Patent Application
20130183761
The pyrrolysl-tRNA synthetase/tRNA (PylRS/tRNACUAPyl) pairs from M. barkeri (Mb) and M. mazei (Mm) are orthogonal in E. coli.1 These pairs have been evolved to direct the site-specific incorporation of a range of unnatural amino acids, including amino acids that are post-translationally modified, amino acids containing bio-orthogonal chemical handles, and amino acids protected with light and acid sensitive groups into proteins in E. coli in response to the amber codon.1-6 In contrast to other aminoacyl-tRNA synthetase/tRNA pairs for the incorporation of unnatural amino acids, which are orthogonal in either eukaryotic or prokaryotic hosts, the PylRS/tRNA pairs are orthogonal in both E. coli and mammalian cells.2,6,7 Several unnatural amino acids have been site-specifically incorporated into proteins in mammalian cells by evolving the synthetase/tRNA pair in E. coli and subsequently transferring it to mammalian cells. This approach has the advantage of bypassing the requirement to evolve the amino acid specificity of the synthetase directly in a eukaryotic host.8,10 Another advantage of said approach is the versatility of the variants of pyrrolysl-tRNA synthetase that can be used in the orthogonal pair to site specifically incorporate a variety of unnatural amino acids.
Many biological processes are more effectively addressed in the yeast Saccharomyces cerevisiae (S. cerevisiae) than in mammalian cells. Yeast has a rapid doubling time, bar-coded libraries of gene knockouts exist, protein interaction and transcriptome data is most complete, tap-tagged strains are readily available and powerful genetic approaches can be simply implemented. However the requirement to evolve the current orthogonal pairs directly in yeast has limited the scope of unnatural amino acids that have been incorporated in yeast.
Preliminary work by Yokoyama and coworkers introduced a PylRS/tRNACUAPyl, pair into yeast and reported very weak phenotypes consistent with poor incorporation of Nε-tert-butyl-oxycarbonyl-
Here we report the creation and characterization of functional and orthogonal tRNA synthetase-tRNA pairs (such as pyrrolysyl-tRNA synthetase/tRNAPyl pairs) in a eukaryote such as yeast. We demonstrate the incorporation of several useful unnatural amino acids using variants of this pair created in E. coli.
In another aspect the invention provides an orthogonal PylRS/tRNAPyl pair that is functional in a eukaryote such as yeast for site-specifically incorporating unnatural amino acids into proteins.
In another aspect, the invention relates to a nucleic acid comprising a nucleotide sequence encoding a tRNA orthogonal to a eukaryotic cell, said nucleotide sequence operably linked to a promoter capable of directing transcription by eukaryotic RNA polymerase III.
At least three aminoacyl-tRNA synthetase/tRNACUA pairs (EcTyrRS/tRNACUA, EcLeuRS/tRNACUA and PylRS/tRNACUA from methanosarcina species) are orthogonal in eukaryotic cells and can be used to incorporate unnatural amino acids. In mammalian, worm (nematode) and fly systems, suitably PylRS/PylT are used. In worm (nematode) and fly systems suitably the M. mazei versions are used. In worm (nematode) and fly systems suitably the synthetase is wild type or the PCKRS mutant, most suitably wild type. In fly systems suitably the PCKRS mutant may be used.
Suitably said orthogonal tRNA is tRNAPyl.
As a practical matter, when said orthogonal tRNA is tRNAPyl, it should be noted that the PylT gene used may however lack the three 3′ bases (CCA), since in eukaryotes these are added post-transcriptionally. Suitably the wild type PylRS is used for multicellular eukaryote systems.
Suitably said eukaryotic cell is a yeast cell and the tRNAPyl comprises sequence at positions 3 and 70 which do not form a 3-70 base pair.
In another aspect, the invention relates to a nucleic acid comprising a nucleotide sequence encoding tRNAPyl operably linked to a promoter capable of directing transcription by yeast RNA polymerase III, wherein the tRNAPyl comprises sequence at positions 3 and 70 which do not form a 3-70 base pair.
Suitably the tRNAPyl comprises adenosine at position 3.
Suitably the yeast is Saccharomyces cerevisiae.
RNA POL III Promoter
Suitably any promoter capable of directing RNA Pol III transcription in eukaryotic cells may be used.
Various options for RNA Pol III promoters are described throughout the specification, including intragenic and extragenic (internal and external) promoters.
Suitably the promoter comprises A and B box consensus sequences.
Suitably said promoter is, or is derived from, the eukaryotic U6 promoter.
An exemplary U6 promoter is described in Das, G., Henning, D., & Reddy, R. (1987). Structure, organization, and transcription of Drosophila U6 small nuclear RNA genes. The Journal of biological chemistry, 262(3), 1187-1193.
An exemplary U6 promter for use in human and/or mouse systems is described in Gautier, A., Nguyen, D. P., Lusic, H., An, W., Deiters, A., & Chin, J. W. (2010). Genetically encoded photocontrol of protein localization in mammalian cells. Journal of the American Chemical Society, 132(12), 4086-4088
C. elegans Pol III promoters which may find application in the invention (also the primer sequences to amplify them from genomic worm DNA) are presented in the following table:
An exemplary RNA Pol III promoter may be used as follows: Drosophila melanogaster U6-2 snRNA gene, complete sequence.
Another exemplary RNA Pol III promoter may be used as follows:
A most preferred nucleic acid of the invention comprises a U6 promoter capable of directing RNA Pol III transcription in mammalian cells such as mouse or human cells operably linked to tRNAPyl, more suitably tRNAPylcua.
Suitably the promoter comprises the yeast sequence encoding tRNAArgUCU.
Suitably the tRNAPyl is tRNAPylCUA.
Suitably the sequence encoding tRNAPylCUA comprises the M. mazei tRNAPylCUA sequence.
