reaction mixture was used to transform electrocompetent POP2136 E. coli cells. The cells were plated onto LB / Amp agar plates. Individual colonies of the transformants were picked, cultured in LB / Amp medium, and plasmids were isolated. The presence of tRNA clusters was confirmed by PCR amplification and sequencing using primers NA3 and NA4.

[0281] ii) poRBS plasmid

[0282] Lambda P L Orthogonal and wild-type rRNA operons under the transcriptional control of promoters and T1 / T2 terminators are converted to pO2 and pAM552 plasmids using primers NA5 and NA6. 4 PCR amplification was performed on each plasmid pKD13.5 Using primers NA7 and NA8, Kan R The gene was amplified using the pCSacB plasmid. 6 The origin of replication for pSC101 was amplified using primers NA9 and NA10. The PCR reaction was treated with DpnI to reduce the background of the parent plasmid. The PCR product was purified, confirmed by electrophoresis, and mixed with Gibson assembly reaction (40 ng each). After incubation at 50°C for 1 hour, 3 μl of the reaction mixture was transformed into electrocompetent POP2136 E. coli cells. The cells were plated on LB / Kan agar plates. After incubation at 37°C for 24 hours, individual colonies were picked, cultured in LB / Kan medium, and plasmids were isolated and confirmed by restriction digestion and sequencing.

[0283] iii) poGFP plasmid

[0284] The o-GFP gene, possessing 5'UTR, 3'UTR, and T1 / T2 terminators, is introduced into the plpp5-oGFP plasmid. 4 PCR amplification was performed using primers NA11 and NA12. LuxR repressor and P Lux promoter 7 Then, using primers NA13 and NA14, the pJDO75 plasmid was modified. 8 PCR amplification was performed from Spc. R The marker (aadA) was added to the ptRNA67 plasmid using primers NA15 and NA16. 6PCR amplification was performed from the p15A replication origin. The p15A replication origin was PCR amplified from the ptRNA67 plasmid using primers NA17 and NA18. The PCR reaction product containing the plasmid template was treated with DpnI. The purified PCR products (40 ng each) were mixed into the Gibson assembly reaction. After incubation at 50°C for 1 hour, 3 μl of the reaction mixture was used to transform electrocompetent JM109 E. coli cells (Promega). The cells were plated on LB / Spc agar plates. After incubation at 37°C for 24 hours, individual colonies were picked, cultured in LB / Spc medium, and the plasmid was isolated. The presence of luxR gene insertion was confirmed by PCR using primers NA19 and NA20. Restriction enzyme digestion of the obtained plasmid showed that its size was ~1 kb larger than expected. Subsequent restriction enzyme analysis and sequencing showed that the luxR gene had undergone replication (Figure 5c). This replication is not expected to affect the expression of the o-gfp reporter.

[0285] iv) poRFP / oGFP plasmid

[0286] Spc R The marker (aadA) and the origin of p15A replication are located in the ptRNA67 plasmid. 6 The reaction was amplified using [a specific method]. The PCR reaction product was treated with DpnI. plpp5-oGFP plasmid. 4 From P lpp5 The o-GFP gene, containing a promoter, 5'UTR, 3'UTR, and T1 / T2 terminators, was amplified by PCR. Purified PCR products (~40 ng each) were mixed into a Gibson assembly. After incubation at 50°C for 1 hour, 3 μl of the reaction mixture was used to transform electrocompetent JM109 E. coli cells (Promega). The cells were plated onto LB / Spc agar plates. After incubation at 37°C for 24 hours, individual colonies were picked, cultured in LB / Spc medium, and the plasmid was isolated. The structure of the obtained plasmid plpp5-oGFP-pA15-Spec was confirmed by restriction digestion and sequencing. Plasmid pRYG 9 From, PT5 The rfp gene, including the promoter and T0 transcription terminator, was amplified by PCR. An orthogonal SD sequence was introduced by PCR, and the resulting o-rfp construct was inserted into the unique SphI site of the plpp5-oGFP-pA15-Spc plasmid.

[0287] v) pOLuc plasmid

[0288] A plasmid containing an orthogonal luciferase gene, poLuc, was constructed based on poGFP (Figure 5c). The 1653 bp gene luc, which encodes firefly luciferase, was PCR amplified from the pBESTluc plasmid (Promega) using primers NA21 and NA22. The resulting PCR products and poGFP plasmid were cleaved with restriction enzymes BglII and SalI, and then ligated. The ligated mixture was transformed into E. coli JM109 competent cells, and luc gene-positive clones were identified by colony PCR. The integrity of the cloned luc gene was confirmed by sequencing.

[0289] vi)poGFP-TnaC plasmid

[0290] To construct reporter poGFP-TnaC plasmids (wild-type or W12R mutant), the GFP-coding sequence in the poGFP plasmid was replaced with a sequence encoding a chimeric wild-type or mutant GFP-TnaC protein. DNA inserts containing an orthogonal ribosome binding site and the GFP-TnaC or GFP-TnaC(W12R) coding sequence were prepared by PCR using primers NA23 and NA24 and a template used for in vitro translation (described later). After purification, the inserts were introduced into poGFP plasmids cut with restriction enzymes BglII and SalI using Gibson assembly. After transformation, the presence of the correct inserts in individual colonies was confirmed by colony PCR using primers NA25 and NA26, and by sequencing of the corresponding segments of the plasmid.

[0291] b. Modified Ribo-T expressing cells

[0292] Chromosomal rRNA alleles 10 Lacking Ribo-T 4 SQ171 FG cells (table in Figure 18) containing mutations in the ybeX and rpsA genes that promote proliferation upon expression were used as the host (Figure 6). Since 5-fluorouracil negative selection may be used in the future, the upp gene was inactivated by recombineering.

[0293] The recipient cells initially had the following two plasmids: rrnB operon, counter-selective sacB marker, and Kan R pCSacB plasmid containing the gene, and ptRNA67 plasmid containing the deleted tRNA gene removed during chromosomal rRNA operon deletion. 6The cells were electrocompetent, and then 50 μl of the cell suspension was transformed with 50 ng of pRibo-Tt plasmid, which contains the Ribo-T rRNA gene but lacks the tRNA gene, isolated from POP2136 cells (Figure 5). The transformed cells were diluted in 1 ml of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 10 mM MgSO4, 10 mM MgCl2, 20 mM glucose) and incubated at 37°C for 6 hours with shaking. 150 μl aliquots of the culture were diluted to 2 ml of fresh SOC medium supplemented with 50 μg / ml ampicillin, 25 μg / ml spectinomycin, and 0.25% sucrose, and incubated at 37°C for 12 hours with constant shaking. Cells were spun down (1 min, 5000 g) and seeded on LB / agar plates containing 50 μg / ml ampicillin, 25 μg / ml spectinomycin, 5% sucrose, and 1 mg / ml erythromycin (Ery). The plates were incubated at 37°C for 48 hours. The absence of the pCSacB plasmid was confirmed by the sensitivity of transformants to kanamycin, which was tested by replica plating colonies onto LB / agar plates supplemented with or without 50 μg / ml kanamycin, 50 μg / ml ampicillin, and 25 μg / ml spectinomycin. The transformants were then cultured in LB medium supplemented with 50 μg / ml ampicillin and 25 μg / ml spectinomycin, the plasmid was isolated, and confirmed by restriction enzyme analysis. Furthermore, the absence of wild-type rRNA was confirmed by total RNA isolation using the RNeasy Mini Kit (Qiagen) and agarose gel electrophoresis.

