Patent Application
20230117150
This invention pertains to 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 may be utilized to prepare sequence defined polymers.
The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established paradigm of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed essential for biogenesis since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation [Karbstein 2013]. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation.
The ribosome is an extraordinary complex machine. This large particle, in which RNA is the main structural and functional component, is invariably comprised of two subunits that coordinate distinct but complementary functions: the small subunit decodes the mRNA, while the large subunit catalyzes peptide-bond formation and provides the exit tunnel for the polypeptide. The association of the subunits is tightly regulated throughout the cycle of translation. First, several assembly factors prevent the two subunits from associating during maturation of the ribonucleoproteins. Later on, the initiation of translation is also strictly controlled such that initiation factors, mRNA and fMet-tRNAfMet sequentially join the small subunit to form a pre-initiation complex before recruiting the large subunit. During elongation, the subunits ratchet relative to each other with an angle of about 6 degrees. Upon termination, the newly synthesized protein is released from the ribosome and the subunits dissociate during an active process called ribosome recycling to prepare for additional rounds of translation. Thus, the requirement for programmed subunit association and dissociation at specific stages of translation likely explains why the ribosome has been maintained as two subunits during the course of evolution. Although initiation at the leaderless mRNAs was suggested to be carried out by the 70S ribosome with pre-associated subunits, no experimental evidence exists showing that the full cycle of protein synthesis could be accomplished by the ribosome with inseparable subunits.
The random exchange of ribosomal subunits between recurrent acts of protein biosynthesis presents an obstacle for making fully orthogonal ribosomes, a task with important implications for both fundamental science and bioengineering. Previously, it was possible to redirect a subpopulation of the small ribosomal subunits from translating indigenous mRNA to translation of a specific mRNA by placing an alternative Shine-Dalgarno (SD) sequence in a reporter mRNA and introducing the complementary changes in the anti-SD region in 16S rRNA [Hui 1987; Rackham 2005], which enabled selection of mutant 30S subunits with new decoding properties [Wang 2007]. However, because large subunits freely exchange between native and orthogonal small subunits, creating a fully orthogonal ribosome has been impossible thereby limiting the engineering of the 50S subunit, including the peptidyl transferase center (PTC) and the nascent peptide exit tunnel, for specialized new properties.
The engineering of a tethered ribosome, in which the subunits are linked to each other, could open new venues preparing orthogonal translation systems, evolving the ribosome for the incorporation of unnatural amino acids in synthetic biology, and molecularly characterizing dominant lethal mutations. Previously, we and others disclosed tethered ribosomes and methods of making and using tethered ribosomes. (See International Published Application WO 2015/184283, “Tethered Ribosomes and Methods of Making and Using Thereof,” and Orelle et al., “Protein synthesis by ribosomes with tethered subunits,” Nature, 6 Aug. 2015, Vol. 524, page 119). Here, we disclose further improvements to systems and methods that incorporate ribosomes with tethered subunits.
Disclosed herein are engineered polynucleotides, engineered ribosomes, and engineered cells and systems. The engineered polynucleotides, engineered ribosomes, and engineered cells and systems may be utilized in methods for preparing sequence defined polymers. In some embodiments, the engineered ribosomes comprise a small subunit, a large subunit, and a linking moiety comprising a polynucleotide sequence, wherein the linking moiety tethers the small subunit with the large subunit and wherein the engineered ribosome is capable of supporting translation of a sequence defined polymer.
In certain embodiments, the small subunit of the engineered ribosomes comprises rRNA and protein, the large subunit of the engineered ribosomes comprises rRNA and protein, and the linking moiety tethers the rRNA of the small subunit with the rRNA of the large subunit. In certain embodiments, the large subunit comprises a permuted variant of a 23S rRNA. In certain embodiments, the small subunit comprises a permuted variant of a 16S rRNA. As such, in certain embodiments, the engineered ribosomes comprise an engineered polynucleotide comprising a fusion of: (a) 16S rRNA, a permuted variant thereof, or fragments thereof; and (b) 23S rRNA, a permuted variant thereof, or fragments thereof.
The rRNA of the small subunit of the disclosed engineered ribosomes may comprise an anti-Shine-Dalgarno (anti-SD) sequence. In some embodiments, the anti-SD sequence of the rRNA of the small subunit of the engineered ribosomes corresponds or is identical to the native anti-SD sequence of an engineered host cell that comprises the engineered ribosome. In such embodiments, the anti-SD sequence of the rRNA of the small subunit of the engineered ribosomes exhibits reverse complementarity to the Shine-Delgarno (SD) sequence of the native mRNA's of the engineered host cell. In some embodiments of the disclosed engineered ribosomes, the rRNA of the small subunit of the disclosed engineered ribosomes is linked to the rRNA of the large submit via a linking moiety comprising a polynucleotide sequence, where the engineered ribosomes may be described as having tethered large subunits and small subunits and comprising a native anti-SD sequence of the engineered host cell that comprises the engineered ribosome which exhibits reverse complementarity to the SD sequence of native mRNA's of the engineered host cell. As such, the engineered ribosomes having tethered large subunits and small subunits may support translation using native mRNA's of the engineered host cell.
In other embodiments, the anti-SD sequence of the rRNA of the small subunit of the engineered ribosome is modified to include base substitutions relative to the anti-SD sequence of native mRNA's of an engineered host cell that comprises the engineered ribosome (or relative to the anti-SD sequence of the first engineered ribosome). In such embodiments, the engineered host cell may be engineered to comprise modified mRNA's having a modified anti-SD sequence that exhibits reverse complementarity to the modified anti-SD sequence of the rRNA of the small subunit of the engineered ribosome permitting translation of the modified mRNA's by the engineered ribosome having an rRNA with a modified anti-SD sequence.
The disclosed engineered ribosomes may be combined for use in engineered host cells. In some embodiments, the disclosed combination of ribosomes may include a first engineered ribosome and a second engineered ribosome. The first engineered ribosome may comprise: i) a small subunit comprising ribosomal RNA (rRNA) and protein, ii) a large subunit comprising ribosomal RNA (rRNA) and protein, and iii) a linking moiety; where the linking moiety comprises a polynucleotide sequence and tethers the rRNA of the small subunit with the rRNA of the large subunit. In some embodiments, the rRNA of the small subunit of the first engineered ribosome comprises an anti-SD sequence corresponding to the SD sequence of native mRNA's of the engineered host cell permitting translation of native mRNA's of the engineered host cell and preferably not permitting translation of mRNA's having a modified SD sequence (i.e., a modified SD sequence having one or more nucleotide substitutions relative to the anti-SD sequence of native ribosomes of the engineered host cell). The second engineered ribosome may comprise: i) a small subunit comprising rRNA and protein; and ii) a large subunit comprising rRNA and protein; where the second engineered ribosome lacks a linking moiety between the large subunit. In some embodiments, the rRNA of the small subunit of the second engineered ribosome comprises a modified anti-SD sequence having one or more nucleotide substitutions relative to the anti-SD sequence of native ribosomes of the engineered host cell (and/or relative to the anti-SD of the first engineered ribosome). The modified anti-SD sequence preferentially permits translation of mRNA templates having a complementary or cognate SD sequence that is different from the SD sequence of native cellular mRNAs and/or an anti-SD sequence that is different than the anti-SD sequence of the first engineered ribosome (i.e., permitting translation of mRNA's having a modified SD sequence that is complementary to the anti-SD of the rRNA of the small subunit of the second ribosome permitting translation of the mRNA's having a modified SD sequence by the second ribosome, and preferably not permitting translation of native mRNA's of the engineered host cell by the second engineered ribosome) and/or where the second engineered ribosome comprises one or more change-of-function mutations in the large subunit and/or small subunit relative to the native ribosomes of the engineered host cell (or relative to the first engineered ribosome) which change-of-function mutations are not present at the anti-SD sequence.
In certain embodiments where the large subunit and small subunit of the engineered ribosomes are tethered by a linking moiety, the linking moiety covalently bound a helix of the large subunit to a helix of the small subunit. In certain embodiments where the large subunit comprises a 23S rRNA (or a permuted variant of 23S rRNA) and the small subunit comprises a 16S rRNA (or a permuted variant of 16S rRNA), the linking moiety covalently bonds helix 10, helix 38, helix 42, helix 54, helix 58, helix 63, helix 78, or helix 101 of 23S rRNA (or a permuted variant of 23S rRNA) to a helix of 16S rRNA (or a permuted variant of 16S rRNA). In certain embodiments where the large subunit comprises a 23S rRNA (or a permuted variant of 23S rRNA) and the small subunit comprises a 16S rRNA (or a permuted variant of 16S rRNA), the linking moiety covalently bonds, the linking moiety covalently bonds helix 11, helix 26, helix 33, or helix 44 of 16S rRNA (or a permuted variant of 16S rRNA) to a helix of 23S rRNA (or a permuted variant of 23S rRNA).
In certain embodiments, the large subunit comprises a L1 polynucleotide domain, a L2 polynucleotide domain, and a C polynucleotide domain, wherein the L1 domain is followed, in order, by the C domain and the L2 domain, from 5′ to 3′. In certain embodiments, the polynucleotide consisting essentially of the L2 domain followed by the L1 domain, from 5′ to 3′, is substantially identical to 23S rRNA (e.g., 23S rRNA of E. coli). In certain embodiments, the polynucleotide consisting essentially of the L2 domain followed by the L1 domain, from 5′ to 3′, is at least 95% identical to a 23S rRNA. In certain embodiments, the C domain comprises a polynucleotide having a length ranging from 1-200 nucleotides. In certain embodiments, the C domain comprises a GAGA polynucleotide.
In certain embodiments, the small subunit comprises a S1 polynucleotide domain and a S2 polynucleotide domain, wherein the S1 domain is followed, in order, by the S2 domain, from 5′ to 3′. In certain embodiments, the polynucleotide consisting essentially of the S1 domain followed by the S2 domain, from 5′ to 3′, is substantially identical to a 16S rRNA (e.g., 16S rRNA of E. coli). In certain embodiments, the polynucleotide consisting essentially of the S1 domain followed by the S2 domain, from 5′ to 3′, is at least 95% identical to a 16S rRNA.
In certain embodiments, the linking moiety comprises a T1 polynucleotide domain and a T2 polynucleotide domain. In certain embodiments, the T1 domain links the S1 domain and the L1 domain and wherein the S1 domain is followed, in order, by the T1 domain and the L1 domain, from 5′ to 3′. In certain embodiments, the T1 domain comprises a polynucleotide having a length ranging from 5 to 200 nucleotides. In certain embodiments, the T1 domain comprises a polynucleotide having a length ranging from 7 to 40 nucleotides. In certain embodiments, the T1 domain comprises a polyadenine polynucleotide. In certain embodiments, the T1 domain comprises a polyadenine polynucleotide having a length of 7 to 12 adenine nucleotides. In certain embodiments, the T2 domain links the S2 domain and the L2 domain and wherein the L2 domain is followed, in order, by the T2 domain and the S2 domain, from 5′ to 3′. In certain embodiments, the T2 domain comprises a polynucleotide having a length ranging from 5 to 200 nucleotides. In certain embodiments, the T2 domain comprises a polynucleotide having a length ranging from 7 to 20 nucleotides. In certain embodiments, the T2 domain comprises a polyadenine polynucleotide. In certain embodiments, the T2 domain comprises a polyadenine polynucleotide having a length of 7 to 12 adenine nucleotides.
In certain embodiments, an engineered ribosome comprises the S1 domain followed, in order, by the T1 domain, the L1 domain, the C domain, the L2 domain, the T2 domain, and the S2 domain, from 5′ to 3′. In certain embodiments, the engineered ribosome comprises a polynucleotide consisting essentially of the S1 domain followed, in order, by the T1 domain, the L1 domain, the C domain, the L2 domain, the T2 domain, and the S2 domain, from 5′ to 3′.
In certain embodiments, the disclosed engineered ribosomes comprise a mutation relative to a wild-type host cell (e.g., relative to wild-type E. coli). In certain embodiments, the mutation is a change-of-function mutation. In certain embodiments, the change-of-function mutation is a gain-of-function mutation. In certain embodiments, the gain-of-function mutation is present in a peptidyl transferase center of the large subunit of the engineered ribosomes. In certain embodiments, the gain-of-function mutation is present in an A-site of the peptidyl transferase center of the large subunit of the engineered ribosomes. In certain embodiments, the gain-of-function mutation is present in the exit tunnel of the large subunit of the engineered ribosomes. In certain embodiments, the engineered ribosome comprise an antibiotic resistance mutation present in the large subunit and/or small subunit of the engineered ribosomes.
Also disclosed herein are polynucleotides, the polynucleotides encoding the rRNA of the engineered ribosomes. In certain embodiments, the polynucleotide is a vector. In certain embodiments, the polynucleotide further comprises a gene to be expressed by the engineered 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 engineered ribosome comprises a modified anti-SD sequence and the gene comprises a complementary modified SD sequence corresponding to the anti-SD sequence of the engineered ribosomes. In certain embodiments, the gene comprises a codon and the codon encodes for an unnatural amino acid. In some embodiments, the ribosome comprising the modified anti-SD sequence is an untethered ribosome.