An exemplary sequence of M. mazei tRNACUA is as follows: the 3′ CCA that is added post-transcriptionally in eukaryotes (and therefore may be omitted as is the case in the gene in the expression constructs in the examples section) is indicated in BOLD:
Suitably the sequence encoding tRNAPylCUA comprises the M. barkeri tRNAPylCUA sequence having a G3A substitution.
In another aspect, the invention relates to an expression system comprising a nucleic acid as described above; said system further comprising a nucleotide sequence encoding a PylRS capable of aminoacylating the tRNAPyl.
Suitably the PylRS comprises M. barkeri PylRS or AcKRS or TfaKRS or PcKRS.
Suitably the synthetase comprises PylRS such as M. mazei PylRS. An exemplary sequence of M. mazei PylRS is shown. The sequence in BOLD is a FLAG tag which is optionally included to be able to easily detect the protein on a western blot.
In another aspect, the invention relates to a eukaryote such as a yeast cell comprising a nucleic acid as described above or an expression system as described above.
Suitably the yeast cell is S. cerevisiae.
In another aspect, the invention relates to use of a nucleic acid as described above or an expression system as described above to incorporate an unnatural amino acid into a protein in a eukaryote such as a yeast cell.
In another aspect, the invention relates to a method for incorporating an unnatural amino acid into a protein in a eukaryote cell such as a yeast cell comprising the following steps:
In another aspect, the invention relates to a method as described above, wherein the unnatural amino acid to be incorporated is an alkyne-containing amino acid or a post-translationally modified amino acid or an amino acid containing bio-orthogonal chemical handles or a photo-caged amino acid or a photo-crosslinking amino acid.
PylRS is pyrrolysyl-tRNA synthetase. This may typically be an Archaea PylRS such as a Methanosarcina PylRS such as a M. barkeri or M. mazei PylRS, or a PylRS derived from same. PylRS derived from a M. barkeri or M. mazei PylRS may include acetyl-lysyl-tRNA synthetase (AcKRS) or Trifluoro-acetyl-lysyl-tRNA synthetase (TfaKRS) or photocaged Lysyl-tRNA synthetase (PcKRS) as discussed below.
Suitably, the PylRS is derived from M. barkeri.
Suitably the nucleotide sequence encoding the PylRS is codon optimised for a eukaryote such as a yeast such as S. cerevisioe.
Suitably the orthogonal tRNA of the invention is tRNAPyl.
A preferred example of a tRNAPyl of the invention is the tRNAPyl of M. mazei.
Suitably the tRNAPyl is tRNACUAPyl. This incorporates unnatural amino acids by amber suppression i.e. by recognition of the amber codon.
Suitably the tRNAPyl is operably linked to a promoter for transcription by eukaryotic RNA polymerase III, such as yeast RNA polymerase III. Suitably the promoter comprises the sequence encoding for yeast tRNAUCUArg (alternatively described in the art as tDNA).
The eukaryote (or eukaryotic cell) may be any eukaryote (or from any eukaryote) such as yeast, flies (e.g. Drosophila such as Drosophila melanogaster), nematodes (e.g. C. elegans), mice (e.g. Mus musculus), humans or other eukaryote.
The RNA Pol III promoter may be from any source such as any eukaryote provided that it retains the ability to direct transcription of the tRNA in a eukaryote (or eukaryotic cell).
The invention also provides a eukaryote cell such as a yeast cell comprising the PylRS/tRNAPyl pair of the invention. Suitably, the yeast cell is S. cerevisiae, more preferably S. cerevisiae MaV203.
The introduction of the PylRS to a eukaryote such as yeast cell may be done according to any method known in the art, suitably by transforming a nucleotide to sequence encoding the PylRS into the eukaryote cell.
The invention also relates to the use of an orthogonal tRNA synthetase/tRNA pair such as a PylRS/tRNAPyl pair to incorporate unnatural amino acids into proteins in a eukaryote such as a yeast cell. This could be alternatively described as a method for incorporating unnatural amino acids into proteins in a eukaryote such as a yeast cell comprising the following steps: transforming the eukaryote such as yeast cell with a nucleotide sequence or sequences encoding an orthogonal tRNA synthetase/tRNA pair such as PylRS and tRNAPyl as described above and then placing the eukaryote such as yeast cell in medium containing the unnatural amino acid to be incorporated.
Given the variants available for PylRS, as detailed above and discussed below, the PylRS/tRNAPyl pair is an especially versatile use or method of incorporating unnatural amino acids in yeast because it can be used to incorporate an alkyne-containing amino acid or a post-translationally modified amino acid or an amino acid containing bio-orthogonal chemical handles or a photo-caged amino acid or a photo-cross linking amino acid.
Preferably, said incorporation is done through amber suppression and thus with a tRNACUAPyl.
Genetic code expansion has been limited to the incorporation of unnatural amino acids in cultured cells and unicellular organisms. Here we report genetic code expansion in eukaryotes. In addition we demonstrate this in multicellular eukaryotic animals, such as the nematode C. elegans
Suitably the pyrrolysyl-tRNA synthetase/tRNAPyl pair function as an orthogonal aminoacyl-tRNA synthetase/tRNA pair to incorporate unnatural amino acids into proteins in a eukaryote such as yeast with site specificity i.e. in response to a codon recognised by the tRNAPyl.
Within the context of the present invention, yeast means a eukaryotic microorganism classified in the Kingdom Fungi, with about 1,500 species described. Most reproduce asexually by budding, although a few reproduce by binary fission. Yeasts generally are unicellular, although some species may become multicellular through the formation of a string of connected budding cells known as pseudohyphae, or false hyphae. Exemplary yeasts that can be used in the disclosed methods and kits include but are not limited to Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, and Saccharomycetales. Most suitably the yeast is Saccharomyces cerevisiae.