[0294] c. Removal of ptRNA67 plasmid

[0295] Next, the ptRNA67 plasmid was removed from the resulting transformants. To do this, the cells were passaged for approximately 100 generations in LB medium supplemented with 100 μg / ml of amp. After plating the cell dilutions, the absence of the ptRNA67 plasmid in individual clones was confirmed by the absence of a visible band of ptRNA67 plasmid in sensitivity to Spc and in restriction digestion of the total plasmid preparation.

[0296] d. Inactivation of the recA gene in Ribo-T expressing cells

[0297] Initial attempts to introduce poRbs into modified cells frequently resulted in the appearance of abnormal plasmids arising from recombination between poRbs and the pRibo-Tt plasmid. To avoid this problem, the recA gene was inactivated in cells containing the pRibo-Tt plasmid (inactivating the recA gene before removing the ptRNA67 plasmid prevented plasmid loss after prolonged cell passage in the absence of Spc).

[0298] To inactivate the recA gene in OSYRIS cells by transduction of P1 phage, first use the pKD3 plasmid. 5 The donor strain BW25113 recA::cat was prepared using a conventional recombinant procedure with a chloramphenicol (Chl)-resistant cassette. The cassette was PCR-amplified using primers NA27 and NA28. The PCR fragments were transformed into the BW25113 strain containing the Red recombinase expression plasmid pDK46. After selecting and validating the recA::cat strain and removing the pKD46 plasmid, the resulting strain was used as a donor for phage transduction. The protocol was standard except that the recovery culture before seeding the transdextrin on an LB / agar plate supplemented with 50 μg / ml ampicillin and 15 μg / ml chloramphenicol was 6 hours instead of 1 hour. 11 P1 phage transduction was performed according to the instructions. The genotypes of the modified strains are shown in the table in Figure 20.

[0299] Introduction of the e.poRbs plasmid

[0300] Next, the SQ171 FG ΔrecA / pRibo-Tt strain was subjected to electroporation and Amp R / Kan R / Chl R Cells were selected and transformed with poRbs (or pRbs, if necessary) plasmids. The only difference from the standard transformation protocol was that transformant recovery in antibiotic-free SOC medium was extended to 6 hours prior to the selection of transformants, and transformants were selected on LB / agar plates supplemented with 50 μg / ml ampicillin, 25 μg / ml kanamycin, and 15 μg / ml chloramphenicol. Transformants were confirmed by restriction enzyme analysis of the whole plasmid and rRNA analysis by agarose gel electrophoresis.

[0301] f. Introduction of reporter plasmid

[0302] Reporter plasmids (poGFP, poRFP / oGFP, poLuc, poGFP-TnaC) are essentially introduced into SQ171 FG ΔrecA / pRibo-Tt / poRbs cells via electroporation and Amp r / Kan r / Chl r / Spc r It was introduced through cell selection.

[0303] Validation of the g.OSYRIS cell genome sequence

[0304] During OSYRIS cell construction, the initial host cells undergo multiple passages and single-colony purification steps, potentially leading to the accumulation of spontaneous mutations. Therefore, the entire genome sequence of fully assembled OSYRIS cells was determined. Analysis of the resulting sequences revealed mutations in several genes (Table in Figure 20). Some of these mutations (e.g., the gene ptsI or ackA) may negatively impact cell proliferation under certain conditions and could potentially be corrected in the future through genome engineering.

[0305] h. Monitoring of orthogonal GFP gene expression in vivo

[0306] OSYRIS cells containing either a poRbs or pRbs plasmid (expressing orthogonal or non-orthogonal ribosomes, respectively) and a poGFP reporter plasmid were incubated overnight at 37°C with constant shaking in LB medium supplemented with 50 μg / ml ampicillin, 25 μg / ml kanamycin, 25 μg / ml spectinomycin, and 15 μg / ml chloramphenicol. The cultures were diluted 1:40 (volt / volt) in fresh LB medium supplemented with the same antibiotics and further containing 1 ng / ml of N-(β-ketocaproyl)-L-homoserine lactone (HSL) (Santa Cruz Biotechnology), an inducer of reporter gene transcription. The culture (120 μl) was placed in the wells of a 96-well flat-bottom polystyrene tissue culture plate (Costar), placed on a plate reader (TECAN Infinite M200 Pro), and incubated at 37°C with constant linear (3 mm) shaking. Cell culture density (A 600 ) and GFP fluorescence (excitation wavelength 485 nm, emission wavelength 520 nm, optimal gain 30% RFU with gain adjustment function applied) were monitored over a period of 24 to 48 hours. Autofluorescence of cells lacking a reporter was subtracted from all recorded values.

[0307] For erythromycin sensitivity testing, cells cultured overnight were diluted 1:40 in fresh LB medium supplemented with either HSL alone (final concentration: 0-16 ng / ml) or 1 ng / ml HSL and various concentrations of erythromycin (final concentration: 0-1 mg / ml). Monitoring of cell proliferation and GFP expression was performed as described in the preceding paragraph.

[0308] When OSYRIS cells possessed a poRFP / poGFP reporter, RFP expression was monitored using an excitation wavelength of 550 nm, an emission wavelength of 675 nm, and an optimal gain of 30% RFU with the gain adjustment function applied.

[0309] 2. In vivo expression of orthogonal luciferase genes

[0310] OSYRIS cells containing the poLuc plasmid were cultured for 24 hours in LB medium supplemented with 50 μg / ml ampicillin, 25 μg / ml kanamycin, 25 μg / ml spectinomycin, and 15 μg / ml chloramphenicol, and then diluted 1:40 in fresh medium containing the same antibiotics and 1 ng / ml HSL. After 6 hours, A 600Each culture was spun down (5 min, 5000 g, 4°C) and the cell pellet was flash-frozen. Luciferase activity was measured using the Luciferase Assay System (Promega) according to the manufacturer's protocol. Specifically, the cell pellet was thawed in a 20°C water bath and resuspended in LB supplemented with 25 μl of 10% (vol / vol) dibasic phosphate buffer (1 M K2HPO4 pH 7.8, 20 mM EDTA). 20 μl of cell suspension was mixed with 60 μl of freshly prepared lysis mix (25 mM trisphosphate pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 1% Triton X-100, 1.25 mg / ml lysozyme, 2.5 mg / ml bovine serum albumin) and dissolved at room temperature for 10 minutes. Then, 10 μl aliquots of the cell lysate were placed in the wells of a 96-well black / clear bottom assay plate (Corning), 50 μl of Luciferase Assay Reagent (Promega) was added, and fluorescence was immediately read using a TECAN microplate reader.

[0311] 3. Comparison of reporter expression using oRibo-T or oRbs

[0312] E. coli BL21 strain was transformed with either poGFP or poLuc plasmid. Transformants were selected on LB / agar plates supplemented with 50 μg / ml Spc, grown from individual colonies, and then electrocompetent. Reporter-containing cells were then subjected to pBR322 ori-based ampoules expressing poRibo-T(oRibo-T rRNA). R Plasmid) 3 , or o-pAM552 plasmid (oRbs rRNA expressing pBR322 ori base, Amp R Plasmid) 3 It transformed.