Also disclosed herein are methods for preparing an engineered ribosome, the method comprising expressing a polynucleotide encoding the rRNA of the engineered ribosome, for example, in an engineered host cell such as E. coli. In certain embodiments, the method further comprises preparing the engineered ribosome in a host cell, expressing a selectable marker, and selecting an engineered ribosome that expresses the selectable marker in the engineered host cell. In some embodiments, the selected engineered ribosome will include one or more mutations relative to the engineered ribosome that was expressed in the engineered host cell (and/or relative to the native ribosomes of the engineered host cell). In certain embodiments, the selection step comprises a negative selection step, a positive selection step, or both a negative and a positive selection step.
Also disclosed herein are engineered cells. The engineered cells are host cells, such as E coli cells, comprising (i) a polynucleotide encoding the rRNA of the engineered ribosome, (ii) the engineered ribosome, or both (i) and (ii). In some embodiments, the engineered host cells comprise a first engineered ribosome having a large subunit and a small subunit which are tethered, where the small subunit comprises rRNA having an anti-SD sequence corresponding to the SD sequence of native mRNA's of the engineered host cell. In some embodiments, the engineered host cells further comprise a second engineered ribosome having a large subunit and a small subunit which are not tethered, where the small subunit comprises rRNA having an anti-SD sequence which is modified relative to the SD sequence of native mRNA's of the engineered host cell and permits translation of mRNA's having a modified SD sequence corresponding to the modified anti-SD sequence of the rRNA of the small subunit of the second ribosome.
In some embodiments, the engineered cells comprise a first protein translation mechanism and a second protein translation mechanism. The first protein translation mechanism may comprise a first engineered ribosome, wherein the first engineered ribosome includes a linking moiety to tether the first and the second subunits. The second translation mechanism may comprise a second engineered ribosome, wherein the second engineered ribosome lacks a linking moiety between the large subunit and the small subunit. In some embodiments, the second engineered ribosome comprises a modified anti-SD sequence relative to the anti-SD sequence of native ribosomes which is complementary to the SD sequence of native mRNA's (and/or relative to the anti-SD sequence of the first engineered ribosome) and/or a change-of-function mutation other than at the anti-SD sequence relative to the native ribosomes of the engineered cells (and/or relative to the first engineered ribosome).
Also disclosed herein are methods for preparing a sequence-defined polymer, the methods comprising (a) providing an engineered ribosome or an engineered cell comprising one or more engineered ribosomes, and (b) providing an mRNA or DNA template encoding the sequence-defined polymer, and preparing the sequence-defined polymer using the one or more engineered ribosomes, the engineered cell comprising the one or more engineered ribosomes, and the mRNA or DNA template encoding the sequence-defined polymer. The sequence-defined polymer may be prepared in vitro and/or in vivo.
In certain embodiments, the sequence-defined polymer is prepared in vitro, and the methods further comprise providing (c) a ribosome-depleted cellular extract or purified translation system and using the ribosome-depleted cellular extract or purified translation system to preparing the sequence-defined polymer. In certain embodiments, the ribosome-depleted cellular extract comprises an S150 extract prepared from mid- to late-exponential growth phase cell cultures or cultures having an OD600 of at least about 2.0, 2.5, or 3.0 at time of harvest.
In certain embodiments, the sequence defined polymer is prepared in vivo. The sequence defined polymer may be prepared in an engineered cell comprising a first and second translation system comprising engineered ribosomes, wherein the first translation system comprises tethered ribosomes having a wild-type anti-SD sequence (i.e., the native anti-SD sequence of ribosomes of the engineered host cell which is complementary to the SD sequence of native mRNA's of the engineered host cell), and wherein the second translation system comprises untethered ribosomes having (a) a modified anti-SD sequence (e.g., relative to the native anti-SD sequence of ribosomes of the engineered host cell or relative to the anti-SD of the tethered ribosomes of the first translation system), which is not complementary to the SD sequence of native mRNA's of the host cell) and/or (b) a change-of-function mutation other than at the anti-SD sequence, which mutation is relative to the native ribosomes of the engineered host cell or relative to the tethered ribosome of the first translation system. In certain embodiments, the mRNA or DNA encoding the sequence-defined polymer comprises a modified SD sequence and the untethered, engineered ribosome of the second translation system comprises a modified anti-SD sequence complementary to the modified SD sequence of the mRNA or DNA encoding the sequence-defined polymer permitting translation of the mRNA encoding the sequence-defined polymer permitting translation by the second translation system (and preferably permitting translation by the first translation system).
In certain embodiments, the sequence-defined polymer comprises an amino acid. In certain embodiments, the amino acid is a natural amino acid. In certain embodiments, the amino acid is an unnatural or non-canonical amino acid and the untethered, engineered ribosomes of the second translation system comprise one or more mutations relative to the native ribosomes (or relative to the tethered, engineered ribosomes of the first translation system), which permit incorporation of the unnatural or non-canonical amino acid into the sequence-defined polymer.
Ribosomes with tethered and thus inseparable subunits (“Ribo-T”) that are capable of successfully carrying out protein synthesis are disclosed. Ribo-T may be prepared by engineering a ribosome comprising a small subunit, a large subunit, and a linking moiety that tethers the small subunit with the large subunit. The engineered ribosome may comprise a hybrid rRNA comprising a small subunit rRNA sequence, a large subunit rRNA sequence, and RNA linkers that may covalently link the small subunit rRNA sequence and the large subunit rRNA sequence into a single entity. The engineered ribosome may be prepared by expressing a polynucleotide encoding the rRNA of the engineered ribosome. The engineered ribosome may also be evolved by positively or negatively selecting mutations. Strikingly, Ribo-T is not only functional in vitro, but is able to support cell growth even in the absence of wild-type (“wt”) ribosomes. As a result, Ribo-T has many uses. For example, Ribo-T may be used to prepare sequence-defined polymers, such as naturally occurring proteins or unnaturally occurring amino-acid polymers; create fully orthogonal ribosome-mRNA systems in vitro or in vivo; explore poorly understood functions of the ribosome; and engineer ribosomes with new functions.
Tethered Ribosome
Reference is made to U.S. Publication No. 2017/0073381, which discloses tethered ribosomes and methods of making and using tethered ribosomes and which content is incorporated herein by reference in its entirety. The engineered ribosome comprises a small subunit, a large subunit, and a linking moiety, wherein the linking moiety tethers the small subunit with the large subunit. The engineered ribosome is capable of supporting translation of a sequence-defined polymer.
In contrast to a naturally occurring ribosome, the engineered ribosome has a large and a small subunit that are not separable.
An embodiment of the engineered tethered ribosome is illustrated in
Large Subunit
The large subunit 301 comprises a subunit capable of joining amino acids to form a polypeptide chain. The large subunit 301 may comprise 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, wherein the L1 domain is followed, in order, by the C domain and the L2 domain, from 5′ to 3′.
The large subunit rRNA 301 may be a permuted variant of a separable large subunit rRNA. In certain embodiments, the permuted variant is a circularly permuted variant of a separable large subunit rRNA. The separable large subunit may be any functional large subunit. In certain embodiments, the separable large subunit may be a 23S rRNA. In certain embodiments, the separable large subunit is a wild-type large subunit rRNA. In specific embodiments, the separable large subunit is a wild-type 23S rRNA.
If the large subunit 301 is a permuted variant of a large subunit rRNA, then the polynucleotide consisting essentially of the L2 domain followed by the L1 domain, from 5′ to 3′, may be substantially identical to a large subunit rRNA. In certain embodiments, the polynucleotide consisting essentially of the L2 domain followed by the L1 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 large subunit rRNA.
In certain embodiments where the large subunit 301 is a permuted variant of a separable large subunit rRNA, the large subunit 301 may further comprise a C domain 304 that connects the native 5′ and 3′ ends of the separable large subunit rRNA. The C domain may comprise a polynucleotide having a length ranging from 1-200 nucleotides. In certain embodiments, the C domain 304 comprises a polynucleotide having a length ranging from 1-150 nucleotides 1-100 nucleotides, 1-90 nucleotides, from 1-80 nucleotides, 1-70 nucleotides, 1-60 nucleotides, 1-50 nucleotides, 1-40 nucleotides, 1-30 nucleotides, 1-20 nucleotides, 1-10 nucleotides, 1-9 nucleotides, 1-8 nucleotides, 1-7 nucleotides, 1-6 nucleotides, 1-5 nucleotides, 1-4 nucleotides, 1-3 nucleotides, or 1-2 nucleotides. In certain embodiments, the C domain comprises a GAGA polynucleotide.
Small Subunit
The small subunit 302 is capable of binding 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”), wherein the S1 domain is followed, in order, by S2 domain, from 5′ to 3′. Referring again to
The small subunit rRNA 302 may be a permuted variant of a separable small subunit rRNA. In certain embodiments, the permuted variant is a circularly permuted variant of a separable small subunit rRNA. The separable small subunit may be any functional small subunit. In certain embodiments, the separable small subunit may be a 16S rRNA. In certain embodiments, the separable small subunit is a wild-type small subunit rRNA. In specific embodiments, the separable small subunit is a wild-type 23S rRNA.
If the small subunit 302 is a permuted variant of a small subunit rRNA, then the polynucleotide consisting essentially of the S1 domain followed by the S2 domain, from 5′ to 3′, may be substantially identical to a small subunit rRNA. In certain embodiments, the polynucleotide consisting essentially of the S1 domain followed by the S2 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.
The small subunit may further comprise a modified-anti-Shine-Dalgarno sequence. The modified anti-Shine-Dalgarno sequence allows for translation of templates having a complementary Shine-Dalgarno sequence different from an endogenous cellular mRNA.
Linking Moiety
Referring again to
The linking moiety may also comprise a first tether domain (“T1 polynucleotide domain” or “T1 domain”) and a second tether domain (“T2 polynucleotide domain” or “T2 domain”). Referring again to
The T1 domain links that S1 domain and the L1 domain, wherein the S1 domain is followed, in order, by the T1 domain and the L1 domain, from 5′ to 3′. The T1 domain may comprise a polynucleotide having a length ranging from 5-200 nucleotide, 5-150 nucleotides, 5-100 nucleotides, 5-90 nucleotide, 5-80 nucleotides, 5-70 nucleotides, 5-60 nucleotides, 5-50 nucleotides, 5-40 nucleotides, 5-30 nucleotides, or 5-20 nucleotides, including polynucleotides having 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides. In certain embodiments, T1 comprises polyadenine. In certain embodiments, T1 comprises polyuridine. In certain embodiments, T1 comprises an unstructured polynucleotide. In certain embodiments, T1 comprises nucleotides that base-pairs with the T2 domain.
The T2 domain links that L2 domain and the S2 domain, wherein the L2 domain is followed, in order, by the T2 domain and the S2 domain, from 5′ to 3′. The T2 domain may comprise a polynucleotide having a length ranging from 5-200 nucleotides, 5-150 nucleotides, 5-100 nucleotides, 5-90 nucleotide, 5-80 nucleotides, 5-70 nucleotides, 5-60 nucleotides, 5-50 nucleotides, 5-40 nucleotides, 5-30 nucleotides, or 5-20 nucleotides, including polynucleotides having 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides. In certain embodiments, T1 comprises polyadenine. In certain embodiments, T2 comprises polyuridine. In certain embodiments, T2 comprises an unstructured polynucleotide. In certain embodiments, T2 comprises nucleotides that base-pairs with the T1 domain.
In embodiments having a T1 domain and a T2 domain, the T1 domain and the T2 domain may have the same number of polynucleotides. In other embodiments, the T1 domain and the T2 domain may have a different number of polynucleotides.
In certain embodiments, the engineered ribosome may comprise a S1 domain followed, in order, by a T1 domain, a L1 domain, a C domain, a L2 domain, a T2 domain, and a S2 domain, from 5′ to 3′. In specific embodiments, the engineered ribosome may consist essentially of a S1 domain followed, in order, by a T1 domain, a L1 domain, a C domain, a L2 domain, a T2 domain, and a S2 domain, from 5′ to 3′.
Mutations
In certain embodiments, an engineered ribosome may comprise one or more mutations. In specific embodiments the mutation is a change-of-function mutation. A change-of-function mutation may be a gain-of-function mutation or a loss-of-function mutation. A gain-of-function mutation may be any mutation that confers a new function. A loss-of-function mutation may be any mutation that results in the loss of a function possessed by the parent.
In certain embodiments, the change-of-function mutation may be in the peptidyl transferase center of the ribosome. In specific embodiments, the change-of-function mutation may be in an A-site of the peptidyl transferase center. In other embodiments, the change-of-function mutation may be in the exit tunnel of the engineered ribosome.
In certain embodiments the change-of-function mutation may be an antibiotic resistance mutation. The antibiotic resistance mutation may be either in the large subunit or the small subunit. In certain embodiments antibiotic resistance mutation may render the engineered ribosome resistant to an aminoglycoside, a tetracycline, a pactamycin, a streptomycin, an edein, or any other antibiotic that targets the small ribosomal subunit. In certain embodiments antibiotic resistance mutation may render the engineered ribosome resistant to a macrolide, a chloramphenicol, a lincosamide, an oxazolidinone, a pleuromutilin, a streptogramin, or any other antibiotic that targets the large ribosomal subunit.