The tRNAPyl suitably comprises sequence at positions 3 and 70 which do not form a 3-70 base pair; more suitably the tRNAPyl comprises adenosine at position 3 to achieve this. The absence of this base pair has the advantage of avoiding interference with yeast alanyl-tRNA synthetase. Thus this feature provides orthogonality. Suitably the tRNA” derives from M. mozei as this is an example of tRNAPyl that works with pyrrolysyl-tRNA synthetase (or its variants).
The expression “derived from” means that the nucleic acid or protein is based on or corresponds to the nucleic acid or protein recognised in the art as wild-type for the sequence of interest. The actual origin of the nucleic acid or protein is immaterial to the scope of the invention. There are many alternatives in the art to produce or isolate sequences of nucleic acid or protein, and the person skilled in the art is capable of choosing the most advantageous or suitable for his needs. Suitably derived from means at least 70% sequence identity to, more suitably at least 80% sequence identity to, more suitably at least 90% sequence identity to, more suitably at least 95% sequence identity to, more suitably at least 97% sequence identity to, more suitably at least 98% sequence identity to, more suitably at least 99% sequence identity to the sequence from which it is derived.
Within the context of the present invention pyrrolylsyl-tRNA synthetase or its variants (PylRS) is a group of aminoacyl-tRNA synthetases that possess a common protein structure but which may have been adapted (mutated) to carry different unnatural amino acids. The common protein structure is wild-type pyrrolylsyl-tRNA synthetase derived from M. barkeri (MbPylRS). Suitable PylRS species include AcKRS (a variant of MbPylRS that has been evolved to use 23), TfaKRS (a variant of MbPylRS that can use 3, see text), PcKRS (a variant of MbPylRS that has been evolved to use 42. Thus the invention advantageously allows the incorporation of a wide variety of unnatural amino acids into proteins made in a eukaryote such as yeast with site specificity.
The use of a pyrrolylsyl-tRNA synthetase or its variants (PylRS) derived from M. barkeri and the use of amber suppression system permits the variation pyrrolylsyl-tRNA synthetases as discussed above.
This pairing of aminoacyl-tRNA synthetase and tRNA is advantageously used in a eukaryote such as yeast cells. Preferably said yeast cells are S. cerivisiae. These are the most studied yeast cells and most used in current molecular biology and biotechnology experimentation—fields where the present invention finds applications. The invention relates to the provision of eukaryotic cells such as yeast cells with an orthogonal pairing that comprises tRNAPyl. A preferred method of providing said eukaryotic cell with the orthogonal pairing is by providing nucleotides, preferably deoxyribonucleotides, that encode the synthetase protein and the tRNAPyl. Within the context of the present invention, “encode” refers to any process whereby the information in the sequence of a polymeric macromolecule is used to direct the production of a second molecule or sequence. This process can include transcription or translation, the operation of which in eukaryotic cells such as yeast cells is well known.
Both the synthetase protein and the tRNAPyl are preferably derived from prokaryotes or archaea. Methods of inducing the expression of a heterologous protein in eukaryotic cells are well known and therefore can be easily done for the synthetase. tRNAPyl is derived from prokaryotes and is transcribed in eukaryotic cells according to the present invention. A tRNA is suitably transcribed in eukaryotic cells by RNA Polymerase III. Thus another aspect of the present invention is the provision of a nucleotide that allows for the transcription of tRNAPyl in eukaryotic cells. This is suitably done according to the present invention by operably linking the nucleotide sequence encoding tRNAPyl to a promoter for RNA Polymerase
Suitably the nucleotides encoding the orthogonal pairing according to the invention, especially tRNAPyl, are deoxyribonucleotides.
Within the context of the present invention, promoter means a region of DNA that generally is located upstream (towards the 5′ region of a gene) that is needed for transcription. The promoter of the present invention for the tRNA is suitably for eukaryotic RNA Polymerase III. When the transcript comprises a leader RNA sequence, suitably the leader is subsequently cleaved post-transcriptionally from the primary transcript to yield the mature RNA product. The leader sequence may comprise one in which A- and B-boxes are internal to the primary transcript, but are external to the mature RNA product. As shown herein, internal promoters can be exploited to express E. coli tRNAs in eukaryotes such as yeast.
However, in other aspects of the invention the RNA Pol III promoter is suitably external to the transcribed RNA sequence. Incorporation of internal RNA polymerase III promoters into the transcribed section of a tRNA gene can affect the tertiary structure of the resulting tRNA. This can be by insertion and/or by substitution (mutation) but clearly in either case the resulting tRNA sequence has been altered when the RNA Pol III promoter is incorporated internally to the tRNA sequence. For this reason, it is advantageous to avoid altering the DNA sequence encoding the tRNA sequence to incorporate internal promoter(s). Suitably the RNA Pol III promoter is external to the tRNA coding sequence. Suitably the RNA Pol III promoter operably linked to the tRNA coding sequence is an extragenic promoter. Suitably the RNA Pol III promoter is 5′ to the tRNA coding sequence. The use of RNA Pol III promoters which are external to the tRNA sequence offers the advantage that the sequence of the tRNA is not affected by being operably linked to the RNA Pol III promoter.
Suitably said RNA Pol III promoters may include the SNR52 promoter, the RPR1 promoter or the SNR6 promoters. More suitably the promoter comprises tDNAUCUArg tDNAUCArg is a deoxyribonucleotide sequence which is part of a dicistronic gene which derives from yeast and codes for two mature tRNAs in yeast: tRNAUCUArg-tRNAGUCAsp. tDNAUCUArg is easily separable from the tDNAGUCAsp for example as described herein.
The promoter is operably linked to the deoxyribonucleotide encoding the tRNAPylvia any method known in the art. Preferably, it is attached by a 10-15 nucleotide bridge, for example as disclosed in
As already mentioned above, the PylRS/tRNAPyl pairing in a yeast cell is another aspect of the invention. The PylRS/tRNAPyl are preferably created in E. coli. Any method known in the art can be used to introduce them to yeast cells, either together or separately. It is preferable if both are introduced into the yeast cell as deoxyribonucleotide sequences encoding for the PylRS protein and tRNAPyl, and that then these are transcribed by the yeast cell. Most suitably the sequences may be present on the same nucleic acid such as a plasmid.