[0313] The expression of the o-GFP or o-luc reporter was measured as described above.

[0314] 4. Analysis of contained mutant rRNAs

[0315] The presence or absence of modified mutations in the 23S rRNA of orthogonal ribosomes was analyzed using primer extension. For this purpose, total RNA was isolated from OSYRIS cells using the RNeasy Mini Kit (Qiagen). The primers and dNTP and ddNTP combinations for the analysis of each mutation are shown in the table in Figure 21. In each assay, the appropriate 5'[ 32 A P-labeled primer (0.5 pmol) was annealed to 1 μg of total RNA in 1× hybridization buffer (50 mM K-HEPES, pH 7.0, 100 mM KCl). The annealed primer was extended at 42°C for 20 minutes using 2 units of AMV reverse transcriptase (Roche) in the presence of 0.25 mM appropriate ddNTPs and 0.2 mM of the remaining dNTPs (see table in Figure 21) (final reaction volume 8 μl). The reaction was stopped by adding 120 μl of stop buffer (84 mM NaOAc, 0.8 mM EDTA, pH 8.0, 70% EtOH), cooling at -80°C for 15 minutes, and centrifugation at 15000 g (4°C) for 1 hour to pelletize the nucleic acids. The supernatant was removed, the pellet was dried, and dissolved in formamide loading dye. cDNA products were degraded in a 12% denatured polyacrylamide gel and visualized by phosphoimaging. The intensity of the toe print bands was measured using ImageJ software. 12 Measurements were taken using [tool / method]. Background noise was subtracted.

[0316] 5. Expression of GFP-TnaC (wild-type) or GFP-TnaC (W12R) protein in a cell-free translation system

[0317] DNA templates containing the T7 RNA polymerase promoter, a ribosome binding site derived from bacteriophage T7 gene 10, and a GFP-TnaC or GFP-TnaC(W12R) coding sequence (see Appendix I) were constructed by crossover PCR. First, the T7 promoter and GFP coding sequence were used to construct the pY71-T7-GFP plasmid. 13 PCR amplification was performed using either the T7 promoter forward primer NA29 (table in Figure 19) and either NA30 complementary to wild-type tnaC or NA31 complementary to the W12R mutant of the tnaC gene. Independently, plasmids pGF2500-tnaC-wt or pGF2500-tnaC-mut were used. 14 Therefore, the 3' segment of the wild-type or mutant tnaC gene containing a 3' untranslated region was PCR amplified using forward primer NA32 for the wild-type, NA33 for the W12R mutant, and a common reverse primer NA34.

[0318] Next, two PCR products corresponding to either the wild-type or mutant gfp-tnaC construct were combined at 400 pg / μl (final concentration) and re-amplified using T7 and TnaC(rev) primers.

[0319] In vitro translation of the gfp-tnaC template was performed using the PURExpress (New England Biolab) cell-free translation system, which consists of purified components including Δ-ribosomes, Δ-tRNA, and Δ-amino acids, with some modifications. 15The procedure was carried out as described. The reaction was supplemented with a mixture of 19 amino acids (final concentration: 0.3 mM each amino acid) and 50 μM L-tryptophan (reaction under low tryptophan conditions) or 5 mM Trp (reaction under high tryptophan conditions). A DNA template prepared by PCR was added to a final concentration of 5 ng / μl. The reaction was carried out at 37°C for 3 hours with a total volume of 5 μl in a 384-well plate (Falcon) with a black wall and clear bottom in a plate reader (TECAN Infinite M200 Pro). GFP fluorescence (excitation wavelength 485 nm, emission wavelength 520 nm, optimal gain 30% RFU with gain adjustment function applied) was monitored over time.

[0320] 6. Preparation of a PTC variant library

[0321] pT7rrnB Library 16 A PTC mutant library was constructed by transferring individual mutations from the poRbs plasmid to the 23S rRNA gene.

[0322] To prepare the plasmid backbone, the poRbs plasmid was digested with SgsI and Bst1107I restriction enzymes, and a 1546nt fragment was excised from the 23S rRNA gene. The reaction products were separated by agarose gel electrophoresis, and a 7483bp backbone fragment was purified from the gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research) and the DNA Clean & Concentrator Kit (Zymo Research) in sequence.

[0323] To generate a 1606 bp insert containing the PTC mutation, individual plasmids from the pT7rrnB plasmid library were used as templates for PCR reactions catalyzed by Q5 High-Fidelity DNA polymerase (New England Biolab) and using primers NA35 and NA36. The PCR products were cleaned up using the DNA Clean & Concentrator Kit (Zymo Research).

[0324] A plasmid backbone (35 ng) and a DNA insert (60 ng) were mixed with a total volume of 5 μl of Gibson assembly reaction solution and incubated at 50°C for 1 hour.

[0325] Chemically competent POP2136 cells were transformed using individual Gibson assembly reaction products. High-throughput transformation was performed using a low-evaporation, clear, lidded, flat-bottomed 96-well tissue culture plate (Falcon). 20 μl of competent cells were mixed with 2 μl of individual Gibson assembly reaction products in each well of the plate. The plate was incubated on ice for 30 minutes, then at 42°C for 50 seconds, and again on ice for 15 minutes. 100 μl of SOC medium was added to each well, and the cells were recovered on a shaker at 30°C for 2 hours. The plate was rotated in a swing bucket rotor at 6000g for 6 minutes, and the amount of culture medium was reduced to 40μl by removing 80μl of supernatant. Then, 6μl of each of the remaining cell suspensions was spot-plated using a multichannel pipette into LB / agar rectangular OmniTray Single-Well plates (Nunc) supplemented with 50μg / ml kanamycin. The plates were incubated at 30°C for 20 hours.

[0326] Individual colonies were inoculated into fresh LB medium supplemented with 50 μg / ml kanamycin and incubated at 30°C for 12 hours. Plasmids were isolated, and the presence of desired mutations in the PCR-amplified 23S rRNA segment, along with the absence of off-target mutations, was confirmed by capillary sequencing.

[0327] Next, individual PTC variant library plasmids were introduced into OSYRIS cells by transforming SQ171 FG / pRibo-Tt / poGFP-TnaC cells using the above high-throughput transformation approach with the following modifications. i) 20 ng of purified individual plasmid was used for transformation. ii) Transformants were recovered in SOC medium at 37 °C for 6 hours and patched onto LB / agar plates supplemented with 50 μg / ml ampicillin, 25 μg / ml kanamycin, 25 μg / ml spectinomycin, and 15 μg / ml chloramphenicol. iii) Plates were incubated at 37 °C for 48 hours. iv) Glycerol stocks were made in 96-well plates from cultures grown from individual colonies of transformants.

[0328] 7. PTC Library Screening

[0329] Individual colonies of OSYRIS cells with PTC library variants were inoculated into wells of 96-well plates containing 120 μl of LB medium supplemented with 50 μg / ml ampicillin, 25 μg / ml kanamycin, 25 μg / ml spectinomycin, and 15 μg / ml chloramphenicol and cultured at 37 °C for 24 hours with constant shaking. Cultures were diluted 1:40 (volume / volume) into 120 μl of fresh LB medium supplemented with the same antibiotics, 0.35 mg / ml 1-methyl-tryptophan (Sigma), and 0.016 ng / ml of HSL. Plates were placed in a TECAN Infinite M200 Pro plate reader and incubated at 37 °C with constant linear (3 mm) shaking. The optical density (A 600 ) and oGFP fluorescence of the cultures were monitored as described above.