Designing the Tethered Ribosome
A successful chimeric construct that tethers a large subunit and a small subunit must i) properly interact with the ribosomal proteins and biogenesis factors for functional ribosome assembly; ii) avoid ribonuclease degradation; and iii) have a linker(s) sufficiently short to ensure subunit cis-association, yet long enough for minimal inhibition of subunit movement required for translation initiation, elongation, and peptide release. The native ends of the large subunit and the small subunit are unsuitable given the design constraints outlined above. For example, in a native prokaryotic ribosome, for example, the 5′ and 3′ ends of 16S and 23S rRNA are too far apart (>170 Å) to be connected with a nuclease resistant RNA linker. As a result, alternative designs are needed if functioning engineered ribosome are to be realized.
One approach for designing a tethered ribosome is to permute a large subunit to generate new 5′ and 3′ termini. In certain embodiments, a circular permutation (CP) approach is employed because the native ends on the large subunit are proximal to each other. Circular permutation can be illustrated in the following scheme:
As such, in circular permutations of a polynucleotide, the sequence of the polynucleotide is maintained in each permutation but each nucleotide is at the end of an individual permutation. Circular permutations are utilized to replace the end of a polynucleotide at a different position while maintaining the secondary structure of the polynucleotide.
The CP approach has been pioneered in vitro by Polacek and coworkers [Erlacher 2005], and a subsequent pilot study showed that three 23S rRNA circularly permuted variants could assemble into a functional subunit in vivo [Kitahara 2009]. This approach is illustrated in
Continuing the approach outlined above, new termini for the small subunit need to be prepared so that the new termini for the small unit can be joined with the new termini of the large subunit by the linking moiety, as shown in
The approach outlined above can be used to generate collections of circularly permuted mutants with new termini. The new termini may be prepared at any location in the native subunit. Although some new termini result in permuted mutants may not be viable, the process disclosed herein is capable of generating and testing collections of permuted mutants.
In some embodiments, the location of the new termini of a small subunit or large subunit may be selected based on the secondary structure of a subunit, the proximity to the other subunit, the ribosome viability, or any combination thereof.
The secondary structure of either or both of the large subunit and the small subunit may be used to determine the location for new termini. In certain embodiments, the new termini are prepared in a helix of a native subunit. In some specific embodiments the new termini are prepared in hairpin of a native subunit.
The proximity to the other subunit may be used to select the location of the new termini in either or both of the large subunit or the small subunit. In certain embodiments, the new termini are located in the subunit solvent side of the native subunit. In some other embodiments the new termini are located close to the subunit interface rim. In certain specific embodiments the new termini are located in the subunit solvent side and close to the subunit interface rim.
Ribosome viability may be used to select the location of the new termini in either or both of the large subunit or the small subunit. For example, polynucleotide sequences or secondary structures that are in either or both of the large subunit or the small subunit that are not highly conserved in populations may be used to select the location for new termini.
In certain embodiments where the engineered ribosome is a 23S construct, the linking moiety may covalently bond helix 10, helix 38, helix 42, helix 54, helix 58, helix 63, helix 78, or helix 101 of a permuted variant of the 23S rRNA. In certain embodiments where the engineered ribosome is a 16S rRNA construct, the linking moiety may covalently bond helix 11, helix 26, helix 33, or helix 44 of a permuted variant of the 16S rRNA. In certain other embodiments where the engineered ribosome is a 16S construct, the linking moiety may covalently bond close to the E-site of a permuted variant of the 16S rRNA. In specific embodiments where the engineered ribosome is a 16S-23S construct, the linking moiety may covalently bond helix 44 of a permuted variant 16S rRNA with helix 101 of a permuted variant 23S rRNA, the linking moiety may covalently bond helix 26 of a permuted variant 16S rRNA with helix 10 of a permuted variant 23S rRNA, the linking moiety may covalently bond helix 33 of a permuted variant 16S rRNA with helix 38 of a permuted variant 23S rRNA, the linking moiety may covalently bond helix 11 of a permuted variant 16S rRNA with helix 58 of a permuted variant 23S rRNA, the linking moiety may covalently bond helix 44 of a permuted variant 16S rRNA with helix 58 of a permuted variant 23S rRNA, the linking moiety may covalently bond helix 26 of a permuted 1 variant 6S rRNA with helix 54 of a permuted variant 23S rRNA, the linking moiety may covalently bond helix 11 of a permuted variant 16S rRNA with helix 63 of a permuted variant 23S rRNA, or the linking moiety may covalently bond helix 44 of a permuted variant 16S rRNA with helix 63 of a permuted variant 23S rRNA.
As explained above, the linking moiety must be sufficiently short to prevent degradation and to ensure subunit cis-association while long enough for minimal inhibition of subunit movement required for translation initiation, elongation, and peptide release. As a result, the linking moiety must span tens of Angstroms between the new termini on the large subunit and the short subunit.
Polynucleotides Encoding the Tethered Ribosome
Polynucleotides encoding the tethered ribosome are also disclosed. The polynucleotide encoding for the tethered ribosome may be any polynucleotide capable of being expressed to produce the rRNA of the tethered ribosome.
The polynucleotides encoding for the tethered ribosome may further comprise genes encoding for other rRNA subunits of the ribosome or ribosomal proteins. For example, the polynucleotide encoding for an engineered ribosome comprising a permuted 23S rRNA tethered to a permuted 16S rRNA, the polynucleotide may further comprise a gene encoding for a 5S rRNA.
In certain embodiments the polynucleotide is a vector that may introduce foreign genetic material into a host cell. The vector may be a plasmid, viral vector, cosmid, or artificial chromosome.
Optionally the plasmid encoding a prokaryotic ribosome having separable subunits comprises one or more additional genes. The additional gene(s) may comprise a modified Shine-Dalgarno sequence that is complimentary with a modified anti-Shine-Dalgarno sequence of the small subunit of the untethered ribosome.
In contrast to the plasmid encoding the ribosome having separable subunits, the plasmid encoding a tethered ribosome 700 has a chimeric gene encoding for a large subunit, a small subunit, and a linking moiety connecting the large subunit with the small subunit 701-707. Plasmid comprises the genes for the expression of the tethered ribosome 720. Optionally, the plasmid may further comprise one or more addition genes 740.
The gene encoding for the tethered subunits comprises the sequence that encodes for the rRNA of a S1 domain 701 followed, in order, by a sequence that encodes for the rRNA of a T1 linker 705, a sequence that encodes for the rRNA of a L1 domain 702, a sequence that encodes for the rRNA of a C domain 706, a sequence that encodes for the rRNA of a L2 domain 703, a sequence that encodes for the rRNA of a T2 linker 707, and a sequence that encodes for the rRNA of a S2 domain 704, from 5′ to 3′. The processing sequences of a small subunit flanking the chimeric gene, indicated by the small rectangles, may be retained for proper maturation of the small subunit termini, whereas the processing sequences for the large subunit 716 may be moved to another location in the plasmid or eliminated entirely to prevent cleavage of the large subunit out of the hybrid.
In certain embodiments, the plasmid encoding the tethered subunits further comprises a gene encoding a 5S subunit 711, a gene encoding antibiotic resistance 714, and an origin of replication gene 715.
Optionally, the plasmid encoding the tethered subunits may comprise a modified anti-Shine-Dalgarno sequence 708 (circle). Although the modified anti-SD sequence is shown in
Optionally, the plasmid encoding the tethered subunits comprises one or more additional genes 740. The additional gene may comprise a modified Shine-Dalgarno sequence that is complimentary with a modified anti-Shine-Dalgarno sequence of the tethered ribosome. In certain embodiments that additional gene may be a reporter gene. In specific embodiments, the reporter gene is a green fluorescent protein. In some embodiments, the additional gene comprise a wild-type anti-Shine-Dalgarno sequence.
Preparing the Polynucleotide
Methods of preparing the polynucleotide are also disclosed herein. The method comprises preparing a plasmid encoding a permuted subunit rRNA construct, identifying a viable permuted subunit rRNA construct, and preparing a polynucleotide encoding the engineered ribosome comprising a large subunit, a small subunit, and a linking moiety that tethers the small subunit with the large subunit.
Preparation of a plasmid encoding a permuted subunit rRNA construct may be accomplished by the circular permutation approach that connects the native ends of the subunit and prepares new termini
To identify permuted subunit rRNA viable constructs, the plasmid encoding the permuted subunit rRNA may be introduced in to host cell strains and a screening mechanism is used to identify transformants. The host cells comprise the plasmid as well as a plasmid encoding for the wild-type rRNA operon and may be spotted onto an agar plate along with an antibiotic. The selection mechanism includes identifying transformants resistant to the antibiotic. By way of example, the plasmids may be transformed into Δ7 rrn SQ171 strain carrying pCSacB plasmid with wild-type rRNA operon and transformants resistant to ampicillin, erythromycin and sucrose are selected. To confirm complete replacement of the wild-type rRNA operon with the plasmid encoding for the permuted subunit rRNA, a three-primer diagnostic PCR check may be performed on the total plasmid extract.
Preparing a polynucleotide encoding the engineered ribosome comprising a large subunit, a small subunit, and a linking moiety that tethers the small subunit with the large subunit comprises grafting the permuted subunit rRNA construct and the linking moiety into the other subunit. In certain embodiments the preparation step may also include preparing a plasmid comprising the polynucleotide encoding the engineered ribosome comprising a large subunit, a small subunit, and a linking moiety that tethers the small subunit with the large subunit. In other embodiments, the preparation step may also include preparing a plasmid comprising the polynucleotide encoding the engineered ribosome comprising a large subunit, a small subunit, and a linking moiety that tethers the small subunit with the large subunit and a polynucleotide encoding for an additional gene.
Preparing the Tethered Ribosome
Also disclosed are methods for preparing the tethered ribosome. The tethered ribosome may be prepared by expressing a polynucleotide encoding the engineered ribosome. In certain embodiments preparation of the tethered ribosome further comprises preparing the polynucleotide encoding the engineered ribosome. In other embodiments the preparation of the tethered ribosome further comprises transforming a cell with the polynucleotide encoding the engineered ribosome. In some specific embodiments, the preparation of the tethered ribosome further comprises preparing the polynucleotide and transforming a cell with the polynucleotide.
Tethered Ribosome Evolution
Also disclosed are methods for evolving the tethered ribosome. Methods for tethered ribosome evolution include expressing a polynucleotide encoding for the engineered ribosome and selecting a mutant. The selection step may comprise a negative selection step, a positive selection step, or both a negative and a positive selection step. The mutant selected may comprise a tethered ribosome having a change-of-function mutation. The change-of-function mutation may be a gain-of-function mutation or a loss-of-function mutation.
Utility and Applications of Tethered Ribosomes
Some uses and applications of the tethered ribosomes are described below.
Artificial Cells
Artificial cells are disclosed. The artificial cell may comprise a polynucleotide encoding an engineered ribosome, the engineered ribosome comprising a small subunit, a large subunit, and a linking moiety, wherein the linking moiety tethers the small subunit with the large subunit. The artificial cell comprising a polynucleotide encoding the engineered ribosome may be capable of expressing the polynucleotide to prepare the engineered ribosome. In other embodiments, the artificial cell comprises the engineered ribosome. In some specific embodiments the artificial cell comprises a polynucleotide encoding the engineered ribosome and the engineered ribosome.
Artificial cells may comprise one or more translation mechanism. In a first embodiment, the artificial cell has one translation mechanism comprising an engineered ribosome, the engineered ribosome comprising a small subunit, a large subunit, and a linking moiety, wherein the linking moiety tethers the small subunit with the large subunit.
In another embodiment, the artificial cell may comprise two translation mechanisms. The first translation mechanism may comprise a ribosome wherein the ribosome lacks a linking moiety between the large subunit and the small subunit. The second translation mechanism comprises an engineered ribosome, the engineered ribosome comprising a small subunit, a large subunit, and a linking moiety, wherein the linking moiety tethers the small subunit with the large subunit. In some embodiments the first translation mechanism or the second translation mechanism is an orthogonal translation mechanism. In some embodiments the first translation mechanism and the second translation mechanism are orthogonal translation mechanisms. An orthogonal translation mechanism may be prepared by modifying the anti-Shine Dalgarno sequence of the ribosome to permit translation of templates having a complementary Shine-Dalgarno sequences different from the endogenous cellular mRNAs.
In another embodiment, a cell comprising a first mechanism and a second mechanism for protein translation is disclosed. The first mechanism comprises tethered ribosomes with a wild-type anti-Shine-Dalgarno sequence, wherein mRNA is translated by the ribosomes in accordance with the natural genetic code (that is, triplet code endogenous to the cell). The second mechanism includes an artificial mechanism derived from untethered ribosomes that functions to allow for expression of a heterologous gene. The second mechanism, in some embodiments, comprises ribosomes having a modified anti-Shine-Dalgarno sequence.
Preparation of Sequence-Defined Polymers
Methods for preparing sequence-defined polymers are also provided. In certain embodiments the method for preparing a sequence defined polymer comprises providing an engineered ribosome and providing an mRNA or DNA template encoding the sequence-defined polymer. In some embodiments, the engineered ribosome comprises a small subunit, a large subunit, and a linking moiety and wherein the linking moiety tethers the small subunit with the large subunit, and wherein the engineered ribosome comprises a modified anti-Shine-Dalgarno sequence. In some embodiments, the engineered ribosome comprises a small subunit, a large subunit, no linking moiety, and a modified Shine-Dalgarno sequence. In one aspect of the method, one of any of the steps includes adding at least one exogenous DNA template encoding an mRNA for the sequence-defined polymer.