The yeast cell could also be a cell that is part of a stable yeast cell line with the orthogonal pair according to the invention or nucleotides encoding for said pairing present in the yeast cell line.
The methods described herein rely upon the introduction of foreign or exogenous nucleic acids into yeast. Methods for yeast transformation with exogenous deoxyribonucleic acid, and particularly for rendering cells competent to take up exogenous nucleic acid, are well known in the art. The preferred method is the lithium acetate method.
The present invention allows incorporation of unnatural amino acids site specifically into proteins in yeast. The advantages of said orthogonal system include that it allows such incorporation to be done without otherwise disrupting the cell and thus to study the effects of the incorporation in vivo in yeast cells. Some examples of said uses are:
Given the growing list of amino acids that can be incorporated using MbPylRS and its variants,1-6 it is clear that the introduction of a wide range of chemical functional groups into proteins in yeast via the invention is of wide industrial application.
Further Applications
The cells are suitably in vitro cells. Suitably the methods of the invention are in vitro methods. Suitably any test animals used are used in laboratory setting. Suitably the methods of the invention are not methods of treatment or surgery of the human or animal body.
In some embodiments, the cells may be comprised by a whole organism. In some embodiments the methods of making polypeptide incorporating unnatural amino acids may take place in the cells within an organism. Suitably such an organism is a multicellular eukaryote.
The invention also relates to systems and/or kits comprising the elements for incorporation of unnatural amino acids into polypeptides in eukaryotes according to the present invention. In particular such a system or kit may have three components:
In one embodiment (i) and (iii) may be provided on the same nucleic acid.
The coding sequence of (ii) is suitably operably linked to its own promoter. This promoter is suitably a promoter for RNA pol II, i.e. the conventional RNA polymerase use to express polypeptide coding sequences in eukaryotes. The coding sequence of (ii) may further be linked to a stabilising 3′ untranslated region (3′UTR) to stabilise the RNA in a eukaryotic cell. Thus in one embodiment the nucleic acid of (ii) comprises in the order 5′ to 3′; promoter, suitably RNA pol II promoter; coding sequence for polypeptide of interest comprising orthogonal codon at position for incorporation of unnatural amino acid; stabilising sequence such as stabilising 3′UTR sequence.
The invention also relates to new selectable marker constructs in nematodes such as C. elegans. In another aspect, the invention relates to a method for producing a nematode comprising a recombinant nucleic acid, said method comprising:
A challenge presented by multicellular eukaryotes is getting the unnatural amino acid into their cells to be available for incorporation. One method is to include the unnatural amino acid in the medium in which the multicellular eukaryotes live or grow. Another approach is to include the unnatural amino acid in their food. In this embodiment suitably the food comprises bacteria and suitably the unnatural amino acid is contacted with the bacteria; in this manner the unnatural amino acid is introduced to the multicellular eukaryote via the bacteria taking it up and being consumed by the multicellular eukaryote.
(A) Western blot of lysates from mixed populations of worms grown in the absence or presence of (1). Antibodies against GFP were used to detect full length GFP-mCherry protein (upper panel) and GFP truncated at the amber stop codon (lower panel). (B) affinity purification of GFP-mCherry used in
Supplementary Movies 1-4.
Animals carrying the Ex1[Prps-0::mGFP-TAG-mcherry-HA-NLS, Prps-0::FLAG-MmPylRS, PCeN74-1::MmPylT, Prps-0::hpt)] extra-chromosomal array were grown for 48 h in the presence (movies 1 & 2) or absence (movies 3 & 4) of (1). Movies were acquired using filter sets for mCherry and GFP. Channels were switched during movie acquisition, active filter sets are indicated.
To investigate the amber suppressor activity and potential orthogonality of the MbPylRS/tRNACUAPyl pair in S. cerevisiae we used MaV203:pGADGAL4(2TAG) cells.8,9 This yeast strain contains a GAL4 transcriptional activator gene bearing amber codons, is auxotrophic for histidine, and contains HIS3 and LacZ genes on GAL4 activated promoters. When a functional amber suppression system, such as the EeTyrRS/tRNACUAPyl pair,8,9 is transformed into this strain, full length GAL4 is produced, leading to activation of LacZ and HIS3 genes. Transcription of these genes allows cells to grow in the absence of histidine and turn blue in the presence of X-Gal.
Experimental Section
General methods Nε-[(2-Propynyloxy)carbonyl]-
Phenotyping yeast cells Phenotyping was performed as described in Chin et al.,8 Briefly, S. cerevisiae MaV203 (Invitrogen) was transformed by the lithium acetate method with the pGADGAL4(2TAG) reporter, pMbPylRS and tDNACUAPyl constructs. Overnight cultures were serially diluted and replica plated onto selective media in the presence or absence of 2 mM Nε-[(2-propynyloxy)carbonyl]-
Protein Expression, Purification, Western Blot Analysis and Mass Spectrometry
Appropriate selective medium±unnatural amino acid was inoculated with a stationary phase culture to give an O.D.600˜0.2. Cultures were grown at 30° C. for 24-48 h. Proteins were extracted from yeast cells using Y-PER reagent (Thermo Scientific) containing complete, EDTA-free inhibitor cocktail (Roche). Clarified supernatants were separated by SDS-PAGE and western blots were performed using anti-His6 (Qiagen). Human superoxide dismutase was purified using Ni2+-NTA resin (Qiagen) as previously described.26 For expressions with N68 acetyl-
The nucleotide sequences here below discussed are listed in a separate annex, Annex 1, at the end of the description.
Plasmid Construction
PCR reactions were carried out with Pfu or Pfu turbo polymerases (Stratagene) unless otherwise stated.