[0330] The termination arrest bypass score was calculated by comparing the GFP expression efficiency of OSYRIS cells with the GFP-TnaC(W12R) mutant construct with the expression efficiency of OSYRIS cells with the wild-type GFP-TnaC construct. The stalled bypass (SB) score was calculated using the following formula based on the readings obtained at 48 hours.

number

[0331] The average SB score was calculated using data obtained from two independent experiments.

[0332] 8. Isolation of 50S ribosome subunits derived from OSYRIS cells

[0333] Ohashira 17 Ribosomes were isolated from OSYRIS cells according to the protocol reported by [research organization name]. Specifically, OSYRIS cells expressing ribosomes with 23S rRNA mutations U2500G, A2060C, or A2450U were cultured overnight at 37°C in LB medium supplemented with 50 μg / ml ampicillin, 25 μg / ml kanamycin, 25 μg / ml spectinomycin, and 15 μg / ml chloramphenicol. The cultures were then transferred to 1 L of fresh LB medium supplemented with the same antibiotics. 600 = Diluted to 0.003, and the optical density is A 600The cells were cultured for approximately 15 hours with vigorous shaking until the pH reached 0.35. The cells were collected by centrifugation at 5000g (4°C) for 15 minutes, and the cell pellet was flash-frozen in liquid nitrogen and stored at -80°C. The frozen cell pellet was resuspended in 20 ml of lysis buffer (10 mM HEPES-KOH, pH 7.6, 50 mM KCl, 10 mM Mg(OAc)2, 7 mM β-mercaptoethanol), dissolved at 15000 psi for 5 minutes using an EmulsiFlex-C3 homogenizer (AVESTIN Inc.), and the lysate was then clarified by centrifugation at 20000g (4°C) for 30 minutes and transferred to a new centrifuge tube. Ammonium sulfate was added to a final concentration of 1.5 M, and the tube was centrifuged at 20000g (4°C) for 1 hour. The supernatant containing ribosomes (Ribo-T+ dissociable ribosomes) was filtered through a 0.22-μm φ30mm polyethersulfone (PES) membrane filter (CELLTREAT Scientific Products). The ribosome material was purified by hydrophobic chromatography using a 5 ml HiTrap Butyl FF column (GE Healthcare Life Sciences) equilibrated with 20 mM HEPES-KOH, pH 7.6, 10 mM Mg(OAc)2, 7 mM β-mercaptoethanol, and 1.5 M (NH4)2SO4 on an AKTApurifier UPC 10 (GE Healthcare). After loading the material, the column was washed with 20 mM HEPES-KOH, pH 7.6, 10 mM Mg(OAc)2, 7 mM β-mercaptoethanol, and 1.2 M (NH4)2SO4. Subsequently, the ribosomes were eluted with a buffer containing 20 mM HEPES-KOH, pH 7.6, 10 mM Mg(OAc)2, 7 mM β-mercaptoethanol, and 0.75 M (NH4)2SO4. The eluted fractions containing ribosomes were combined and placed in a 35 ml centrifuge tube on a 16 ml 30% sucrose cushion prepared in buffer 20 mM HEPES-KOH, pH 7.6, 10 mM Mg(OAc)2, 30 mM NH4Cl, and 7 mM β-mercaptoethanol. Ribosomes were pelletized by centrifugation at 36,000 rpm and 4°C for 18 hours using a Type 70 Ti rotor (Beckmann).The ribosome pellet was resuspended in a dissociation / storage buffer (20 mM HEPES-KOH pH 7.6, 30 mM KCl, 1.5 mM Mg(OAc)2, 7 mM β-mercaptoethanol), aliquots were flash-frozen, and stored at -80°C.

[0334] To isolate individual 50S ribosome subunits, ribosome preparations were placed in centrifuge tubes for an SW41 rotor (Beckmann) on a 10-40% sucrose gradient prepared in buffer (20 mM Tris-HCl, pH 7.5, 1.5 mM Mg(OAc)2, 100 mM NH4Cl, 2 mM β-mercaptoethanol). The gradient was centrifuged at 27,000 rpm at 4°C for 16 hours, and A 254 Fractionation was performed using a gradient fractionator (BioComp) while monitoring. The fractions corresponding to the major ribosome subunits were pooled and concentrated in a Vivaspin 2 ml concentrator (Sartorius Stedim Biotech GmbH) with a cellulose triacetate membrane, and then collected in ribosome preservation buffer (20 mM HEPES-KOH pH 7.6, 30 mM KCl, 6 mM Mg(OAc)2, 7 mM β-mercaptoethanol). Aliquots were flash-frozen and stored at -80°C.

[0335] 9. Isolation of ribosomes with non-lethal 23S rRNA mutations.

[0336] Ribosomes with non-lethal mutations in 23S rRNA (A2503G, A2062G, C2611G, and C2611U) are treated with pAM552 plasmids containing the corresponding mutations. 4 It was isolated from SQ171 cells possessing the mutant ribosome. A corresponding strain expressing a pure population of mutant ribosomes has been previously described. 18 The preparation was as described above. After sucrose cushion centrifugation, the ribosome pellet was resuspended in ribosome preservation buffer (20 mM HEPES-KOH pH 7.6, 30 mM KCl, 6 mM Mg(OAc)2, 7 mM β-mercaptoethanol) to isolate the ribosomes, except that the pellet was then resuspended. Aliquots were flash-frozen and stored at -80°C.

[0337] 10. Tryptophan printing analysis

[0338] Primer extension inhibition (tryptophan printing) analysis 19 was performed as previously described 20 as follows. If necessary, 5'-O-[N-(L-prolyl)-sulfamoyl]adenosine (L-PSA), a prolyl-tRNA synthetase inhibitor 21 was added to the reaction at a final concentration of 50 μM. After separating the primer extension products on a sequencing gel and performing phosphorimaging, ImageJ software 12 was used to determine the intensity of the tryptophan print bands. The efficiency of translation arrest induced by TnaC at the tnaC stop codon was calculated using the following equation by comparing the intensity (SB) of the tryptophan print band at the stop codon (arrow in Fig. 16c) with the intensity (PB) of the tryptophan print band at the preceding codon in the L-PSA-containing sample (open white arrow in Fig. 16c).

Equation

[0339] 11. Structural analysis and figure preparation

[0340] To calculate the distance between the 23S rRNA nucleotide and the attacking α-amino group of the A-site amino acid, the crystal structure of Thermus thermophilus ribosome (PDB 1VY4) with P-site and A-site tRNAs in the pre-attack state 22 was partially rotated and superimposed on the high-resolution structure of the vacant Escherichia coli ribosome (PDB 4YBB) 23Alignment was performed based on full-length 23S rRNA. Distance measurements and plotting were performed using PyMOL (Molecular Graphics System, version 2.0 Schrodinger, LLC). Figure 4g shows the cryo-electron microscope structure of an E. coli ribosome stalled at the P-site TnaC-tRNA (PDB 4UY8). 24 And the crystal structure of T. thermophilus sliposomes complexed with RF2 (PDB 4V67) 25 It was created by aligning them.