In one aspect of the method, the sequence-defined polymer is a natural biopolymer. In another aspect of the method, the sequence-defined polymer is a non-natural biopolymer. In certain embodiments, the sequence-defined polymer comprises an amino acid. In certain embodiments the amino acid may be a natural amino acid. As used herein a natural amino acid is a proteinogenic amino acid encoded directly by a codon of the universal genetic code. In certain embodiments the amino acid may be an unnatural amino acid. As used here an unnatural amino acid is a nonproteinogenic amino acid. Examples of unnatural amino acids include, but are not limited to a p-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, an O-methyl-L-tyrosine, a p-propargyloxyphenylalanine, a p-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcpβ-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an unnatural analogue of a methionine amino acid; an unnatural analogue of a leucine amino acid; an unnatural analogue of a isoleucine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an α-hydroxy containing acid; an amino thio acid; an α,α disubstituted amino acid; a β-amino acid; a γ-amino acid, a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan. In certain embodiments the sequence-defined polymer is a polypeptide or protein.
In one aspect of the method, the tethered subunit arrangement comprises a linking moiety between the 23S and 16S rRNAs. In one respect of this aspect, the linking moiety covalently bonds helix 101 of the 23S rRNA to helix 44 of the 16S rRNA. In another respect of this aspect, the linking moiety comprises a polynucleotide having a length ranging from 5 nucleotides to 200 nucleotides. The linked ribosome can further include an engineered 16S rRNA having a modified anti-Shine-Dalgarno sequence to permit translation in vitro of translation templates having a complementary SD sequence differing from endogenous cellular mRNAs. In this way, selective translation in vitro of mRNA to produce sequence defined biopolymers with high efficiency is possible.
In one aspect of the method, an engineered ribosome is untethered, and comprises a modified anti-Shine-Dalgarno (SD) 16S sequence to permit translation in vitro or in vivo of translation templates having a complementary SD sequence differing from endogenous cellular mRNAs. In this way, selective translation of mRNA to produce sequence defined biopolymers with high efficiency is possible.
In one aspect of the method, the mRNA or DNA template encodes a modified Shine-Dalgarno sequence. In certain embodiments the engineered ribosome comprises an anti-Shine-Dalgarno sequence complementary to the Shine-Dalgarno sequence encoded by the mRNA or DNA template.
In some embodiments, the mRNA or DNA template is provided to a modified cell (e.g., a cell comprising two different protein translation mechanisms), an extract from such a cell, or a purified translation system from such a cell.
Sequence-defined polymers may be prepared in vitro. In some embodiments, the method for preparing a sequence-defined polymer in vitro further comprises providing a ribosome-depleted cellular extract or a purified translation system. In certain embodiments, the ribosome-depleted cellular extract comprises an S150 extract prepared from mid- to late-exponential growth phase cell cultures or cultures having an O.D.600˜3.0 at time of harvest. In one aspect of the method, the ribosome-depleted extract is prepared with one or more polyamines, such as spermine, spermidine and putrescine, or combinations thereof. In one aspect of the method, the ribosome-depleted extract is prepared with a concentration of salts from about 50 mM to about 300 mM.
The preparation of ribosome-depleted cellular extracts and methods of using them for supporting translation in vitro of sequence-defined polymers is disclosed in International Patent Application No. PCT/US14/35376 to Michael Jewett et al., entitled IMPROVED METHODS FOR MAKING RIBOSOMES, filed Apr. 24, 2014, the contents of which are incorporated by reference herein in its entirety.
In one aspect of the method, mRNA encodes a modified Shine-Dalgarno sequence differing from endogenous cellular mRNAs present in the ribosome-depleted cellular extract. In one respect of this aspect, an engineered ribosome includes an altered 16S rRNA having a modified anti-Shine-Dalgarno sequence complementary to the modified Shine-Dalgarno sequence to permit translation in vitro of the mRNA to prepare the sequence defined biopolymer in vitro.
In one aspect, the method is configured for fed-batch operation or continuous operation. In another aspect of the method, at least one substrate is replenished during operation.
In one aspect of the method, at least one step includes a DNA-dependent RNA polymerase. In one aspect of the method, at least one macromolecular crowding agent is included in one of the steps. In one aspect of the method, at least one reducing agent (e.g., dithiothreitol, tris(2-carboxyethyl) phosphine hydrochloride, etc.) is included in one of the steps.
Sequence-defined polymers may be prepared in vivo. The method for preparing a sequence-defined polymer in vivo may occur in an artificial cell as disclosed above. The artificial cell may have a translation mechanism comprising an engineered ribosome, wherein the engineered ribosome comprises a small subunit, a large subunit, and a linking moiety and wherein the linking moiety tethers the small subunit with the large subunit. In certain embodiments the artificial cell has one translation mechanism. In other embodiments the cell has two translations mechanisms. In some embodiments, the cell has two protein translations mechanisms, the first protein translation mechanism comprising ribosomes, wherein the ribosomes lack a linking moiety between the large subunit and the small subunit and the second protein translation mechanism comprises ribosomes, wherein the ribosomes include a linking moiety linking the large subunit and the small subunit. In some embodiments, the ribosomes of the first translations system comprises a modified anti-Shine-Dalgarno sequence and the ribosomes of the second translation system include a wild-type (unmodified) anti-Shine-Dalgarno sequence.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the invention pertains. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use an aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
The terms “nucleic acid” and “oligonucleotide,” as used herein, refer to polydeoxyribonucleotides (containing 2-deoxy-DRibose), polyribonucleotides (containing DRibose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. For use in the present invention, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs.
A “fragment” of a polynucleotide is a portion of a polynucleotide sequence which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides of a reference polynucleotide. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides of a reference polynucleotide. Fragments may be preferentially selected from certain regions of a molecule. The term “at least a 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.
Regarding polynucleotide sequences, percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Regarding polynucleotide sequences, “variant,” “mutant,” or “derivative” may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's 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 a pair of nucleic acids may show, 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 greater sequence identity over a certain defined length.
A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques known in the art. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
The nucleic acids disclosed herein may be “substantially isolated or purified.” The term “substantially isolated or purified” refers to a nucleic acid that is removed from its natural environment, and is at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which it is naturally associated.
Oligonucleotides can be prepared by any suitable method, including direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al., 1981, Tetrahedron Letters 22:1859-1862; and the solid support method of U.S. Pat. No. 4,458,066, each incorporated herein by reference. A review of synthesis methods of conjugates of oligonucleotides and modified nucleotides is provided in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187, incorporated herein by reference.
The term “primer,” as used herein, refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of four different nucleoside triphosphates and an agent for extension (for example, a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
A primer is preferably a single-stranded DNA. The appropriate length of a primer depends on the intended use of the primer but typically ranges from about 6 to about 225 nucleotides, including intermediate ranges, such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25 to 150 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for the amplification of a given target sequence is well known in the art and described in the literature cited herein.
Primers can incorporate additional features which allow for the detection or immobilization of the primer but do not alter the basic property of the primer, that of acting as a point of initiation of DNA synthesis. For example, primers may contain an additional nucleic acid sequence at the 5′ end which does not hybridize to the target nucleic acid, but which facilitates cloning or detection of the amplified product, or which enables transcription of RNA (for example, by inclusion of a promoter) or translation of protein (for example, by inclusion of a 5′-UTR, such as an Internal Ribosome Entry Site (IRES) or a 3′-UTR element, such as a poly(A)n sequence, where n is in the range from about 20 to about 200). The region of the primer that is sufficiently complementary to the template to hybridize is referred to herein as the hybridizing region.
The term “promoter” refers to a cis-acting DNA sequence that directs RNA polymerase and other trans-acting transcription factors to initiate RNA transcription from the DNA template that includes the cis-acting DNA sequence.
The terms “target, “target sequence”, “target region”, and “target nucleic acid,” as used herein, are synonymous and refer to a region or sequence of a nucleic acid which is to be amplified, sequenced or detected.
The term “hybridization,” as used herein, refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. Conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”. Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e.g., Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; 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).
The term “amplification reaction” refers to any chemical reaction, including an enzymatic reaction, which results in increased copies of a template nucleic acid sequence or results in transcription of a template nucleic acid. Amplification reactions include reverse transcription, the polymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), and the ligase chain reaction (LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary “amplification reactions conditions” or “amplification conditions” typically comprise either two or three step cycles. Two-step cycles have a high temperature denaturation step followed by a hybridization/elongation (or ligation) step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.
As used herein, a “polymerase” refers to 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, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase, among others. “RNA polymerase” catalyzes the polymerization of ribonucleotides. The foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase, which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerase (“RNAP”) include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and E. coli RNA polymerase, among others. The foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase. The polymerase activity of any of the above enzymes can be determined by means well known in the art.
As used herein, the term “sequence defined polymer” refers to a polymer having a specific primary sequence. A sequence defined polymer can be equivalent to a genetically-encoded defined polymer in cases where a gene encodes the polymer having a specific primary sequence.
As used herein, a primer is “specific,” for a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer hybridizes primarily to the target nucleic acid. Typically, a primer is specific for a target sequence if the primer-target duplex stability is greater than the stability of a duplex formed between the primer and any other sequence found in the sample. One of skill in the art will recognize that various factors, such as salt conditions as well as base composition of the primer and the location of the mismatches, will affect the specificity of the primer, and that routine experimental confirmation of the primer specificity will be needed in many cases. Hybridization conditions can be chosen under which the primer can form stable duplexes only with a target sequence. Thus, the use of target-specific primers under suitably stringent amplification conditions enables the selective amplification of those target sequences that contain the target primer binding sites.
As used herein, “expression template” refers to a nucleic acid that serves as substrate for transcribing at least one RNA that can be translated into a polypeptide or protein. Expression templates include nucleic acids composed of DNA or RNA. Suitable sources of DNA for use a nucleic acid for an expression template include genomic DNA, plasmid DNA, cDNA and RNA that can be converted into cDNA. Genomic DNA, cDNA and RNA can be from any biological source, such as a tissue sample, a biopsy, a swab, sputum, a blood sample, a fecal sample, a urine sample, a scraping, among others. The genomic DNA, cDNA and RNA can be from host cell or virus origins and from any species, including extant and extinct organisms. As used herein, “expression template” and “transcription template” have the same meaning and are used interchangeably.
As used herein, “tethered,” “conjoined,” “linked,” “connected,” “coupled” and “covalently-bonded” have the same meaning as modifiers.
As used herein, “tethered ribosome,” and “Ribo-T” will be used interchangeably.
As used herein, the term “engineered ribosome” refers to a ribosome that has been modified. Exemplary modifications may include, but are not limited to one or more of tethering subunits, altering or subunits, and altering one or more rRNA sequence. Exemplary, non-limiting modification may include one or more of a modified: 16S rRNA; 23S rRNA; anti-Shine-Dalgarno sequence, peptidyl transferase center; nascent exit tunnel; ecoding center of the ribosome; interaction site with elongation factors; tRNA binding site; chaperone binding site; nascent chain modifying enzyme binding sites; GTPase center; introduction of antibiotic resistance sequence, etc.).
As used herein, the term “wild-type,” “native,” or “endogeneous” refer to a substance or condition typically found in a given organism.
As used herein, the term “mutant,” exogenous,” “orthagnol,” and “non-native” refer to a substance or conditions typically not found in a given organism.
As used here, “CP” refers to a circularly permuted subunit. As used herein, when CP is followed by “23S” that refers to a circularly permuted 23S rRNA. As used herein, when CP followed by a number may refer to the location of the new 5′ end in a secondary structure, e.g. CP101 means the new 5′ end is in helix 101 of the 23S rRNA, or to the location of the new 5′ nucleotide, e.g. CP2861 means the new 5′ nucleotide is the nucleotide 2861 of the 23 rRNA, depending on context.
As used herein, “translation template” refers to an RNA product of transcription from an expression template that can be used by ribosomes to synthesize polypeptide or protein.
As used herein, a “ribosomal binding site” or “RBS” is a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of protein translation. The RBS may include the Shine-Dalgarno sequence. The Shine-Dalgarno (SD) sequence is a ribosomal binding site in prokaryotic messenger RNA, which generally is located approximately 8 bases upstream of the start codon AUG. The SD sequence helps recruit the ribosome to the messenger RNA (mRNA) to initiate protein synthesis by aligning the ribosome with the start codon. The six-base consensus sequence is AGGAGG and in E. coli the sequence is AGGAGGU.
Miscellaneous
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
The following embodiments are illustrative and should be interpreted to limit the scope of the claimed subject matter.
Embodiment 1. An engineered ribosome, the engineered ribosome comprising a small subunit, a large subunit, and a linking moiety, a. wherein the linking moiety tethers the small subunit with the large subunit and b. wherein the engineered ribosome is capable of supporting translation of a sequence defined polymer.
Embodiment 2. The engineered ribosome of embodiment 1, wherein the small subunit comprises rRNA and protein, wherein the large subunit comprises rRNA and protein, and wherein the linking moiety tethers the rRNA of the small subunit with the rRNA of the large subunit.
Embodiment 3. The engineered ribosome of embodiment 1 or 2, wherein the large subunit comprises a permuted variant of a 23S rRNA (e.g., a circularly permuted variant of 23 rRNA).