MbtDNACUAPyl cassettes: The MbtDNACUAPyl cassette was synthesized (Geneart). Site-directed mutagenesis was carried out using primers: P84/P85 (A box mutant: A11C/U24G/U15G); P82/P83 (B box mutant: A56C); P59/P60 (addition 3′-CCA). SNR52-MbtDNACUAPyl-SUP4 cassette: The tRNA cassette described in Wang et al.13 was synthesized (Geneart) with E. coli tDNACUAPyl replaced with MbtDNACUAPyl.
SNR6up-MbtDNACUAPyl-SNR6down cassette: MbtDNACUAPyl was constructed from primers P88/P186. SNR6 upstream and downstream sequences were amplified from S. cereviasiae S288C genomic DNA with P183/184 and P187/P188 respectively. PCR fragments were assembled by overlap PCR.
Ile{TAT}LRI, Pro{TGG}FL and Asp{GTC}KR cassettes: MbtDNACUAPyl and SNR6 downstream sequences were amplified as above. The upstream sequences, as discussed in Dieci et al.15 were constructed with primers P189/P190 (Ile), P190 (pro) and P192 (Asp) and assembled with MbtDNACUAPyl and SNR6 downstream sequences by overlap PCR with Phusion polymerase (New England Biolabs).
SctDNAUCUArg-MbtDNACUAPyl cassette: The cassette was built by consecutive overlapping PCR with ten primers (P164-P173) using Phusion polymerase. The SctDNAUCUArg-MmtDNACUAPyl cassette was made by introducing the G3A mutation using primers P202/P203.
The tRNA cassettes were cloned into the XmaI/SpeI restriction sites of pRS426 (URA3, ATCC) using the AgeI/NheI restriction sites of the cassette.
Primers P217/P218 were cloned into the AgeI/NheI restriction sites of pEcTyrRS/EctRNACUATyr to replace the EctDNACUATyr. The codon-optimized gene for M. barkeri pyrrolysl-tRNA synthetase was cloned into EcoRI/NotI restriction sites of the resulting plasmid to replace E. coli tyrosyl-tRNA synthetase, giving plasmid pMbPylRS.
Primers P217/P218 were cloned into the AgeI/NheI restriction sites of pEcTyrRS/EctRNACUATyr 9 to replace the EctDNACUATyr. The codon-optimized gene for M. barkeri pyrrolysl-tRNA synthetase was cloned into the EcoRI/NotI restriction sites of the resulting plasmid to replace E. coli tyrosyl-tRNA synthetase, giving plasmid pMbPylRS.
Variant Pyrrolysl-tRNA Synthetase
tRNA synthetases that aminoacylate MmtDNACUAPyl with Nε-Acetyl-
Mutations from Wild-Type MbPylS:
We replaced the functional EcTyrRS/tRNACUATyr pair with the MbPylRS/MbtRNACUAPyl pair (
To address the challenge of creating new promoter elements to direct the transcription of MbtRNACUAPyl, we investigated strategies to introduce A and B box sequences into our tRNA expression construct. We first mutated the sequence of the MbtRNACUAPyl gene to contain either near-consensus A box sequences (A11C/U15G/T24G,
Since enhancing the transcription of MbtRNACUAPyl by mutation of the A and B box sequences within the structural gene did not produce a functional amber suppressor, we next investigated the potential of constructs that might augment the transcription of MbtRNACUAPyl using extragenic sequences. The 5′-leader sequence of the yeast SNR52 primary transcript contains A and B box promoters that are post-transcriptionally removed to produce mature SNR52 snoRNA.12 A previous report suggested that adding 5″-SNR52 and 3′-SUP4 flanking sequences to EctDNACUATyr and EctDNACUALeu enhanced their amber suppression in yeast.13 When MbtRNACUATyr was cloned between 5’-SNR52 and 3′-SUP4 flanking sequences (
The yeast U6 (SNR6) gene assembles the same RNA polymerase III transcriptional machinery as tRNA genes but possesses an additional TATA-box promoter element 30 base pairs upstream of the transcription start site that binds TFIIIB.14 The TATA-box enables TFIIIC-independent RNA polymerase III recruitment and is proposed to overcome the large separation (240 bp) of the A and B-box promoter elements of this gene.15 Several yeast tRNAs, some of which contain large introns between the A and B-boxes, have TATA boxes that allow TFIIIC-independent RNA polymerase transcription.15 We reasoned that by incorporating the flanking sequences of these genes into our tRNA cassettes it may be possible to compensate for the poor A and B-box consensus of MbtRNACUAPyl. We created constructs where the 5′-flanking region of SNR6, Ile{TAT}LR1, Pro{TGG}FL and Asp{GTC}KR and the 3′-flanking region of SNR6 sandwich MbtRNACUAPyl (
We constructed a SctDNAUCUArg-MbtDNACUAPyl cassette containing the natural 5′-, 3 and 10 base pair linker sequences (
The tRNA constructs we discovered that are both transcribed, as judged by northern blot, and functional, as judged by phenotyping (constructs 5 and 7), showed amber suppression phenotypes even in the absence of added amino acid 1: construct 5 is blue on X-Gal in the presence and absence of 1, and construct 7 is blue in the presence and absence of 1 and grows on media lacking histidine and containing 3-aminotriazole (3AT) in the presence and absence of 1. These experiments revealed that MbtRNACUAPyl is not orthogonal in yeast.
