WO2014122231A1 - Cell-free translation system - Google Patents
Cell-free translation system Download PDFInfo
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- WO2014122231A1 WO2014122231A1 PCT/EP2014/052369 EP2014052369W WO2014122231A1 WO 2014122231 A1 WO2014122231 A1 WO 2014122231A1 EP 2014052369 W EP2014052369 W EP 2014052369W WO 2014122231 A1 WO2014122231 A1 WO 2014122231A1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
Definitions
- the present invention relates to a new cell-free translation system.
- the invention relates to a cell-free reaction system for translating in vitro a RNA into a protein, said reaction system comprising a ribosome-depleted red blood cell lysate and ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes.
- the invention also pertains to a method for translating in vitro a ribonucleic acid template into an amino acid sequence of interest using the cell-free reaction system of the invention.
- the invention also relates to the use of (i) a ribosome-depleted red blood cell lysate, and (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes, for producing a cell-free translation system.
- CFPS Cell free protein synthesis systems
- CFPS are excellent tools to rapidly and efficiently produce proteins, for instance proteins with a pharmaceutical interest such as vaccine components and cytokines (Kanter et al. Blood, 109(8) : 3393-3399, 2007; Yang et al. Biotechnol Prog., 20(6) : 1689- 1696, 2004; Yang et al. Biotechnol Bioeng, 89(5) : 503-51 1 , 2005; Zawada et al. Biotechnol Bioeng, 108(7) : 1570-1578, 201 1 ). Further, CFPS are routinely exploited to perform high throughput production of protein librairies (Goshima et al. Nat Methods, 5(12): 101 1 -1017, 2008). In vitro translational assays represent a good alternative for directly sampling and screening molecules due to the absence of cell wall. In addition, expression of proteins in vitro enables synthesis of toxic proteins that can not be produced in live cells.
- CFPS are prepared from crude cell extracts containing all necessary components for energy generation and the complete apparatus required for translation of RNA (e.g. ribosomes, tRNAs and aminoacyl-tRNA synthetases, initiation, elongation and termination factors, chaperones, amino acids etc.).
- RNA e.g. ribosomes, tRNAs and aminoacyl-tRNA synthetases, initiation, elongation and termination factors, chaperones, amino acids etc.
- cell extracts are supplemented with amino acids, energy sources (e.g. ATP, GTP), energy regenerating systems (e.g. creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E.
- energy sources e.g. ATP, GTP
- energy regenerating systems e.g. creatine phosphate and creatine phosphokinase for
- CFPS coli lysate
- Mg 2+ , K + , etc. co-factors
- An advantage of CFPS is that the concentration of the endogenous components can be manipulated by chemicals, enzymes, or modified by the addition of recombinant proteins (Ohlmann et al. EMBO J., 16(4): 844-855, 1997; Ohlmann et al. J Mol Biol., 318(1 ) : 9-20, 2002; Ziegler et al. Virology, 213(2): 549-557, 1995; Ziegler et al. J Virol., 69(6): 3465-3474, 1995).
- the short incubation time and the good level of standardization of commercially available CFPS render experimental procedures very easy to use and reproduce. All these features make in vitro systems very powerful tools for research and protein production.
- reticulocyte lysate does not recapitulate some important translation characteristics that are found in a cellular environment.
- One of them concerns the lack of cap- and poly(A) dependence which is a critical determinant in translational control (Beilharz et al. Prog Mol Subcell Biol., 50: 99-1 12, 2010; Lemay et al. RNA Biol., 7(3): 291 -295, 2010; Tomek & Wollenhaupt, Anim Reprod Sci., 134(1 -2): 2-8, 2012).
- a mammalian in vitro translation system that could combine efficient protein production with features of translational control usually found in a cellular competitive environment
- the inventors have for the first time designed a highly adaptable in vitro system which relies on mixing i) components obtained from a red blood cell lysate (for instance a reticulocyte lysate, and in particular a rabbit reticulocyte lysate) depleted from its ribosomes, with ii) ribosomes that have been purified from eukaryotic cultured cells, in particular mammalian cells such as HeLa, Jurkat, BHK, mouse stem cells, undifferentiated myoblasts and differentiated myotubes.
- a red blood cell lysate for instance a reticulocyte lysate, and in particular a rabbit reticulocyte lysate
- ribosomes that have been purified from eukaryotic cultured cells, in particular mammalian cells such as HeLa, Jurkat, BHK, mouse
- Such a reconstituted in vitro lysate retains the high efficiency of the parental RRL and recapitulates translational characteristics observed in cells from which the ribosomes have been isolated.