Embodiment 4. The engineered ribosome of any of embodiments 1-3, wherein the small subunit comprises a permuted variant of a 16S rRNA (e.g., a circularly permuted variant of 23 rRNA).
Embodiment 5. The engineered ribosome of any of embodiments 1-4, wherein the small subunit comprises a modified anti-Shine-Dalgarno sequence to permit translation of templates having a complementary Shine-Dalgarno sequence different from endogenous cellular mRNAs (e.g., wherein the modified anti-Shine-Dalgarno sequence of the small subunit is complementary to the Shine-Dalgarno sequence different from endogenous cellular mRNAs).
Embodiment 6. The engineered ribosome of any of embodiments 1-5, wherein the linking moiety covalently bonds a helix of the large subunit to a helix of the small subunit.
Embodiment 7. The engineered ribosome of any of embodiments 3-6, wherein the linking moiety covalently bonds helix 10, helix 38, helix 42, helix 54, helix 58, helix 63, helix 78, or helix 101 of the permuted variant of the 23S rRNA.
Embodiment 8. The engineered ribosome of any of embodiments 4-7, wherein the linking moiety covalently bonds helix 11, helix 26, helix 33, or helix 44 of the permuted variant of the 16S rRNA.
Embodiment 9. The engineered ribosome of any of embodiments 1-8, wherein the large subunit comprises or consists essentially of a L1 polynucleotide domain (e.g., a fragment of 23S rRNA), a L2 polynucleotide domain (e.g., a fragment of 23S rRNA), and a C polynucleotide domain, wherein the L1 domain is followed, in order, by the C domain and the L2 domain, from 5′ to 3′.
Embodiment 10. The engineered ribosome of embodiment 9, wherein the polynucleotide comprising or consisting essentially of the L2 domain followed by the L1 domain, from 5′ to 3′, is substantially identical to 23S rRNA or a fragment of 23S rRNA.
Embodiment 11. The engineered ribosome of embodiment 9 or 10, wherein the polynucleotide comprising or consisting essentially of the L2 domain followed by the 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).
Embodiment 12. The engineered ribosome of any of embodiments 9-11, wherein the C domain comprises a polynucleotide having a length ranging from 1-200 nucleotides.
Embodiment 13. The engineered ribosome of any of embodiments 9-12, wherein the C domain comprises a GAGA polynucleotide.
Embodiment 14. The engineered ribosome of any of embodiments 1-13, wherein the small subunit comprises or consists essentially of a S1 polynucleotide domain (e.g., a fragment of 16S rRNA) and a S2 polynucleotide domain (e.g., a fragment of 16S rRNA), wherein the S1 domain is followed, in order, by the S2 domain, from 5′ to 3′.
Embodiment 15. The engineered ribosome of embodiment 14, wherein the polynucleotide comprising or consisting essentially of the S1 domain followed by the S2 domain, from 5′ to 3′, is substantially identical to a 16S rRNA (or a fragment of 16S rRNA).
Embodiment 16. The engineered ribosome of embodiment 14 or 15, wherein the polynucleotide comprising or consisting essentially of the S1 domain followed by the S2 domain, from 5′ to 3′, is at least 95% identical to a 16S rRNA (or at least 96%, 97%, 98%, or 99% identical to 23S rRNA or a fragment of 23S rRNA).
Embodiment 17. The engineered ribosome of any of embodiments 1-16, wherein the linking moiety comprises a T1 polynucleotide domain and a T2 polynucleotide domain.
Embodiment 18. The engineered ribosome of embodiment 17, wherein the T1 domain links the S1 domain and the L1 domain and wherein the S1 domain is followed, in order, by the T1 domain and the L1 domain, from 5′ to 3′.
Embodiment 19. The engineered ribosome of embodiment 17 or 18, wherein the T1 domain comprises a polynucleotide having a length ranging from 5 to 200 nucleotides.
Embodiment 20. The engineered ribosome of embodiment 19, wherein the T1 domain comprises a polynucleotide having a length ranging from 7 to 20 nucleotides.
Embodiment 21. The engineered ribosome of any of embodiments 17-20, wherein the T1 domain comprises a polyadenine polynucleotide.
Embodiment 22. The engineered ribosome of any of embodiments 17-20, wherein the T1 domain comprises a polyadenine polynucleotide having a length of 7 to 12 adenine nucleotides.
Embodiment 23. The engineered ribosome of any of embodiments 17-22, wherein the T2 domain links the S2 domain and the L2 domain and wherein the L2 domain is followed, in order, by the T2 domain and the S2 domain, from 5′ to 3′.
Embodiment 24. The engineered ribosome of any of embodiments 17-24, wherein the T2 domain comprises a polynucleotide having a length ranging from 5 to 200 nucleotides.
Embodiment 25. The engineered ribosome of embodiment 17, 23, or 24, wherein the T2 domain comprises a polynucleotide having a length ranging from 7 to 20 nucleotides.
Embodiment 26. The engineered ribosome of any of embodiments 17-25, wherein the T2 domain comprises a polyadenine polynucleotide.
Embodiment 27. The engineered ribosome of any of embodiments 17-26, wherein the T2 domain comprises a polyadenine polynucleotide having a length of 7 to 12 adenine nucleotides.
Embodiment 28. The engineered ribosome of any of embodiments 17-27, wherein the ribosome comprises the S1 domain followed, in order, by the T1 domain, the L1 domain, the C domain, the L2 domain, the T2 domain, and the S2 domain, from 5′ to 3′.
Embodiment 29. The engineered ribosome of any of embodiments 17-28, wherein the ribosome comprises a polynucleotide consisting essentially of the S1 domain is followed, in order, by the T1 domain, the L1 domain, the C domain, the L2 domain, the T2 domain, and the S2 domain, from 5′ to 3′.
Embodiment 30. The engineered ribosome of any of embodiments 1-29, wherein the engineered ribosome comprises a mutation.
Embodiment 31. The engineered ribosome of embodiment 30, wherein the mutation is a change-of-function mutation.
Embodiment 32. The engineered ribosome of embodiment 31, wherein the change-of-function mutation is in a peptidyl transferase center.
Embodiment 33. The engineered ribosome of embodiment 31, wherein the change-of-function mutation is in an A-site of the peptidyl transferase center.
Embodiment 34. The engineered ribosome of embodiment 31, wherein the change-of-function mutation is in one or more of the exit tunnel of the engineered ribosome, the interaction site with the translocon, or the interaction sites with the auxiliary proteins facilitating translation.
Embodiment 35. The engineered ribosome of any of embodiments 1-35, wherein the engineered ribosome has an antibiotic resistance mutation.
Embodiment 36. A polynucleotide, the polynucleotide encoding the rRNA of the engineered ribosome of any of embodiments 1-35.
Embodiment 37. The polynucleotide of embodiment 36, wherein the polynucleotide is a vector.
Embodiment 38. The polynucleotide of embodiment 36 or 37, wherein the polynucleotide further comprises a gene to be expressed by the engineered ribosome.
Embodiment 39. The polynucleotide of embodiment 38, wherein the gene is a reporter gene.
Embodiment 40. The polynucleotide of embodiment 39, wherein the reporter gene is a green fluorescent protein gene.
Embodiment 41. The polynucleotide of any of embodiments 36-40, wherein the engineered ribosome comprises a modified anti-Shine-Dalgarno sequence and the gene comprises a complementary Shine-Dalgarno sequence to the engineered ribosome.
Embodiment 42. The polynucleotide of any of embodiments 36-41, wherein the gene comprises a codon and the codon encodes for an unnatural amino acid.
Embodiment 43. A method for preparing an engineered ribosome, the method comprising expressing the polynucleotide of any of embodiments 36-42.
Embodiment 44. The method of embodiment 43, the method further comprising selecting a mutant.
Embodiment 45. The method of embodiment 44, wherein the selection step comprises a negative selection step, a positive selection step, or both a negative and a positive selection step.
Embodiment 46. An engineered cell, the engineered cell comprising (i) the polynucleotide of any of embodiments 36-42, (ii) the engineered ribosome of any of embodiments 1-35, or both (i) and (ii).
Embodiment 47. A engineered cell, the engineered cell comprising a first protein translation mechanism and a second protein translation mechanism, a. wherein the first protein translation mechanism comprises a ribosome, wherein the ribosome lacks a linking moiety between the large subunit and the small subunit and b. wherein the second protein translation mechanism comprises the engineered ribosome of any of embodiments 1-35.
Embodiment 48. A method for preparing a sequence-defined polymer, the method comprising (a) providing the engineered ribosome of any of embodiments 1-35 and (b) providing an mRNA or DNA template encoding the sequence-defined polymer.
Embodiment 49. The method of embodiment 48, wherein the sequence-defined polymer is prepared in vitro.
Embodiment 50. The method of embodiment 49, the method further comprising providing a ribosome-depleted cellular extract or purified translation system.
Embodiment 51. The method of embodiment 50, wherein the ribosome-depleted cellular extract comprises an S150 extract prepared from mid- to late-exponential growth phase cell cultures or cultures having an O.D.600˜3.0 at time of harvest.
Embodiment 52. The method of embodiment 48, wherein the sequence defined polymer is prepared in vivo.
Embodiment 53. The method of embodiment 48 or 52, wherein the sequence defined polymer is prepared in the cell of any of embodiments 46 or 47.
Embodiment 54. The method of any of embodiments 48-53, wherein the mRNA or DNA encodes a modified Shine-Dalgarno sequence and the engineered ribosome comprises an anti-Shine-Dalgarno sequence complementary to the modified Shine-Dalgarno sequence.
Embodiment 55. The method of any of embodiments 48-54, wherein the sequence-defined polymer comprises an amino acid.
Embodiment 56. The method of embodiment 55, wherein the amino acid is a natural amino acid.
Embodiment 57. The method of embodiment 55, wherein the amino acid is an unnatural amino acid.
Embodiment 58. The engineered cell of embodiment 47, wherein the ribosomes of the first protein translation mechanism comprise a modified anti-Shine-Dalgarno sequence, and wherein the ribosomes of the second protein translation system comprise an unmodified (e.g., wild-type) anti-Shine-Dalgarno sequence.
Embodiment 59. The method of any one of embodiments 48-53, further comprising untethered ribosomes comprising a modified anti-Shine-Dalgarno sequence.
Embodiment 60. The method of embodiment 59, wherein the mRNA or DNA encodes a modified Shine-Dalgarno sequence and the untethered ribosomes comprise an anti-Shine-Dalgarno sequence complementary to the modified Shine-Dalgarno sequence.
Embodiment 61. The method of embodiment of 60, wherein the sequence defined polymer comprises a natural or an unnatural amino acid.
Embodiment 62. An engineered cell comprising two or more protein translation mechanisms, wherein: (a) a first mechanism is the natural translation mechanism wherein mRNA is translated by a tethered, or stapled, ribosome in accordance with the natural genetic code; (b) a second mechanism is an artificial mechanism derived from a dissociable ribosome that tunes host metabolic burden or in which orthogonal mRNA comprising orthogonal codons is translated by this orthogonal ribosome.
Embodiment 63. An engineered cell comprising two or more protein translation mechanisms, wherein: (a) a first mechanism is the natural translation mechanism wherein mRNA is translated by a tethered, or stapled, ribosome that sustains the life of the cell; (b) a second mechanism is an artificial mechanism derived from a dissociable ribosome that carries out an orthogonal function.
Embodiment 64. An engineered cell comprising two or more protein translation mechanisms, wherein the orthogonal dissociable ribosomes outperforms an orthogonal tethered ribosomes in the context of protein expression.
Embodiment 65. An engineered cell in which not only the o-30S, but also the free 50S subunit is engineered to achieve new functionalities.
Embodiment 66. An engineered cell in which not only the o-30S, but also the free 50S subunit are engineered to achieve new functionalities without interfering with the expression of the cellular proteome not only is the o-30S, but also the free 50S subunit is engineered to achieve new functionalities without interfering with the expression of the cellular proteome.
Embodiment 67. An engineered cell in which not only the o-30S, but also the free 50S subunit is engineered to achieve gain of function ribosome mutations.
Embodiment 68. An engineered cell in which not only the o-30S, but also the free 50S subunit is engineered to achieve gain of function ribosome mutations, wherein these mutations specifically overcome the translation of problematic polymer sequences.
Embodiment 69. An engineered cell comprising a first protein translation mechanism and a second protein translation mechanism, the first protein translation mechanism comprising a first engineered ribosome, the first engineered 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 linking moiety, wherein the linking moiety comprises a polynucleotide sequence and tethers the rRNA of the small subunit with the rRNA of the large subunit; the second protein translation mechanism comprising a second engineered ribosome, the second engineered ribosome comprising: i) a small subunit comprising rRNA and protein, ii) a large subunit comprising rRNA and protein, and iii) wherein the second engineered ribosome lacks a linking moiety between the large subunit and the small subunit; and wherein small subunit of the second engineered ribosome comprises a modified anti-Shine-Dalgarno sequence to permit translation of templates having complementary and/or cognate Shine-Dalgarno sequence different from endogenous cellular mRNAs, and/or wherein the second engineered ribosome comprises one or more change-of-function mutations, wherein the change-of-function mutation is not at the anti-Shine Dalgarno sequence.
Embodiment 70. The engineered cell of embodiment 69, wherein the first and the second protein translation mechanisms are capable of supporting translation of a sequence defined polymer.