To identify the molecular basis of the non-orthogonality of MbtDNACUAPyl we examined the sequence of MbtRNACUAPyl for nucleotides that match the positive identity elements within yeast tRNAs that are specifically recognized by yeast synthetases.20 We realized that MbtRNACUAPyl contains an unusual G3-U70 base pair and wanted to test whether this caused the non-orthogonality in yeast cells. To test this hypothesis we expressed human superoxide dismutase (hSOD) bearing an amber codon at position 33 (from pC1 hSOD33TAG-His6 in MJY125-derived strain SCY422). Expression of hSOD was dependent on the presence of SctDNAUCUArg-MbtDNAPCUAPyl, but did not decrease substantially in the absence of 1 (
To begin to demonstrate the range of amino acids that can be incorporated in yeast using our approach, we incorporated the important post-translational modification Nε-acetyl-
Conclusions
In summary we have solved the key challenges of producing a functional and orthogonal tRNACUAPyl in yeast. We have discovered an MbPylRS/tRNACUAPyl pair that is orthogonal in yeast, and described a simple system through which variant MbPylRS/tRNACUAPyl pairs created in E. coli can be transplanted to expand the genetic code of yeast for a wide range of unnatural amino acids. Using our approach we have incorporated the alkyne-containing amino acid Nε-[(2-propynyloxy)carbonyl]-
Genetic code expansion methods, utilizing orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs, have facilitated the site-specific incorporation of unnatural amino acids into proteins in E. coli, in yeast and in mammalian cells1-6. The application of unnatural amino acid mutagenesis to the production of recombinant proteins allows access to modified proteins, including proteins bearing defined post-translational modifications, for structural biology, enzymology, and single molecule studies6-13. The genetically encoded incorporation of photocaged amino acids in living cells allows the photo-control of protein interactions, protein localization, enzymatic activity and signaling3,14-16, while the incorporation of photocrosslinking amino acids allows the mapping of weak or transient protein interactions, including those in membranes, that are hard to trap by traditional non-covalent approaches14,17-20, and the incorporation of bio-orthogonal chemical handles and biophysical probes are providing new approaches for imaging and spectroscopy21,22. Despite these advances, genetic code expansion methods are currently limited to unicellular systems.
Approaches to site-specifically incorporating unnatural amino acids into proteins in multicellular organisms may ultimately facilitate the extension of the approaches developed for the real time, molecular dissection of biological process inside cells3,14-16,23 to the study of complex processes that require interactions between cells in an organism, such as development and neural processing.
C. elegans is our first target for a multicellular genetic code expansion. The genome of C. elegans is sequenced24 and the lineage of every cell during embryogenesis and post-embryonic development has been mapped in this organism25,26 , which is invaluable in understanding mutant phenoypes at the cellular level. The organism has around 1000 somatic cells that make up a variety of tissues including muscles, nerves and intestines. The entire organism is transparent at every stage of life, making it possible to visualize expression in individual cells using fluorescent proteins. This will facilitate light mediated intervention in biological processes using genetically encoded photo-responsive amino acids, including photocrosslinkers and photcaged amino acids. Many biochemical and signalling pathways involved in disease are conserved between C. elegans and humans, which makes C. elegans an important organism for identifying the molecular mechanisms of disease27. Moreover, C. elegans is the only multicellular organism where amber suppressors have been isolated and introduced into the germ line by classical genetics approaches28-31, and suppression efficiencies exceeding 30% have been reported32. These observations suggest that amber suppression is not problematic for the organism through development and reproduction.
The site-specific incorporation of unnatural amino acids into target proteins poses a number of challenges (
We created a reporter for amber suppression (Prps-0::mGFP-TAG-mcherry-HA-NLS) in which a 5′ mGFP is separated from a 3′ mCherry gene by a linker region containing an amber stop codon (
We reasoned that the low GFP expression was likely due to the degradation of reporter mRNA through nonsense mediated decay (NMD), a surveillance mechanism present in eukaryotic cells that is responsible for detecting and destroying transcripts with premature stop codons35,36. When we crossed worms expressing the reporter with smg-2(e2008) worms that are deficient in NMD35,37, but otherwise healthy, we observed a striking increase of GFP signal (
To address the problem of low transmission levels we tested transformation markers that use a gene conferring resistance to specific antibiotics. Recent reports use puromycin38 and G-41839 resistance genes respectively for antibiotic based selection in worms. However, puromycin efficiently kills wild type animals only in the presence of the permeabilizing detergent, Triton X-100, and G-418 does not kill all wild type worms in a population. We therefore investigated a further antibiotic, hygromycin B40, which is used for selection in eukaryotic cell culture, but has not been used as a selectable marker in C. elegans. We found that hygromycin B (0.5 mg/ml) kills 100% of wild type worms without the addition of Triton X-100 (data not shown). When the hygromycin B phosphotransferase gene (hpt) fused to the rps-0 promoter (Prps-0::hpt) was injected into worms it conferred resistance to the antibiotic. Using this approach we were able to isolate transgenic lines that appear to have transmission rates of 100% in the presence of hygromycin B (data not shown). In all subsequent experiments we used hygromycin B resistance as a marker for introducing DNA constructs into C. elegans.
Three aminoacyl-tRNA synthetase/tRNACUA pairs (EcTyrRS/tRNACUA, EcLeuRS/tRNACUA and PylRS/tRNACUA from methanosarcina species) are orthogonal in eukaryotic cells and have been used to incorporate unnatural amino acids41. We and other have demonstrated that the PylRS/tRNACUA pairs from methanosarcina species including M. barkeri (Mb) and M. mazei (Mm), which naturally uses pyrrolysine, can be used to incorporate a range of unnatural amino acids, including Nε-(1-butyloxycarbonyl)-L-lysine (6)13. Moreover, the PylRS/tRNACUA pairs can be evolved in E. coli to recognize new amino acids6, and then be transplanted into eukaryotic cells41. This is in contrast to the other pairs that need to be evolved for new amino acid specificity directly in a eukaryotic host. Since the library construction methods for synthetase evolution are straightforward in E. coli it is especially attractive to develop the PylRS/tRNACUA system for incorporating unnatural amino acids in animals.