[0341] 12.Statistical analysis

[0342] Statistical values ​​can be found in the legend of the figures as appropriate. The mean of the values ​​was taken as the arithmetic mean. Depending on the number of independent biological copies (n), the range of deviation represents either the standard deviation (n≧3) or the experimental error (n=2). All statistical values ​​were calculated and all graphs were plotted using Microsoft Excel 365 software. Student's t-test was performed using GraphPad Prism version 8.00 for Windows (GraphPad software, La Jolla, California, USA).

[0343] References on materials and methods

[0344] 1 Gibson, DG et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6, 343-345 (2009).

[0345] 2 Kusters, JG, Jager, EJ & van der Zeijst, BA Improvement of the cloning linker of the bacterial expression vector pEX. Nucleic Acids Res 17, 8007 (1989).

[0346] 3 Carlson, E. D. et al. Engineered ribosomes with tethered subunits for expanding biological function. Nat Commun 10, 3920 (2019).

[0347] 4 Orelle, C. et al. Protein synthesis by ribosomes with tethered subunits. Nature 524, 119-124 (2015).

[0348] 5 Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97, 6640-6645 (2000).

[0349] 6 Zaporojets, D., French, S. & Squires, C. L. Products transcribed from rearranged rrn genes of Escherichia coli can assemble to form functional ribosomes. J Bacteriol 185, 6921-6927 (2003).

[0350] 7 Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-793 (2008).

[0351] 8 Davis, J. H. et al. Modular assembly of the bacterial large ribosomal subunit. Cell 167, 1610-1622 (2016).

[0352] 9 Monk, J. W. et al. Rapid and inexpensive evaluation of nonstandard amino acid incorporation in Escherichia coli. ACS Synth Biol 6, 45-54 (2017).

[0353] 10 Quan, S., Skovgaard, O., McLaughlin, R. E., Buurman, E. T. & Squires, C. L. Markerless Escherichia coli rrn deletion strains for genetic determination of ribosomal binding sites. G3 5, 2555-2557 (2015).

[0354] 11 Thomason, L. C., Costantino, N. & Court, D. L. E. coli genome manipulation by P1 transduction. Curr Protoc Mol Biol / edited by Frederick M. Ausubel ... [et al.] Chapter 1, Unit 1 17 (2007).

[0355] 12 Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9, 671-675 (2012).

[0356] 13 Bundy, B. C. & Swartz, J. R. Site-specific incorporation of p-propargyloxyphenylalanine in a cell-free environment for direct protein-protein click conjugation. Bioconjug Chem 21, 255-263 (2010).

[0357] 14 Gong, F. & Yanofsky, C. Reproducing tna operon regulation in vitro in an S-30 system. Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA. J Biol Chem 276, 1974-1983 (2001).

[0358] 15 Martinez, A. K. et al. Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan. Nucleic Acids Res 42, 1245-1256, doi:10.1093 / nar / gkt923 (2014).

[0359] 16 d’Aquino, A. E., Azim, T., Aleksashin, N.A., Hockenberry, A.H., Jewett, M.C. Mutating the ribosomal peptidyl transferase center in vitro. Nucleic Acids Res., in press (2020).

[0360] 17 Ohashi, H., Shimizu, Y., Ying, B. W. & Ueda, T. Efficient protein selection based on ribosome display system with purified components. Biochem Biophys Res Commun 352, 270-276 (2007).

[0361] 18 Vazquez-Laslop, N., Thum, C. & Mankin, A. S. Molecular mechanism of drug-dependent ribosome stalling. Mol Cell 30, 190-202 (2008).

[0362] 19 Hartz, D., McPheeters, D. S., Traut, R. & Gold, L. Extension inhibition analysis of translation initiation complexes. Methods Enzymol 164, 419-425 (1988).

[0363] 20 Orelle, C. et al. Identifying the targets of aminoacyl-tRNA synthetase inhibitors by primer extension inhibition. Nucleic Acids Res 41, e144 (2013).

[0364] 21 Heacock, D., Forsyth, C. J., Shiba, K. & MusierForsyth, K. Synthesis and aminoacyl-tRNA synthetase inhibitory activity of prolyl adenylate analogs. Bioorg Chem 24, 273-289 (1996).

[0365] 22 Polikanov, Y. S., Steitz, T. A. & Innis, C. A. A proton wire to couple aminoacyl-tRNA accommodation and peptide-bond formation on the ribosome. Nat Struct Mol Biol 21, 787-793 (2014).

[0366] 23 Noeske, J. et al. High-resolution structure of the Escherichia coli ribosome. Nat Struct Mol Biol 22, 336-341 (2015).

[0367] 24 Bischoff, L., Berninghausen, O. & Beckmann, R. Molecular basis for the ribosome functioning as an L-tryptophan sensor. Cell Rep 9, 469-475 (2014).

[0368] 25 Korostelev, A. et al. Crystal structure of a translation termination complex formed with release factor RF2. Proc Natl Acad Sci USA 105, 19684-19689 (2008).

[0369] 26 Keseler, I. M. et al. The EcoCyc database: reflecting new knowledge about Escherichia coli K-12. Nucleic Acids Res 45, D543-D550 (2017).

[0370] 27 Sato, N. S., Hirabayashi, N., Agmon, I., Yonath, A. & Suzuki, T. Comprehensive genetic selection revealed essential bases in the peptidyl-transferase center. Proc Natl Acad Sci USA 103, 15386-15391 (2006).

[0371] 28 Gong, F. & Yanofsky, C. Instruction of translating ribosome by nascent peptide. Science 297, 1864-1867 (2002).

[0372] 29 Cruz-Vera, L. R., Rajagopal, S., Squires, C. & Yanofsky, C. Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression. Mol Cell 19, 333-343 (2005).

[0373] 30 Vazquez-Laslop, N., Ramu, H., Klepacki, D., Kannan, K., Mankin, A. S. The key role of a conserved and modified rRNA residue in the ribosomal response to the nascent peptide. EMBO J. 29, 3108-3117 (2010)

[0374] 31 Yanisch-Perron, C., Vieira, J. & Messing, J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119 (1985).

[0375] 33 Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343-345 (2009).

[0376] 34 Kusters, J. G., Jager, E. J. & van der Zeijst, B. A. Improvement of the cloning linker of the bacterial expression vector pEX. Nucleic Acids Res. 17, 8007 (1989).

[0377] 35 Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Nat.l Acad. Sci. USA 97, 6640-6645 (2000).

[0378] 36 Zaporojets, D., French, S. & Squires, C. L. Products transcribed from rearranged rrn genes of Escherichia coli can assemble to form functional ribosomes. J. Bacteriol. 185, 6921-6927 (2003).

[0379] 37 Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat. Biotechnol. 26, 787-793 (2008).

[0380] 38 Davis, J. H. et al. Modular assembly of the bacterial large ribosomal subunit. Cell 167, 1610-1622 (2016)

[0381] 39 Monk, J. W. et al. Rapid and inexpensive evaluation of nonstandard amino acid incorporation in Escherichia coli. ACS Synth. Bio.l 6, 45-54 (2017).

[0382] 40 Thomason, L. C., Costantino, N. & Court, D. L. E. coli genome manipulation by P1 transduction. Curr. Protoc. Mol. Biol. / edited by Frederick M. Ausubel ... [et al.] Chapter 1, Unit 1 17 (2007).

[0383] 41 Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671-675 (2012).