Embodiment 71. The engineered cell of any one embodiments 69-70, wherein the first protein translation mechanism is capable of supporting translation of native, endogenous RNAs.
Embodiment 72. The engineered cell of any one embodiments 69-71, wherein the second protein translation mechanism is capable of supporting translation of non-native, exogenous RNAs.
Embodiment 73. The engineered cell of any one embodiments 69-72, wherein the small subunit of the second engineered ribosome comprises 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′.
Embodiment 74. The engineered cell of any one embodiments 69-74, wherein the second engineered ribosome comprises a change-of-function mutation in one or more of: a) peptidyl transferase center (PTC); b) nascent peptide exit tunnel (NPET); c) interaction site with elongation factors; d) tRNA binding sites; e) chaperone binding sites; f) nascent chain modifying enzyme biding sites; g) GTPase center.
Embodiment 75. The engineered cell of any one embodiments 69-74 wherein the large subunit of the second engineered ribosome comprises a change-of-function mutations at one or more of the following residues of a 23S rRNA: G2061, C2452, U2585, G2251, G2252, A2057, A2058, C2611, A2062, A2503, U2609, G2454, and G2455.
Embodiment 76. The engineered cell of any one embodiments 69-75, wherein the first, the second, or both the first and the second engineered ribosomes comprises an antibiotic resistance mutation.
Embodiment 77. The engineered cell of any one embodiments 69-76, wherein the large subunit of the first engineered ribosome comprises a permuted variant or mutant of a 23SrRNA and/or the small subunit comprises a permuted variant or mutant of a 16S rRNA.
Embodiment 78. The engineered cell of any one embodiments 69-77, wherein the linking moiety covalently bonds a 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, helix, 101, to a helix of the small subunit selected from the group consisting of helix 11, helix, 26, helix 33, and helix 44.
Embodiment 79. A method for preparing a sequence-defined amino acid polymer, the method comprising (a) providing one or more of: (i) the cell of any one of embodiments 69-78; (ii) a cell extract derived from the cell of any one of embodiments 69-78; (iii) purified translation system derived from the cell of any one of embodiments 69-78; b) providing an mRNA encoding the sequence-defined polymer to the cell or the cell extract.
Embodiment 80. The method of embodiment 79, wherein the sequence-defined amino acid polymer is prepared in vivo.
Embodiment 81. The method of embodiment 79, wherein the sequence-defined amino acid polymer is prepared in vitro.
Embodiment 82. The method of any one of embodiments 79-81, wherein the sequence-defined amino acid polymer comprises one or more unnatural amino acids.
The following Examples are illustrative and should not be interpreted to limit the claimed subject matter.
A. Abstract
The ribosome synthesizes genetically-encoded polypeptides from proteinogenic amino acids. Ribosome engineering is emerging as a powerful approach for expanding the catalytic potential of the protein synthesis apparatus and for elucidating its origin, evolution and function. Because the properties of the engineered ribosome might be detrimental for the general protein synthesis, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. The initial solution to this problem has been offered by Ribo-T, an engineered ribosome with the tethered subunits which, while translating a desired protein, could be excluded from translation of the cellular proteome. Here, we provide a conceptually different design of an engineered cell with two orthogonal translation systems, whereby the cellular proteins are translated by Ribo-T, while the native ribosome operates as a segregated protein synthesis machine with its both subunits committed to the translation of specific kind of mRNA. We show that both subunits of the specialized ribosome retain autonomy from Ribo-T, excluded from translation of the cellular proteome and thus, could be engineered for new functions. We illustrate the utility of this system by generating a comprehensive collection of mutants with variations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function mutations that enable the ribosome to overcome the translation termination blockage imposed by the arrest peptide.
B. Introduction
The ribosome performs distinct, complex, and highly coordinated functions during protein synthesis. It is composed of two subunits, small and large, which in bacteria are the 30S and 50S, respectively (
The ribosome has evolved to operate with its natural substrates (mRNAs, tRNAs, and proteinogenic amino acids) enabling it to synthesize genetically-encoded proteins. Nevertheless, its synthetic capabilities could be expanded by molecular engineering to allow the use of alternative genetic codes, polymerization of a wider variety of amino acids, or even carry out a programmable synthesis of non-proteinaceous polymers4. Ribosome engineering could be also employed for elucidating the origin, evolution and function of the protein synthesis apparatus. All such endeavors, however, require altering the intrinsic properties of the ribosome5 that inevitably diminish or even abolish the ribosome's ability to synthesize cellular proteins6,7. Although interesting solutions to this problem could be offered by cell-free translation systems8, the efficiency and scalability issues limit their current application.
The ribosome engineering predicament can be overcome by creating an orthogonal protein synthesis apparatus within the cell that does not participate in the production of the cellular proteome and is exclusively dedicated to the translation of only one or several specific mRNAs9. By mutating the ASD in the 16S rRNA and introducing a complementary SD sequence into an mRNA, it has been possible to direct a fraction of small subunits to the translation of only the cognate mRNA10,11, a strategy that has been exploited for expanding the decoding capacity of the ribosome12. The orthogonality of this set-up, however, is limited to only the small subunit because, due to the stochastic nature of the association of large and small ribosomal subunits in multiple rounds of translation, both the wild-type (wt) and the orthogonal 30S subunits share the same pool of 50S subunits. The inability to create orthogonal 50S subunits has limited the efforts to remodel the PTC and the NPET, the most critical sites for designing a translation apparatus with modified or expanded catalytic capacity. The engineering of the first fully orthogonal translation system became possible with the advent of the ribosome with tethered subunits, Ribo-T13. In Ribo-T, and in subsequent similar designs14-16, circularly-permutated 23S rRNA is embedded into the 16S rRNA, yielding a ribosome whose subunits are tethered by two RNA linkers (
C. Development and Testing of a “Flipped” Orthogonal System for Protein Synthesis in Bacterial Cells
In order to overcome the shortcomings of the original oRibo-T-based approach for engineering cells with two functionally-independent translation machineries, we have now created a conceptually new design of an in vivo system that utilizes dissociable, yet fully segregated, ribosomes dedicated to translation of only specialized mRNAs. By ‘flipping’ the roles of Ribo-T and dissociable ribosomes, we engineered bacterial cells where translation of the proteome is carried out by Ribo-T, whereas the ribosome, composed of the dissociable orthogonal 30S (o-30S) subunit and wt 50S subunit functions as a fully orthogonal translation machine (
The components of the system (
The expression of the o-reporters in the OSYRIS cells relies on the o-ribosomes: in their absence, the reporter proteins (GFP, RFP or luciferase) encoded by the o-mRNAs are produced at low levels, whereas the presence of the dissociable o-ribosomes, greatly stimulates the o-reporter expression (
To test whether only the small subunits or both the small and large subunits of the dissociable o-ribosomes in the OSYRIS cells remain functionally isolated from Ribo-T, we took advantage of the A2058G mutation present in Ribo-T that renders it resistant to the antibiotic erythromycin (Ery)13. If the free 50S subunits, which are Ery-sensitive, could somehow cooperate with the small subunits of Ribo-T in translating the proteome, Ery would inhibit general protein synthesis and interfere with cell growth. However, OSYRIS cells continue to grow even at the highest tested concentration of the antibiotic (1 mg/ml), demonstrating the functional autonomy of the dissociable 50S subunit and Ribo-T (
A more rigorous proof for the orthogonality of the dissociable 50S subunits in the OSYRIS cells was obtained by introducing mutations into its 23S rRNA that are known to be dominantly lethal in wt E. coli cells20,21. Two of these mutations, A2451C and A2602U, alter critical nucleotides of the PTC active site, while mutation G2553C disrupts essential rRNA-tRNA interactions required for the proper placement of the A-site aminoacyl-tRNA for peptide bond formation22,23 (
Altogether, the results of the o-reporter expression and tolerance to dominantly lethal mutations show that in the OSYRIS cells, the dissociable o-ribosomes translate o-mRNAs but do not significantly contribute to translation of the proteome. Therefore, both subunits of the dissociable o-ribosomes in OSYRIS cells are suitable for biomolecular engineering.
Having established the orthogonality of the dissociable ribosome in the OSYRIS cells, we carried out a proof-of-principle experiment to test the potential of the system for selecting mutations in the rRNA of the large subunit that would enable the ribosome to carry out otherwise problematic tasks. Specifically, we aimed to engineer a ribosome capable of efficient release of difficult-to-terminate proteins. In general, release of a fully synthesized polypeptide is a highly-nuanced reaction catalyzed by the PTC with the assistance of class 1 release factors24,25. While most proteins are efficiently released at the stop codons, termination of others can be more problematic26,27. An extreme case of inefficient termination in E. coli is represented by programmed translation arrest at the stop codon of the mRNA encoding the regulatory protein TnaC28-30. At high concentrations of tryptophan, the release of the fully-translated TnaC is inhibited and the resulting stalling of the ribosome at the tnaC stop codon leads to the activation of the expression of the downstream genes of the tna operon31. The termination arrest at the tnaC stop codon is mediated by unfavorable interactions of the nascent TnaC with rRNA nucleotides of the NPET and the PTC30,31. The TnaC-mediated termination arrest represents a paradigm of inefficient protein release and illustrates one of the issues that could curb the expression of bioengineered polypeptides carrying, for example, non-canonical amino acids.
In order to identify mutations that could alleviate the inefficient termination of TnaC, we constructed a reporter in which the TnaC-coding sequence (lacking its own start codon) was appended at the end of the gfp gene (
We then generated a comprehensive library of 120 single-nucleotide 23S rRNA mutants in the poRbs plasmid (
We characterized the ability of the individual mutants to successfully terminate the GFP-TnaC polypeptide by estimating the stalling bypass (SB) score. The SB score reflects the relative expression of the hard-to-terminate GFP-TnaC reporter in comparison with the GFP-TnaC(W12R) variant that terminates efficiently28. In addition, the expression level of the GFP-TnaC(W12R) construct was used to evaluate the effect of the PTC mutations on the general translation activity of the mutant ribosome. Strikingly, a number of the mutants with alterations in the PTC rRNA residues exhibited a notably higher bypass score than the OSYRIS cells with wild type 50S subunit (
A unique opportunity offered by the OSYRIS cells is the possibility of isolating individual ribosomal subunits with even lethal mutations because dissociable 30S or 50S subunits can be separated from Ribo-T by sucrose gradient centrifugation13 (
Our proof-of-principle experiments demonstrated that the OSYRIS design, based on the ability of Ribo-T to sustain cellular growth while compelling the dissociable subunits of the o-ribosome to interact with each other, presents a viable conceptually new approach for generating a fully-orthogonal cellular translation system. Engineering of OSYRIS was possible because Ribo-T is sufficiently active to translate the entire cellular proteome13,17. However, translation driven by Ribo-T is sluggish and RiboT assembly is inefficient, which likely is one of the factors that contributes to the slow growth rate of the OSYRIS cells (doubling time τ˜300 min in 96-well plates in comparison with τ˜45 min for the BL21 strain) (
An obvious possible application of OSYRIS is engineering ribosomes capable of incorporation of non-canonical amino acids into polypeptides that the ribosome discriminates against (such as backbond modified D- and Beta-amino acids32). Although expanding ribosome's synthetic potential requires many components, from a specialized aminoacylation system to a designer genetic code, the fully orthogonal dissociable ribosome operating in the OSYRIS cells could accelerate the achievement of this goal. Importantly, OSYRIS makes possible many other endeavors, from employing ribosome retro-engineering for elucidating the origin of the translation apparatus to evolving new catalytic functions for programmable synthesis of polymers of non-protein nature.