To express MmPylRS from an RNA Polymerase II (Pol II) promoter we created Prps-0::FLAG-MmPylRS, in which Prps-0 directs expression throughout the animal, the FLAG tag allows the expression of PylRS to be detected by western blot. Western blots demonstrate that the synthetase is expressed in the worm (
MmtRNACUA requires RNA polymerase III transcription. Transcription of eukaryotic tRNAs by RNAP III is directed by A and B box sequences that are internal to the tRNA gene. Theses sequences are not present in the orthogonal MmtRNACUA gene and it is challenging to introduce such sites without disrupting the three dimensional structure and functionality of the mature tRNA4. We therefore investigated extragenic RNA polymerase III promoters for the transcription of MmtRNACUA. To direct the transcription of MmtRNACUA we created PCeN74-1::MmPylT::sup-7 3′, in which the selected Pol III promoter, derived from the stem-bulge non coding RNA (ncRNA) CeN74-1 is fused to the 5′ end of the MmtRNACUA gene and transcription of the tRNA is terminated by the region found immediately 3′ of the sup-7 C. elegans tryptophanyl tRNA gene. We chose the CeN74-1 promoter, since it shows a high level of expression in adult animals, and some expression in larval stages42,43; we reasoned that these properties would enable us to more efficiently screen for cells or animals expressing a functional tRNA, since worms are in the adult stage for up to several weeks but are only in the larval stages for a short period. Northern blots, using a probe specific for MmtRNACUA4, demonstrate that the tRNA is efficiently produced from this promoter in C. elegans (
We constructed lines containing all genetic components by biolistic bombardment44 of sing-2(e2008) worms with plasmids encoding the reporter, synthetase, tRNA and hygromycin B phosphotransferase gene (Prps-0::mGFP-TAG-mcherry-HA-NLS, Prps-0::FLAG-MmPylRS, PCeN74-1::MmPylT, Prps-0::hpt). The transformants were grown on plates supplemented with hygromycin B for 2 weeks to kill off all non transgenic worms, resulting in populations where all worms contained the extra-chromosomal transgenic array Ex1[Prps-0::mGFP-TAG-mcherry-HA-NLS; Prps-0::FLAG-MmPylRS; PCeN74-1::MmPylT; Prps-0::hpt]. Surviving worms were grown on 5 mM (6) and inspected by fluorescence microscopy for the presence of mCherry in the nucleus of cells within the worm. This step allowed us to select for animals expressing the reporter as well as functional MmPylRS and MmtRNACUA.
We examined several thousand worms and saw a few (1 to 5) mCherry positive worms per hundred worms examined. Individual worms showed mCherry expression in different tissues, including intestinal cells, pharyngeal cells, neurons and body wall muscle. The mosaicism of expression from these extrachromosomal arrays is well documented and results either from loss of the array during mitosis or partial or complete silencing of the array.
We singled out 13 mCherry positive worms and grew them in the absence of (6) and the presence of hygromycinB, to select for inheritance of the array in the resulting lines. We examined these lines for mCherry fluorescence in the presence and absence of (6). While all lines selected showed amino acid dependent mCherry fluorescence, we focused in subsequent experiments on two lines (1.3.1 and 1.8.1). These lines were singled out from distinct plates, and showed the strongest mCherry fluorescence in the presence of amino acid (6). In the absence of amino acid (6) we did not find any worms expressing mCherry, in the several thousand animals we screened by fluorescence microscopy. In contrast, when amino acid (6) was added to the lines we saw strong mCherry fluorescence that was easily detectable under a dissection microscope by eye (
To further demonstrate that the unnatural amino acid is incorporated in response to the amber codon, leading to the production of the full length GFP-mCherry-HA-NLS, we lysed worms from each line grown in the presence and absence of (6) for western blotting. Anti-HA, and anti-GFP western blots confirmed the unnatural amino acid dependent production of GFP-mCherry-HA-NLS in worms (
In conclusion we have demonstrated the first genetically encoded incorporation of unnatural amino acids in a multicellular organism. Since we see mCherry expression throughout the organism our data suggest that the MmPylRS/MmtRNACUA pair can function in diverse tissues to incorporate unnatural amino acids. Since the PylRS/tRNACUA pair and its derivatives that have been evolved in E. coli can be used to direct the incorporation of a range of unnatural amino acids, extensions of the approach reported here should allow the introduction of post-translational modifications, photocaged amino acids, bioorthgonal chemical handles, and photocrosslinkers into proteins in C. elegans.
Materials and Methods
C. elegans Strains and Maintenance
Worms were grown at 20° C. on NGM agar plates according to standard protocols, unless otherwise indicated. The following alleles were used: LGI: smg-2(e2008); LGX: lin-15B(n765).
C. elegans Transformation
Transgenic lines were created by biolistic bombardment using a PDS-100/He Biolistic Particle Delivery System (Bio-Rad)1-3. The bombardment mix contained 10 μg PCeN74-1::MmPylT, 10 μg Prps-0::FLAG-MmPylRS, 5 μg Prps-0::mGFP-TAG-mcherry-HA-NLS and 5 μg Prps-0::hpt. After bombardment worms were allowed to lay eggs for 36 h before adding hygromycin B to plates to a final concentration of 0.5 mg/ml. For the first 4 days bacteria were added to prevent starvation. Plates were scored for transformants after 2 weeks.
Incorporation of Unnatural Amino Acids
Worm lines were maintained on NGM plates supplemented with 1 mg/ml hygromycin B (InvivoGen). To incorporate unnatural amino acids the animals were transferred onto NGM plates without hygromycin B, supplemented with 7.5 mM amino acid (1) for 24 h to 48 h in the presence of food. Incorporation of (1) was determined by the expression of the mGFP-mCherry fusion from Prps-0::mGFP-TAG-mcherry-HA-NLS by direct fluorescence imaging or western blot of whole worm lysates.