[0384] 42 Bundy, B. C. & Swartz, J. R. Site-specific incorporation of p-propargyloxyphenylalanine in a cell-free environment for direct protein-protein click conjugation. Bioconjug. Chem. 21, 255-263 (2010).

[0385] 43 Gong, F. & Yanofsky, C. Reproducing tna operon regulation in vitro in an S-30 system. Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA. J. Biol. Chem. 276, 1974-1983 (2001).

[0386] 44 d’Aquino, A. E., Azim, T., Aleksashin, N.A., Hockenberry, A.H., Jewett, M.C. Mutational characterization and mapping of the 70S ribosome active site. Nucleic Acids Res 48, 2777-2789 (2020).

[0387] 45 Ohashi, H., Shimizu, Y., Ying, B. W. & Ueda, T. Efficient protein selection based on ribosome display system with purified components. Bioch. Biophys. Res. Commun. 352, 270-276 (2007).

[0388] 46 Vazquez-Laslop, N., Thum, C. & Mankin, A. S. Molecular mechanism of drug-dependent ribosome stalling. Mol. Cell 30, 190-202 (2008).

[0389] 47 Hartz, D., McPheeters, D. S., Traut, R. & Gold, L. Extension inhibition analysis of translation initiation complexes. Methods Enzymol. 164, 419-425 (1988).

[0390] 48 Orelle, C. et al. Identifying the targets of aminoacyl-tRNA synthetase inhibitors by primer extension inhibition. Nucleic Acids Res. 41, e144 (2013).

[0391] 49 Heacock, D., Forsyth, C. J., Shiba, K. & MusierForsyth, K. Synthesis and aminoacyl-tRNA synthetase inhibitory activity of prolyl adenylate analogs. Bioorg. Chem. 24, 273-289 (1996).

[0392] 50 Noeske, J. et al. High-resolution structure of the Escherichia coli ribosome. Nat. Struct. Molec. Biol. 22, 336-341 (2015).

[0393] 51 Korostelev, A. et al. Crystal structure of a translation termination complex formed with release factor RF2. Proc. Natl. Acad. Sci. USA 105, 19684-19689 (2008).

[0394] Additional references

[0395] Rackham, O.; Chin, JW, Compositions and methods relating to orthogonal ribosome mRNA pairs. US 11 / 982,877: Filing date Nov 6, 2007.

[0396] Chin, J.; Wang, K.; Neumann, H., Orthogonal Q-Ribosomes. US 13 / 517,372: Filing date Dec 20, 2010.

[0397] Chin, J.; Wang, K.; Neumann, H., Evolved orthogonal ribosomes. US 12 / 516,230: Filing date Nov 28, 2007.

[0398] E. Applications and advantages of the "flipped" orthogonal translation system of Example 1

[0399] 1.Application

[0400] For example, but not limited to, applications of the compositions or methods disclosed herein include: ribosome evolution / engineering (e.g., towards more efficient non-canonical amino acid uptake); extended genetic codes for non-canonical amino acid uptake; enabling detailed in vivo studies of antibiotic resistance mechanisms and facilitating antibiotic development processes; biopharmaceutical manufacturing; intracellular orthogonal circuits; synthetic biology; creating artificial peptides by incorporating novel functionalities that cannot be obtained from peptides synthesized by native (or wild-type) ribosomes or their post-translational modified derivatives; creating novel protease-resistant peptides that could transform medicinal chemistry; and enabling the evolution of modified ribosomes within cells.

[0401] 2. Advantages

[0402] For example, but not limited to, the advantages of the compositions and methods disclosed herein include the following:

[0403] The unusual design of Ribo-T limits its function as an orthogonal translation system (oRiboT). Specifically, Ribo-T translates proteins at half the rate of dissociable ribosomes. Compared to wild-type ribosomes, it is slower to detach from the start codon. Furthermore, even "wild-type" Ribo-T is quite slow and inefficient to biogenesis, and if the ribosome's functional center is subjected to further modification, assembly problems may worsen.

[0404] To overcome the shortcomings of the original oRibo-T-based method, which modifies cells using two functionally independent translation machinery, we have now created a conceptually novel in vivo system design utilizing ribosomes that are dissociable and even completely isolated, and specialized for translating only specific mRNAs. By "reversing" the roles of Ribo-T and dissociable ribosomes, we created bacterial cells in which proteome translation is performed by Ribo-T, and ribosomes composed of dissociable orthogonal 30S (o-30S) subunits and wild-type 50S subunits function as a fully orthogonal translation machine. In this resulting setup, which we have named OSYRIS (Orthogonal translation SYstem based on Ribosomes with Isolated Subunits), the tethered nature of Ribo-T prevents binding to the o-30S or 50S of dissociable ribosomes, thus achieving complete orthogonality. Therefore, in OSYRIS cells, the physically unlinked o-30S and 50S ribosome subunits are nevertheless compelled to interact with each other and function as fully orthogonal ribosomes (o-ribosomes). As a result, not only the o-30S but also the free 50S subunit can be modified to achieve new functionalities without interfering with the expression of the cellular proteome.

[0405] When OSYRIS cells were compared side-by-side, the expression of two orthogonal reporters (o-gfp and a newly modified o-luc) was carried out by either dissociable orthogonal ribosomes (oRbs) or orthogonal tethered ribosomes (oRibo-T) in the same host (E. coli BL21). Notably, oRbs outperformed oRibo-T, even though oRbs were expressed from high-copy-number vectors and oRbs were transcribed from low-copy-number plasmids. This result clearly demonstrates the advantages offered by oRbs over oRibo-T in the translation of orthogonal mRNA and reinforces the concept that OSYRIS designs are superior to designs based on oRibo-T.

[0406] Ribosome engineering is of great interest to the fields of biotechnology, chemistry, and materials science, but previous methods have not been able to evolve the large ribosome subunits, including catalytic active sites and protein efflux tunnels. The development of tethered ribosomes removes these limitations and expands the possibilities of ribosome engineering. Ribosomes can be modified to incorporate non-natural amino acids to extend protein function or to achieve new chemistry for the production of non-protein polymers.

[0407] This invention details the first-ever orthogonal ribosome-mRNA system in which mRNA sequencing, polypeptide synthesis catalysis, and protein excretion can all be optimized for novel substrates and functions. A key difference from prior art is that not only the small (sequencing) ribosome subunit but also the large (catalytic) ribosome subunit function as a single, combined, and indivisible orthogonal gene synthesis machine.

[0408] Furthermore, tethered ribosomes are unique in that they utilize ribosomes that can survive cells and dissociate freely in their design.

[0409] The inventors emphasize that this invention is the first of its kind. They anticipate that the innovation reported herein will stimulate larger-scale ribosome construction and engineering efforts, helping to push the limits of modified biological systems and creating new commercial opportunities in research areas that are currently beyond adjacency.

[0410] As will be readily apparent to those skilled in the art, various substitutions and modifications can be made to the inventions disclosed herein without departing from the scope and spirit of the invention. The inventions described exemplary herein can be suitably carried out in the presence of any or more elements, limitations, or restrictions not specifically disclosed herein. The terms and expressions used are for illustrative purposes only and not for limiting purposes, and in the use of such terms and expressions there is no intention to exclude any equivalent or part thereof of the presented and described features, but it is recognized that various modifications are possible within the scope of the invention. Therefore, although the invention is illustrated by specific embodiments and optional features, it should be understood that modifications and / or variations of the concepts disclosed herein can be relied upon to those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention.