D. Materials and Methods
1. Assembly of the OSYRIS Cells
a. Plasmid Construction
Plasmids used for generation and optimization of the OSYRIS set-up are shown in
All the plasmids were constructed using Gibson assembly1, with the plasmid backbone prepared by inverse PCR or restriction nuclease digest and the cloned inserts either PCR-amplified from the respective templates or synthesized chemically by Integrated DNA Technologies. PCR reactions were carried out using Q5 High-Fidelity DNA polymerase (New England Biolabs), and PCR products were purified using DNA Clean and Concentrator kit (Zymo Research). The Gibson assembly reactions for rRNA-encoding plasmids were electroporated into E. coli POP2136 cells (all the bacterial strains are listed in the Table at
i) pRibo-Tt Plasmid
The backbone of the pRibo-T v 2.0 plasmid3, carrying the A2058G erythromycin resistance mutation, was linearized with SgsI restriction enzyme and purified. The cluster of the missing tRNAs genes (encoding tRNAGlu, tRNAAla, tRNAIle, tRNATrp and tRNAAsp), whose transcription is controlled by the Ptac promoter and T1 terminator, was synthesized as a gBlock (Integrated DNA Technology) and PCR-amplified using primers NA1 and NA2 (all primers are listed in the Table at
ii) poRBS Plasmid
Orthogonal and wt rRNA operons under transcriptional control of the lambda PL promoter and T1/T2 terminators were PCR amplified from the pO2 and pAM552 plasmids4, respectively, using the primers NA5 and NA6. The KanR gene was amplified from the plasmid pKD135 using the primers NA7 and NA8. pSC101 origin of replication was amplified from the pCSacB plasmid6 using the primers NA9 and NA10. The PCR reactions were treated with DpnI to reduce the background of the parental plasmids. The PCR products were purified, confirmed by electrophoresis, and mixed (40 ng of each) in the Gibson assembly reaction. After 1 h incubation at 50° C., 3 μl of the reaction mix were transformed into electrocompetent POP2136 E. coli cells. Cells were plated onto LB/Kan agar plates. After 24 h incubation at 37° C., individual colonies were picked, grown in LB/Kan media, and plasmids were isolated and verified by restriction digest and sequencing.
iii) poGFP Plasmid
The o-GFP gene with 5′ UTR, 3′UTR, and T1/T2 terminators was PCR amplified from the plpp5-oGFP plasmid4 using primers NA11 and NA12. The LuxR repressor and the PLux promoter7 were PCR amplified from the pJDO75 plasmid8 using primers NA13 and NA14. SpcR marker (aadA) was PCR amplified from the ptRNA67 plasmid6 using primers NA15 and NA16. The p15A origin of replication was PCR amplified from the ptRNA67 plasmid using primers NA17 and NA18. The PCR reactions involving plasmid templates were treated with DpnI. Purified PCR products (40 ng of each) were mixed in the Gibson assembly reaction. After 1 h incubation at 50° C. 3 μl of the reaction mix were transformed into electrocompetent JM109 E. coli cells (Promega). Cells were plated onto LB/Spc agar plates. After 24 h incubation at 37° C., individual colonies were picked, grown in LB/Spc media and plasmids were isolated. The presence of the luxR gene insert was confirmed by PCR using primers NA19 and NA20. Restriction digest of the resulting plasmid indicated that its size exceeds the expected one by ˜1 kb. Subsequent restriction analysis and sequencing showed that the luxR gene has undergone duplication (
iv) poRFP/oGFP Plasmid
The SpcR marker (aadA) and the p15A origin of replication were PCR amplified from the ptRNA67 plasmid6. The PCR reactions were treated with DpnI. The o-GFP gene with Plpp5 promoter, 5′ UTR, 3′UTR, and T1/T2 terminators was PCR amplified from the plpp5-oGFP plasmid4. Purified PCR products (˜40 ng of each) were mixed in the Gibson assembly reaction. After 1 h incubation at 50° C., 3 μl of the reaction mix were transformed into electrocompetent JM109 E. coli cells (Promega). Cells were plated onto LB/Spc agar plates. After 24 h incubation at 37° C., individual colonies were picked, grown in LB/Spc media and plasmids were isolated. The structure of the resulting plasmid plpp5-oGFP-pA15-Spec was verified by restriction digest and sequencing. The rfp gene with PT5 promoter and TO transcription terminator was PCR-amplified from the plasmid pRYG9, the orthogonal SD sequence was introduced by PCR and the resulting o-rfp construct was inserted into a unique SphI site of the plpp5-oGFP-pA15-Spc plasmid.
v) poLuc Plasmid
The plasmid poLuc carrying the orthogonal luciferase gene was constructed based on poGFP (
vi) poGFP-TnaC Plasmid
For constructing the reporter poGFP-TnaC plasmids (wt or W12R mutant), the gfp-coding sequence in the poGFP plasmid was replaced with the sequences coding for the chimeric wt or mutant GFP-TnaC proteins. The DNA inserts containing the orthogonal ribosome binding site and GFP-TnaC or GFP-TnaC (W12R) coding sequences were generated by PCR using the templates used for in vitro translation (described below) using primers NA23 and NA24. After purification, the inserts were introduced by Gibson assembly into the poGFP plasmid cut with the restriction enzymes BglII and Salt After transformation, the presence of the correct insert in individual colonies was checked by colony PCR using the primers NA25 and NA26 and by sequencing the corresponding segments of the plasmid.
b. Engineering Ribo-T-Expressing Cells
SQ171 FG cells (Table at
The recipient cells initially carried two plasmids: the pCSacB plasmid containing the rrnB operon, counter-selectable sacB marker, and KanR gene, and the ptRNA67 plasmid carrying the missing tRNA genes that were eliminated during deletion of the chromosomal rRNA operons6. Cells were made electrocompetent and then 50 μl of the cell suspension were transformed with 50 ng of the pRibo-Tt plasmid, carrying the Ribo-T rRNA genes and missing tRNA genes (
c. Elimination of the ptRNA67 Plasmid
The obtained transformants were then cured of the ptRNA67 plasmid. For that, the cells were passaged in LB media supplemented with 100 μg/ml Amp for ˜100 generations. After plating cell dilutions, the absence of the ptRNA67 plasmid in individual clones was verified by their sensitivity to Spc and the lack of visible amounts of the ptRNA67 plasmid bands in the restriction digest of the total plasmid preparation.
d. Inactivation of the recA Gene in the Ribo-T-Expressing Cells
Our initial attempts to introduce poRbs into engineered cells frequently led to the appearance of the aberrant plasmids resulting from recombination between the poRbs and pRibo-Tt plasmids. Therefore, to avoid this problem, we inactivated the recA gene in the cells bearing the pRibo-Tt plasmid. (Of note, inactivating the recA gene before curing off the ptRNA67 plasmid prevented the plasmid loss even after prolonged passaging of the cells in the absence of Spc).
To inactivate the recA gene in the OSYRIS cells by P1 phage transduction, we first prepared the donor strain BW25113 recA::cat by the conventional recombineering procedure using chloramphenicol (Chl)-resistance cassette from the pKD3 plasmid5. The cassette was PCR-amplified using the primers NA27 and NA28. PCR fragment was transformed into BW25113 strain carrying the Red recombinase-expressing plasmid pDK46. After the selection and verification of the recA::cat strain, and curing the pKD46 plasmid, the resulting strain was used as a donor for the phage transduction. P1 phages transduction was carried out according to the standard protocol11 except that the recovery incubation was 6 h instead of 1 h before plating the transductants on LB/agar plates supplemented with 50 μg/ml Amp and 15 μg/ml Chl. The genotype of the engineered strain is shown in the table at
e. Introduction of the poRbs Plasmid
The SQ171 FG ΔrecA/pRibo-Tt strain was then transformed with the poRbs (or when needed, pRbs) plasmid by electroporation and selection of the AmpR/KanR/ChlR cells. The only deviation from the standard transformation protocol was that recovery of the transformants in the SOC medium lacking antibiotics was prolonged to 6 h prior and transformants were selected on LB/agar plates supplemented with 50 μg/ml Amp, 25 μg/ml Kan and 15 μg/ml Chl. Transformants were verified by restriction analysis of the total plasmid and analysis of rRNA by agarose gel electrophoresis.
f. Introduction of the Reporter Plasmids
Reporter plasmids (poGFP, poRFP/oGFP, poLuc, poGFP-TnaC) were introduced by electroporation into SQ171 FG ΔrecA/pRibo-Tt/poRbs cells and selection of the Ampr/Kanr/Chlr/Sper cells, essentially as described in the previous section.
g. Verifying the Genome Sequence of the OSYRIS Cells
During the construction of the OSYRIS cells, the original host cells have been passaged multiple times and undergone single-colony purification at multiple steps, possibly leading to the accumulation of spontaneous mutations. Therefore, the total genome of the fully assembled OSYRIS cells was sequenced. Analysis of the resulting sequence showed the presence of mutations in several genes (Table at
h. Monitoring the In Vivo Expression of the Orthogonal gfp Gene
The OSYRIS cells carrying either poRbs or pRbs plasmids (expressing orthogonal or non-orthogonal ribosomes, respectively) and the poGFP reporter plasmid were grown overnight in LB media supplemented with 50 μg/ml Amp, 25 μg/ml Kan, 25 μg/ml Spc and 15 μg/ml Chl at 37° C. with constant shaking. Cultures were diluted 1:40 (v/v) in fresh LB media supplemented with the same antibiotics and additionally containing 1 ng/ml of N-(β-ketocaproyl)-L-homoserine lactone (HSL) (Santa Cruz Biotechnology), the inducer of the reporter gene transcription. The cultures (120 μl) were placed in the wells of the 96-well flat-bottom polystyrene tissue culture plate (Costar) and placed in the plate reader (TECAN Infinite M200 Pro) and incubated at 37° C. with constant linear (3 mm) shaking. Cell culture densities (A600) and GFP fluorescence (an excitation wavelength of 485 nm, an emission wavelength of 520 nm, optimal gain 30% RFU with applying the gain regulation function) were monitored over a time period of 24-48 h. The autofluorescence of cells lacking the reporter was subtracted from all the recorded values.
For the erythromycin sensitivity test, overnight cultures were diluted 1:40 into fresh LB media supplemented with either only HSL (final concentrations: 0-16 ng/ml) or with 1 ng/ml of HSL and varying concentrations of erythromycin (final concentrations: 0-1 mg/ml). Monitoring of cell growth and GFP expression was as described in the previous paragraph.
When OSYRIS cells carried the poRFP/poGFP reporter, the expression of RFP was monitored using an excitation wavelength of 550 nm and an emission wavelength of 675 nm, optimal gain 30% RFU with applying the gain regulation function.
2. In Vivo Expression of the Orthogonal Luciferase Gene
The OSYRIS cells carrying the poLuc plasmid were grown for 24 h in LB media supplemented with 50 μg/ml Amp, 25 μg/ml Kan, 25 μg/ml Spc and 15 μg/ml Chl and then diluted 1:40 into fresh medium containing the same antibiotics and 1 ng/ml of HSL. After 6 hrs, 0.2 A600 of each culture was spun down (5 min, 5000 g, 4° C.), and cell pellets were flash-frozen. Luciferase activity was measured using the Luciferase Assay System (Promega) following the manufacturer's protocol. Specifically: cell pellets were thawed in a 20° C. water bath and resuspended in a 25 μl of LB supplemented with 10% (v/v) of dibasic phosphate buffer (1 M K2HPO4 pH 7.8, 20 mM EDTA). 20 μl of cell suspension were mixed with 60 μl of freshly prepared lysis mix (25 mM Tris-phosphate 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 lysed at room temperature for 10 min. The 10 μl aliquots of cell lysates were then placed into wells of 96-well black/clear bottom assay plate (Corning), 50 μl of Luciferase Assay Reagent (Promega) were added and fluorescence readings were immediately acquired in TECAN microplate reader.
3. Comparison of the Reporter Expression Driven by oRibo-T or oRbs
E. coli BL21 strain was transformed with either poGFP or poLuc plasmids. The transformants were selected on LB/agar plates supplemented with 50 μg/ml of Spc, grown from individual colonies, and then rendered electrocompetent. The reporter-containing cells were then transformed with poRibo-T (the pBR322 ori-based, AmpR plasmid expressing oRibo-T rRNA)3, or with o-pAM552 plasmid (the pBR322 ori-based, AmpR plasmid expressing oRbs rRNA)3.
The expression of o-gfp or o-luc reporters was measured as described above.
4. Analysis of the Mutant rRNA Content
The presence of the engineered mutations in the 23S rRNA of the orthogonal ribosome was analyzed by primer extension. For that, total RNA was isolated from the OSYRIS cells using the RNeasy Mini Kit (Qiagen). The primers and combination of dNTPs and ddNTPs for analysis of each mutation are shown in the table at
5. Expression of the GFP-TnaC (wt) or GFP-TnaC(W12R) Proteins in the Cell-Free Translation System
The DNA templates containing the T7 RNA polymerase promoter, ribosome binding site from bacteriophage T7 gene 10 and GFP-TnaC or GFP-TnaC (W12R) coding sequences (see Appendix I) were generated by cross-over PCR. First, the T7 promoter and the gfp-coding sequence were PCR amplified from the pY71-T7-GFP plasmid13 using the T7 promoter forward primer NA29 (Table at
Two PCR products corresponding to either the wt or mutant gfp-tnaC constructs were then combined together at 400 pg/μl (final concentration) and reamplified using the T7 and TnaC(rev) primers.
In vitro translation of the gfp-tnaC templates was carried out in the PURExpress, ΔRibosome, ΔtRNAs, Δ amino acids cell-free translation system composed of purified components (New England Biolabs), as described in15 with minor modifications. Reactions were supplemented with a 19-amino acids mixture (final concentration: 0.3 mM of each amino acid) and L-tryptophan to a final concentration of 50 μM (for reactions with low-tryptophan conditions) or 5 mM Trp (for high-tryptophan conditions). PCR-generated DNA templates were added to a final concentration of 5 ng/μl. The reactions were carried out at 37° C. for 3 h in a total volume of 5 μl in 384-well plates with black walls and clear bottom (Falcon) in a plate reader (TECAN Infinite M200 Pro). GFP fluorescence (excitation at 485 nm, emission at 520 nm, optimal gain 30% RFU with applying the gain regulation function) was monitored over time.
6. Preparation of the PTC Mutant Library
The PTC mutant library was generated by transferring individual mutations from the pT7rrnB library16 into the 23S rRNA gene in the poRbs plasmid.
To prepare the plasmid backbone, the poRbs plasmid was digested with SgsI and Bst1107I restriction enzymes, resulting in the excision of a 1546 nt fragment from the 23S rRNA gene. The reaction products were separated by agarose gel electrophoresis, and the 7483 bp backbone fragment was purified from the gel using Zymoclean Gel DNA Recovery Kit (Zymo Research) and DNA Clean & Concentrator Kit (Zymo Research) sequentially.