Western Blots & Northern blots
Worms, approximately 2000, were lysed in 100 mL 4× LDS sample buffer (Invitrogen) supplemented with DTT by boiling for 15 min. After gel electrophoresis and transfer to nitrocellulose membrane the blots were probed using the following primary antibodies: anti-HA 3F10 (Roche), anti-GFP 7.1 and 13.1 (Roche), anti-FLAG M2 (Agilent). Secondary antibodies used were goat anti-rat IgG-HRP sc2065 (Santa Cruz Biotechnology) and horse anti-mouse IgG-HRP 7076 (Cell Signaling). All blocking, binding and washing steps were performed in TBS supplemented with 0.1% Tween 20 and 5% milk powder. The blots were incubated with primary antibody over night at 4° C. and with secondary antibody 1 h at room temperature. Northern blots were performed as previously described4, using 40 μg of total extracted RNA.
Protein Purification for Immunoprecipitations
Worms were grown on 9cm egg plates to high density1. They were washed off the plate using M9 buffer. 0.5 ml of packed worm pellet was split equally between fresh egg plates with and without amino acid (1, 10 mM). The worms were grown on the egg plates at 20° C. for 48 h. The animals were then washed off the plates, washed once with M9 buffer, resuspended in RIPA buffer, flash frozen in liquid nitrogen and pulverized using a SPEX SamplePrep 6870 Freezer/Mill (Elvatech). After thawing, the lysate was incubated for a further 30 min at room temperature, centrifuged at 16000 g for 20 min and the supernatant incubated with RFP-trap magnetic particles (Chromotek) over night at 4° C. The particles were washed twice with 10 mM Tris pH 7.5, 300 mM NaCl and bound protein eluted by boiling in 2× LDS sample buffer (Invitrogen).
Plasmid Construction
All PCR reactions were carried out using Phusion polymerase (Finnzymes). Protein encoding constructs were assembled into pDEST R4-R3 or pDEST R4-R3_unc-54 using the Gateway system (Invitrogen). pDEST R4-R3_unc-54 contains an unc-54 3′UTR downstream of the attR3 site. Expression of all protein coding genes was driven by the rps-0 promoter (including the rps-0 ATG codon) consisting of 2.2 kb upstream of the rps-0 coding sequence, the unc-54 3′UTR was added downstream of all protein coding genes. The wild type PylRS gene from Methanosarcina mazei was amplified and an N-terminal FLAG tag introduced using primers P32/P35 and P33. An amber stop codon was introduced at the end of the mGFP coding sequence P44 and P45 and XhoI and AscI restriction enzymes. The mCherry construct was amplified using primers P158, P 159, P160 and P161 introducing a C-terminal HA tag followed by the egl-13 nuclear localization sequence5. The hygromycin B phosphotransferase gene (hpt), which confers resistance to hygromycin B, was amplified using primers P283 and P284.
The plasmid encoding M. mazei PylT was constructed by fusing the promoter of Ce N74.1 to PylT linked by a 2 bp sequence (AT). At the 3′ end PylT was fused to the sequence immediately downstream of C. elegans. sup-7. Primers used were P39, P40, P41, P249, P250 and P251. The PCR product was then cloned into pJet1.2 and the resulting plasmid used for transformation.
The following plasmids were constructed:
Entry Vectors:
pDONR P4-P1R
pDONR221
pENTR P2R-P3
Constructs Used for Transformation:
Demonstration of the invention in Drosophila systems are described.
We have tested constructs in cell culture (Drosophila S2) and subsequently used those constructs to make transgenic lines (with the constructs genomically integrated).
In the cell culture experiment we used a fluorescent reporter (GFP fused to mCherry, separated by an amber codon).
GAL4 drives expression of genes behind UAS; protein expression controlled using pMT - - - GAL4 (GAL4 driven by Metallotheine promoter - - - >expression of GAL4 is induced by addiEon of 0.5 mM Cu2+); aa - - - tRS stands for M. mazei PylRS; fusion protein which is only present in the case of incorporation of the unnatural amino acid is detected by probing with antibodies against a C-terminal HA - - - tag or by detecEng GFP (the fusion protein will be twice the size of GFP alone). Constructs are shown in
Results are shown in
(U6 promoter+PylT+U6 3′ region) cloned into a single vector. For the anE - - - GFP blot, only the samples with PylRS are shown). The amino acid used is Nε - - - (t - - - butyloxycarbonyl) - - - L - - - lysine (BocK)
In the transgenic lines the observations are consistent with incorporation of an unnatural amino acid using a Luciferase based reporter. The PylRS and reporter are cloned behind UAS promoters, it is thus possible to cross these flies with publicly available fly lines expressing GAL4 in different tissues. GAL4 induces expression of genes cloned behind UAS.
Adult flies were fed yeast supplemented with 10 mM amino acid, allowed to lay eggs, the resul/ng embryos lysed and the lysates assayed using luciferase. The expressed reporter consisted of renilla luciferase followed by firefly luciferase (the two luciferases separated by an amber stop codon. The graph shows measurements of firefly luciferase ac/vity normalised using renilla luciferase activity. Data from two independent experiments is shown. (Arbitrary fluorescence units) The animals were stably transformed with a plasmid containing Mm PylRS; luciferase reporter; 4 copies of the PylT expression cassette. PylRS and the reporter were cloned behind the UAS promoter (expression is driven by GAL4). GAL4 expression was driven by an armadillo promoter. Results are shown in
Exemplary nucleic acid constructs are shown in
Included are data showing incorporation in fly cell culture using the wild type PylRS and BocK, as well as using the PCKRS incorporating photocaged lysine (as described above and in Gautier, A., Nguyen, D. P., Lusic, H., An, W., Deiters, A., & Chin, J. W. (2010). Genetically encoded photocontrol of protein localization in mammalian cells. Journal of the American Chemical Society, 132(12), 4086-4088).
Also provided are fluorescence microscopy pictures of the cell culture experiments using GFP and mCherry as reporter.
Also provided are images for the fly embryo studies.
All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1016143.8 | Sep 2010 | GB | national |
| 1111661.3 | Jul 2011 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2011/001392 | 9/23/2011 | WO | 00 | 3/22/2013 |