[0411] This specification cites numerous patent and non-patent references. These cited references are incorporated herein by reference in their entirety. In the event of any conflict between the definitions of terms in this specification and those in the cited references, the terms should be interpreted according to the definitions in this specification. Some aspects of the present invention are described below. 1. Modified cells comprising a first protein translation mechanism and a second protein translation mechanism, a) The first protein translation mechanism includes a first modified ribosome, and the first modified ribosome is i) Small subunits containing ribosomal RNA (rRNA) and proteins, ii) Large subunits including ribosomal RNA (rRNA) and proteins, and iii) Connecting part Includes, In this configuration, the linking portion includes a polynucleotide sequence, and the rRNA of the small subunit is linked to the rRNA of the large subunit. b) The second protein translation mechanism includes a second modified ribosome, and the second modified ribosome is i) Small subunits containing rRNA and protein, and ii) Large subunits containing rRNA and proteins Includes, In this case, the second modified ribosome lacks the connecting portion between the large subunit and the small subunit, and Therein, the small subunit of the second modified ribosome contains a modified anti-Shine-Dalgano sequence, enabling the translation of a template having a Shine-Dalgano sequence that is different from and / or congenerally related to the endogenous cellular mRNA of the cell. Modified cells. 2. The modified cell according to item 1, wherein the first and second protein translation mechanisms are capable of assisting the translation of sequence-controlled polymers. 3. The modified cell described in item 1, wherein the first protein translation mechanism described above can assist in the translation of native endogenous RNA. 4. The modified cell described in item 1, wherein the second protein translation mechanism described above can assist in the translation of non-native exogenous RNA. 5. The modified cell according to item 1, wherein the second modified ribosome comprises one or more functionally transformative mutations, wherein the functionally transformative mutation is not the anti-shine-dalgano sequence. 6. The modified cell according to item 1, wherein the small subunit of the second modified ribosome contains a modified anti-shine-Dalgano sequence selected from the group consisting of 3'-GGUGUU-5', 3'-UGGUGU-5', 3'-GGUGUC-5', 3'-GUUUAG-5', 3'-UGGAAU-5', 3'-GGAUCU-5', 3'-UGGAUC-5', 3'-UGGUAA-5', and 3'-UGGAUC-5'. 7. The second modified ribosome described above, a) Peptidyltransferase center (PTC), b) New peptide exit tunnel (NPET), c) Interaction site with elongation factor, d) tRNA binding site, e) Chaperone binding site, f) nascent chain modification enzyme binding site; g) GTPase center One or more of these include functional conversion mutations. Modified cells as described in item 1. 8. The modified cell according to item 1, wherein the large subunit of the second modified ribosome contains a functionally transformative mutation in one or more of the following residues of 23S rRNA: G2061, C2452, U2585, G2251, G2252, A2057, A2058, C2611, A2062, A2503, U2609, G2454, and G2455. 9. The modified cell described in item 1, wherein the first, second, or both of the first and second modified ribosomes contain antibiotic resistance mutations. 10. The modified cell according to item 1, wherein the large subunit of the first modified ribosome contains a permutation substitution variant or mutant of 23S rRNA, and / or the small subunit contains a permutation substitution variant or mutant of 16S rRNA. 11. The modified cell according to item 1, wherein the connecting portion covalently connects the helix of the large subunit selected from the group consisting of helix 10, helix 38, helix 42, helix 54, helix 58, helix 63, helix 78, and helix 101 to the helix of the small subunit selected from the group consisting of helix 11, helix 26, helix 33, and helix 44. 12. A method for preparing sequence-defined polymers, (a) one or more (i) The cells described in item 1, (ii) Cell-free extract derived from the cells described in any one of item 1, (iii) Purified translation system derived from the cells described in item 1 To prepare, b) Providing mRNA encoding the sequence-defined polymer to the cells or the cell-free extract, and c) Translate the mRNA in the cells or cell-free extract to obtain the sequence-defined polymer. Methods that include... 13. The method according to item 12, wherein the sequence-defined polymer is prepared in vivo. 14. The method according to item 12, wherein the sequence-defined polymer is prepared in vitro. 15. The cell-free extract is obtained from cell cultures in the mid-to-late stages of the exponential growth phase or from cells with an OD of at least approximately 2.0, 2.5, or 3.0 at the time of collection. 600 The method according to item 12, comprising an S150 extract prepared from a culture having [the specified characteristic]. 16. The method according to item 12, wherein the mRNA encoding the sequence-defined polymer comprises a modified Shine-Dalgano sequence, and the modified ribosome of the second translation system comprises an anti-Shine-Dalgano sequence complementary to the modified Shine-Dalgano sequence of the mRNA. 17. The method according to item 12, wherein the sequence-defined polymer comprises an amino acid polymer. 18. The method according to item 17, wherein the amino acid polymer contains canonical amino acids. 19. The method according to item 17, wherein the amino acid polymer comprises one or more non-canonical amino acids. 20. The method according to item 12, wherein the sequence-defined polymer includes a non-amino acid-based polymer. 21. The method according to item 12, wherein the mRNA is prepared by preparing DNA encoding the mRNA and transcribing the DNA in the cell or the cell-free extract to obtain the mRNA. 22. The method according to item 21, wherein the modified cells are modified to express an exogenous RNA transcriptase, such as a T7 RNA transcriptase that transcribes the DNA, thereby producing the mRNA. 23. One or more polynucleotides that a) encode the rRNA of the modified ribosome of the first protein translation mechanism and / or b) encode the rRNA of the modified ribosome of the second protein translation mechanism of the modified cell as described in item 1. 24. The polynucleotide described in item 23, wherein the polynucleotide is a vector. 25. The polynucleotide according to item 23, wherein the polynucleotide further comprises a gene expressed by the modified ribosome. 26. The polynucleotide described in item 25, wherein the gene is a reporter gene or a selection marker. 27. The polynucleotide described in item 26, wherein the reporter gene is the green fluorescent protein gene. 28. The polynucleotide described in item 26, wherein the selection marker is resistant to the antibiotic. 29. The polynucleotide according to item 23, wherein the rRNA of the modified ribosome of the second translation system comprises a modified anti-Shine-Dalgano sequence, and the mRNA comprises a Shine-Dalgano sequence complementary to the modified ribosome of the second translation system. 30. The polynucleotide described in item 23, wherein the mRNA contains a codon, and the codon codes for a non-natural amino acid. 31.a) a polynucleotide according to item 23, comprising a first polynucleotide encoding the rRNA of the modified ribosome of the first translation system, and a) a second polynucleotide comprising a second polynucleotide encoding the rRNA of the modified ribosome of the second translation system. 32. The polynucleotide described in item 31, wherein the first polynucleotide and / or the second nucleotide are present on one or more vectors. 33. A method for preparing modified ribosomes, comprising expressing the polynucleotide described in item 23 in a host cell, wherein the host cell optionally includes the modified cell described in item 1. 34. The method according to item 33, further comprising subjecting the host cells to selection and selecting host cells containing mutant ribosomes. 35. The aforementioned mutant is a) Peptidyltransferase center (PTC), b) New peptide exit tunnel (NPET), c) Interaction site with elongation factor, d) tRNA binding site, e) Chaperone binding site, f) nascent chain modification enzyme binding site; g) GTPase center, h) Interaction sites with the translocon, and i) Interaction sites with translation-promoting accessory proteins The method described in item 34, comprising a mutation in one or more of the following. 36. The method according to item 35, wherein the selection step includes a negative selection step, a positive selection step, or both negative and positive selection steps.