To generate the 1606-bp inserts carrying the PTC mutations, individual plasmids of the pT7rrnB plasmid library were used as a template for the PCR reaction catalyzed by the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and employing the primers NA35 and NA36. PCR products were cleaned up using the DNA Clean & Concentrator Kit (Zymo Research).
The plasmid backbone (35 ng) and the DNA inserts (60 ng) were mixed in a total volume of 5 μl of a Gibson assembly reaction and incubated for 1 h at 50° C.
Individual Gibson-assembly reactions were used to transform chemically-competent POP2136 cells. The high-throughput transformation was carried out in a flat-bottom tissue culture 96-well plates with low evaporation transparent lid (Falcon). In each well of the plate, 20 μl of competent cells were mixed with 2 μl of individual Gibson assembly reactions. Plates were incubated on ice for 30 minutes, at 42° C. for 50 s and again on ice for 15 minutes. One hundred μl of SOC medium were added to each well, and cells were allowed to recover at 30° C. for 2 h on a shaker. Culture volumes were reduced to 40 μl by spinning the plate at 6000 g for 6 min in a swinging bucket rotor and removing 80 μl of supernatant. Six μl of each of the remaining cell suspension were then spot-plated using a multi-channel pipettor on LB/agar rectangular OmniTray Single-Well plates (Nunc) supplemented with 50 μg/ml Kan. Plates were incubated at 30° C. for 20 h.
Individual colonies were inoculated in fresh LB media supplemented with 50 μg/ml Kan and grown for 12 h at 30° C. Plasmids were isolated, and the presence of the desired mutations, as well as the lack of off-target mutations in the PCR-amplified 23S rRNA segments, were confirmed by capillary sequencing.
The individual PTC mutant library plasmids were then introduced into OSYRIS cells by transforming them into SQ171 FG/pRibo-Tt/poGFP-TnaC cells using the high-throughput transformation approach described above with the following modifications: i) 20 ng of the purified individual plasmids were used in transformation; ii) transformants were recovered in SOC medium for 6 h at 37° C. and patched onto LB/agar plates supplemented with 50 μg/ml Amp, 25 μg/ml Kan, 25 μg/ml Spc, and 15 μg/ml Chl; iii) plates were incubated at 37° C. for 48 h; iv) glycerol stocks were prepared in 96-well plates from cultures grown from individual colonies of the transformants.
7. PTC Library Screening
Individual colonies of the OSYRIS cells carrying the PTC library mutants were inoculated in the wells of a 96-well plate containing 120 μl of LB media, supplemented with 50 μg/ml Amp, 25 μg/ml Kan, 25 μg/ml Spc, and 15 μg/ml Chl, and grown for 24 h at 37° C. with constant shaking. Cultures were diluted 1:40 (v/v) in 120 μl of fresh LB supplemented with the same antibiotics, 0.35 mg/ml 1-methyl-tryptophan (Sigma) and 0.016 ng/ml of HSL. Plates were placed into TECAN Infinite M200 Pro plate reader and incubated at 37° C. with constant linear (3 mm) shaking. Optical density (A600) of the cultures and oGFP fluorescence were monitored as described above.
The termination arrest bypass score was calculated by comparing the efficiency of GFP expression in the OSYRIS cells carrying GFP-TnaC(W12R) mutant construct to that in the OSYRIS cells carrying wt GFP-TnaC construct. The stalling bypass (SB) score values were computed based on the readings obtained at the 48 h time point using the following formula:
The mean SB score values were calculated using data obtained in two independent experiments.
8. Isolation of the 50S Ribosomal Subunits from the OSYRIS Cells
The ribosomes were isolated from the OSYRIS cells following the protocol described by Ohashi et al17. Specifically, OSYRIS cells expressing ribosomes with mutations U2500G, A2060C or A2450U in 23S rRNA were grown overnight at 37° C. in LB medium supplemented with 50 μg/ml Amp, 25 μg/ml Kan, 25 μg/ml Spc, and 15 μg/ml Chl. The cultures were diluted to the final A600=0.003 into 1 L of fresh LB media supplemented with the same antibiotics and grown for approximately 15 h with vigorous shaking until optical density reached A600=0.35. Cells were collected by centrifugation for 15 min at 5000 g (4° C.), and cell pellets were flash-frozen in liquid nitrogen and stored at −80° C. Frozen cell pellets were 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), lysed in EmulsiFlex-C3 homogenizer (AVESTIN Inc.) at 15000 psi for 5 min and then lysates were clarified by 30 min centrifugation at 20000 g (4° C.) and transferred to new centrifuge tubes. Ammonium sulfate was added to the final concentration of 1.5 M and tubes were centrifuged for 1 h at 20000 g (4° C.). The ribosome (Ribo-T+dissociable ribosomes)-containing supernatant was filtered through a 0.22-μm Ø 30 mm polyethersulfone (PES) membrane filter (CELLTREAT Scientific Products). 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, 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, 1.2 M (NH4)2SO4, and the ribosomes were then eluted with the buffer containing 20 mM HEPES-KOH, pH 7.6, 10 mM Mg(OAc)2, 7 mM β-mercaptoethanol, 0.75 M (NH4)2SO4. Eluate fractions containing ribosomes were pulled together and loaded onto 16 ml 30% sucrose cushion prepared in the buffer 20 mM HEPES-KOH, pH 7.6, 10 mM Mg(OAc)2, 30 mM NH4Cl, 7 mM β-mercaptoethanol in 35 ml centrifuge tubes. Ribosomes were pelleted by centrifugation at 36000 rpm for 18 h at 4° C. in the Type 70 T1 rotor (Beckman). Ribosome pellets were resuspended in the dissociation/storage buffer (20 mM HEPES-KOH pH 7.6, 30 mM KCl, 1.5 mM Mg(OAc)2, 7 mM β-mercaptoethanol) and aliquots were flash-frozen and stored at −80° C.
To isolate individual 50S ribosomal subunits, the ribosome preparations were loaded on 10-40% sucrose gradients prepared in buffer 20 mM Tris-HCl, pH 7.5, 1.5 mM Mg(OAc)2, 100 mM NH4Cl, 2 mM β-mercaptoethanol in the centrifuge tubes for the SW41 rotor (Beckman). The gradients were centrifuged for 16 h at 27000 rpm at 4° C. and fractionated on a gradient fractionator (BioComp) with A254 monitoring. Fractions corresponding to the large ribosomal subunits were pooled, concentrated on Vivaspin 2 ml concentrators with cellulose triacetate membrane (Sartorius Stedim Biotech GmbH) and recovered in the ribosome storage buffer (20 mM HEPES-KOH pH 7.6, 30 mM KCl, 6 mM Mg(OAc)2, 7 mM β-mercaptoethanol). The aliquots were flash-frozen and stored at −80° C.
9. Isolation of Ribosomes with Non-Lethal 23S rRNA Mutations
Ribosomes carrying non-lethal mutations in the 23S rRNA (A2503G, A2062G, C2611G, and C2611U) were isolated from the SQ171 cells carrying pAM552 plasmids4 with the corresponding mutations. The corresponding strains expressing pure populations of the mutant ribosomes were prepared as described previously18. The ribosomes were isolated as described above except that after sucrose cushion centrifugation, the ribosomal pellets were resuspended in the ribosome storage buffer (20 mM HEPES-KOH pH 7.6, 30 mM KCl, 6 mM Mg(OAc)2, 7 mM β-mercaptoethanol). The aliquots were flash-frozen and stored at −80° C.
10. Toe-Printing Analysis
Primer extension inhibition (toeprinting) analysis19 was performed as described previously20. When needed, the prolyl-tRNA synthetase inhibitor 5′-O-[N-(L-prolyl)-sulfamoyl] adenosine (L-PSA)21 was added to the reactions to the final concentrations of 50 μM. After separation of the primer extension products in the sequencing gel and phosphorimaging, the intensity of the toeprint bands was determined using the ImageJ software12. The efficiency of the TnaC-induced translation arrest at the tnaC stop codon was calculated by comparing the intensity of the stop codon toeprint band (SB) (arrowhead in
where SBBG and PBBG are backgrounds for the corresponding bands.
11. Structural Analysis and Figure Preparation
For calculating the distances of the 23S rRNA nucleotides to the attacking a-amino group of the A-site amino acid, the crystal structure of the Thermus thermophilus ribosomes with P- and A-site tRNAs in the pre-attack state (PDB 1VY4)22 were aligned on the basis of the full-length 23S rRNA with the high-resolution structure of the partially rotated vacant E. coli ribosome (PDB 4YBB)23. The distance measurements and figure rendering were performed in PyMOL (Molecular Graphics System, Version 2.0 Schrödinger, LLC.).
12. Statistical Analysis
Where relevant, statistical values can be found in the figure legends. The mean of the value was defined as the arithmetic mean. Depending on the numbers of the independent biological replicates (n), deviation ranges represent either standard deviation (s.d.) (n≥3) or experimental error (n=2). All statistical values were calculated and all graphs were plotted using the Microsoft Excel 365 software. The Student's t-test was performed using GraphPad Prism version 8.00 for Windows (GraphPad Software, La Jolla Calif. USA).
E. Applications and Advantages of the “Flipped” Orthogonal Translation System of Example 1
1. Applications
By way of example, but not by way of limitation, applications of the compositions and methods disclosed herein include, but are not limited to: Ribosome evolution/engineering (for example towards more efficient non-canonical amino acid incorporation); Expanded genetic codes for non-canonical amino acid incorporation; Enabling detailed in vivo studies of antibiotic resistance mechanisms, enabling antibiotic development process; Biopharmaceutical production; Orthogonal circuits in cells; Synthetic biology; Producing engineered peptide by incorporating new functionality inaccessible to peptides synthesized by native (or wildtype) ribosome or their post-translationally modified derivatives; Producing novel protease-resistant peptides that could transform medicinal chemistry; Allows for the development of engineered ribosomes in cells.
2. Advantages
By way of example, but not by way of limitation, advantages of the compositions and methods disclosed herein include but are not limited to the following.
The unusual design of Ribo-T limits its functionality as an orthogonal translation system (oRiboT). Specifically, Ribo-T translates proteins with only half the rate of the dissociable ribosome. It is slower in departing from the start codons in comparison with the wt ribosomes. Furthermore, the biogenesis of even ‘wt’ Ribo-T is rather slow and inefficient and the assembly problems could be additionally exacerbated if the ribosome's functional centers are subjected to additional alterations.
In order to overcome the shortcomings of the original oRibo-T-based approach for engineering cells with two functionally-independent translation machineries, we have now created a conceptually new design of an in vivo system that utilizes dissociable, yet fully segregated, ribosomes dedicated to translation of only specialized mRNAs. By ‘flipping’ the roles of Ribo-T and dissociable ribosomes, we engineered bacterial cells where translation of the proteome is carried out by Ribo-T, whereas the ribosome, composed of the dissociable orthogonal 30S (o-30S) subunit and wt 50S subunit functions as a fully orthogonal translation machine. In the resulting setup, that we named OSYRIS (Orthogonal translation SYstem based on Ribosomes with Isolated Subunits), complete orthogonality is achieved because the tethered nature of Ribo-T precludes it from associating with either the o-30S or the 50S of the dissociable ribosome. Therefore, in OSYRIS cells, the physically-unlinked o-30S and 50S ribosomal 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 engineered to achieve new functionalities without interfering with the expression of the cellular proteome.
When compared OSYRIS cells in a side by side the expression of two orthogonal reporters (o-gfp and the newly engineered o-luc) driven by either the dissociable orthogonal ribosomes (oRbs) or the orthogonal tethered ribosomes (oRibo-T) in the same host (E. coli BL21). Noteworthy, in spite of oRibo-T being expressed from a high copy number vector while oRbs was transcribed from a low copy number plasmid, oRbs outperformed oRibo-T. This result clearly demonstrates the advantage offered by oRbs over oRibo-T in translating orthogonal mRNAs and solidifies the notion that the OSYRIS design is superior to the one based on oRibo-T.
Ribosome engineering is of great interests to the fields of biotechnology, chemistry, and material science, but previous approaches have not been able to evolve the large subunit of the ribosome, which comprises the catalytic active site and the protein excretion tunnel. The development of a tethered ribosome removes these limitations and expands the possibilities of ribosome engineering. Ribosomes may be engineered to incorporate unnatural amino acids for expanded protein functionality or to perform new chemistry for the production of non-protein polymers.
This invention details the first ever orthogonal ribosome-mRNA system where mRNA decoding, catalysis of polypeptide synthesis, and protein excretion can all be optimized for new substrates and functions. The key difference from the prior art is that not only the small (decoding) ribosomal subunit, but also the large (catalytic) ribosomal subunit function as a single, combined and undividable orthogonal genetic synthetic machine.
Moreover, this is unique in that a tethered ribosome keeps the cell alive and a freely dissociable ribosome is used for engineering.
We stress that this invention represents the first of its kind. We anticipate that the innovations reported here will help to inspire larger ribosome construction and engineering efforts to push the limits of engineered biological systems, opening new commercial opportunities in research areas that are currently beyond the adjacent possible.
In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.
This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/993,860, filed on Mar. 24, 2020, which content is incorporated herein by reference in its entirety.
This invention was made with government support under MCB-1716766 and MCB-1615851 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/024006 | 3/24/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62993860 | Mar 2020 | US |