Claims

1. A modified cell comprising a first protein translation system and a second protein translation system, a) The first protein translation system includes a first modified ribosome, and the first modified ribosome is i) Small subunits containing ribosomal RNA (rRNA) and proteins, ii) Large subunits including rRNA and protein, and iii) Connecting part Includes, In this configuration, the linking portion includes a polynucleotide sequence, and the rRNA of the small subunit is linked to the rRNA of the large subunit. b) The second protein translation system includes a second modified ribosome, and the second modified ribosome is i) Small subunits including rRNA and protein, and ii) Large subunits containing rRNA and proteins Includes, In this case, the second modified ribosome lacks the connecting portion between the large subunit and the small subunit, and Therein, the small subunit of the second modified ribosome contains a modified anti-Shine Dalgano sequence, enabling the translation of a template having a Shine Dalgano sequence that is different from and / or congenerally related to the endogenous cellular mRNA of the cell. Modified cells.

2. The modified cell according to claim 1, wherein the first and second protein translation systems can assist in the translation of a sequence-defined polymer.

3. The modified cell according to claim 1, wherein the first protein translation system can assist in the translation of native endogenous RNA.

4. The modified cell according to claim 1, wherein the second protein translation system can assist in the translation of non-native exogenous RNA.

5. The modified cell according to claim 1, wherein the second modified ribosome comprises one or more functionally transformative mutations, wherein the functionally transformative mutation is not the anti-shine-dalgano sequence.

6. The modified cell according to claim 1, wherein the small subunit of the second modified ribosome comprises a modified anti-shine dalgano sequence selected from the group consisting of 3'-GGUGUU-5', 3'-UGGGUGU-5', 3'-GGUGUC-5', 3'-GUUUAG-5', 3'-UGGAAU-5', 3'-GGAUCU-5', 3'-UGGAUC-5', 3'-UGGUAA-5', and 3'-UGGAUC-5'.

7. The second modified ribosome described above, a) Peptidyltransferase center (PTC), b) New peptide exit tunnel (NPET), c) Interaction site with elongation factor, d) tRNA binding site; e) Chaperone binding site, f) nascent chain modification enzyme binding site; g) GTPase center One or more of these include functional conversion mutations. The modified cell according to claim 1.

8. The modified cell according to claim 1, wherein the large subunit of the second modified ribosome contains a functionally transformative mutation in one or more of the following residues of 23S rRNA: G2061, C2452, U2585, G2251, G2252, A2057, A2058, C2611, A2062, A2503, U2609, G2454, and G2455.

9. The modified cell according to claim 1, wherein the first, second, or both of the first and second modified ribosomes contain an antibiotic resistance mutation.

10. The modified cell according to claim 1, wherein the large subunit of the first modified ribosome contains a permutation substitution variant or mutant of 23S rRNA, and / or the small subunit contains a permutation substitution variant or mutant of 16S rRNA.

11. The modified cell according to claim 1, wherein the connecting portion covalently bonds the helix of the large subunit, selected from the group consisting of helix 10, helix 38, helix 42, helix 54, helix 58, helix 63, helix 78, and helix 101, to the helix of the small subunit, selected from the group consisting of helix 11, helix 26, helix 33, and helix 44.

12. A method for preparing sequence-controlled polymers in vitro, (a) one or more (i) The cell according to claim 1, (ii) A cell-free extract comprising the first and second protein translation systems derived from the cells according to claim 1, (iii) Purified first and second protein translation systems according to claim 1 To prepare, b) Providing mRNA encoding the sequence-controlled polymer to the cells, the cell-free extract, or the purified first and second protein translation systems, and c) Translate the mRNA in the cells, the cell-free extract, or the purified first and second protein translation systems to obtain the sequence-defined polymer. Methods that include...

13. The cell-free extract is a cell culture in the mid-to-late stages of the exponential growth phase or has an OD of at least 2.0, 2.5, or 3.0 at the time of collection. 600 The method according to claim 12, comprising an S150 extract prepared from a culture having the following characteristics.

14. The method according to claim 12, wherein the mRNA encoding the sequence-defined polymer comprises a modified Shine-Dalgano sequence, and the modified ribosome of the second translation system comprises an anti-Shine-Dalgano sequence complementary to the modified Shine-Dalgano sequence of the mRNA.

15. The method according to claim 12, wherein the sequence-controlling polymer includes an amino acid polymer.

16. The method according to claim 15, wherein the amino acid polymer contains canonical amino acids.

17. The method according to claim 15, wherein the amino acid polymer comprises one or more non-canonical amino acids.

18. The method according to claim 12, wherein the sequence-defined polymer includes a non-amino acid-based polymer.

19. The method according to claim 12, wherein the mRNA is prepared by preparing DNA encoding the mRNA and transcribing the DNA in the cell or the cell-free extract to obtain the mRNA.

20. The method according to claim 19, wherein the modified cells are modified to express an exogenous RNA transcriptase, such as T7 RNA transcriptase, that transcribes the DNA, thereby yielding the mRNA.

21. One or more polynucleotides comprising both a) a first polynucleotide encoding the rRNA of the modified ribosome in the first protein translation system of the modified cell according to claim 1, and b) a second polynucleotide encoding the rRNA of the modified ribosome in the second protein translation system of the modified cell according to claim 1.

22. The polynucleotide according to claim 21, wherein the polynucleotide is a vector.

23. The polynucleotide according to claim 21, wherein the one or more polynucleotides further comprise a gene expressed by the modified ribosome.

24. The polynucleotide according to claim 23, wherein the gene is a reporter gene or a selection marker.

25. The polynucleotide according to claim 24, wherein the reporter gene is a green fluorescent protein gene.

26. The polynucleotide according to claim 24, wherein the selection marker is resistant to antibiotics.

27. The polynucleotide according to claim 21, wherein the rRNA of the modified ribosome in the second translation system comprises a modified anti-Shine-Dalgarno sequence complementary to mRNA containing a Shine-Dalgarno sequence different from that of endogenous cell mRNA.

28. The polynucleotide according to claim 27, wherein the mRNA contains a codon, and the codon codes for a non-natural amino acid.

29. The polynucleotide according to claim 21, wherein the first polynucleotide and / or the second polynucleotide are present on one or more vectors.

30. A method for preparing modified ribosomes in vitro, comprising expressing the polynucleotide described in claim 21 in a host cell, wherein the host cell includes the modified cell described in claim 1.

31. The method according to claim 30, further comprising subjecting the host cells to selection and selecting host cells containing mutant ribosomes.

32. The aforementioned mutant is a) Peptidyltransferase center (PTC), b) New peptide exit tunnel (NPET), c) Interaction site with elongation factor, d) tRNA binding site; e) Chaperone binding site, f) nascent chain modification enzyme binding site; g) GTPase center, h) Interaction sites with the translocon, and i) Interaction sites with translation-promoting accessory proteins The method according to claim 31, wherein one or more of the features are modified.

33. The method according to claim 32, further comprising the step of selecting the variant, wherein the selection step comprises a negative selection step, a positive selection step, or a selection step of both negative and positive.

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