- the inventors have shown that addition of ribosomes from a given cell type is sufficient to confer the translational characteristics found in living cells. As such, the inventors recapitulated cap/poly(A) synergy, the selective advantage of IRES-driven translation and, in particular, cellular tropism that is observed in some cell types and with some specialized mRNAs. For instance, the inventors have reproduced translation stimulation of mRNAs that are activated by cell differentiation.
- system of the invention can also be used for the preparation of an in vitro translational assay in which endogenous proteins which are normally associated to the ribosomal pellet has been depleted by RNA interference.
- ribosomes can be obtained from eukaryotic cells previously transfected with a DNA construct (for instance a plasmid comprising a cDNA) coding for a protein of interest, so that transcription of the DNA into mRNA occurs in cellulo with no need to go through an in vitro transcription procedure.
- a DNA construct for instance a plasmid comprising a cDNA
- the ribosomes are isolated together with the mRNAs.
- the advantage of this embodiment of the in vitro translation system of the invention is that it skips the in vitro transcription step and allows studying translation of transcripts that have been synthesized and processed in their native environment.
- Ribosomes can be obtained from eukaryotic cells infected with a virus, enabling studying translation of viral transcripts that have been synthesized and processed in the host cell. Ribosomes can also be obtained from eukaryotic cells infected with viruses, bacteria or protozoan, enabling studying the effect of these infections on the expression of genes from said eukaryotic cells.
- Ribosomes can also be obtained from eukaryotic cells derived from healthy or pathogenic organs or tissues, enabling studying translation of transcripts that have been synthesized and processed in these specific organs and tissues.
- the inventors have also shown that the in vitro translation system of the invention is efficient even with minute amount of RNA. Indeed, as few as 0.14 fmol (i.e. 0.14x10 "15 mol) allows an efficient translation. In contrast, with other in vitro translation systems based on extracts from mammalian cells, in particular that marketed by Pierce Biotechnology Inc., no translation is detected when 0.14 fmol of RNA is used in the translational assay.
- this system enables analysing the actively translated transcriptome of eukaryotic cells of interest from RNAs, produced in said eukaryotic cells of interest and isolated together with the ribosomes from said eukaryotic cells of interest.
- the invention relates to a new cell-free translation system comprising a ribosome-depleted red blood cell lysate and ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes.
- the invention also relates to methods using this new cell-free translation system for producing a protein of interest, as well as the use of a ribosome-depleted red blood cell lysate and ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease rabbit untreated reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes, for producing an in vitro translation system.
- the invention also concerns an in vitro method for analysing an actively translated transcriptome of eukaryotic cells of interest comprising:
- a translation system comprising (i) a ribosome-depleted red blood cells lysate and (ii) ribosomes and RNAs isolated from the eukaryotic cells of interest in step a), for a time sufficient to achieve translation of the RNAs, isolated with the ribosomes in step a), into the corresponding amino acid sequences,
- step b) identifying and optionally quantifying the amino acid sequences obtained in step b).
- the invention relates to a kit for translating in vitro a RNA into a protein of interest comprising a ribosome-depleted red blood cells lysate and ribosomes isolated from eukaryotic cells.
- a kit for translating in vitro a RNA into a protein of interest comprising a ribosome-depleted red blood cells lysate and ribosomes isolated from eukaryotic cells.
- the invention relates to a method for translating in vitro a ribonucleic acid template into an amino acid sequence of interest, the method using a translation reaction mixture comprising: (i) a ribosome-depleted red blood cells lysate, (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome- depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes.
- the present invention provides a method for translating in vitro a ribonucleic acid template into an amino acid sequence of interest, the method comprising: pre-a) optionally preparing a translation reaction mixture comprising: (i) a ribosome- depleted red blood cells lysate, (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome- depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes;
- step (pre-a) to form a translation system, contacting the translation reaction mixture as defined in step (pre-a) with:
- RNA ribonucleic acid template
- a transcription reaction mixture comprising (i) a DNA encoding the amino acid sequence of interest, and (ii) the necessary element for transcribing said DNA into the ribonucleic acid template;
- the present invention provides a method for translating in vitro a ribonucleic acid template into an amino acid sequence of interest, the method comprising:
- a) to form a translation system contacting a translation mixture comprising: (i) a ribosome-depleted red blood cells lysate, (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome- depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes, with :
- RNA ribonucleic acid template
- a transcription reaction mixture comprising (i) a DNA encoding the amino acid sequence of interest, and (ii) the necessary element for transcribing said DNA into the ribonucleic acid template;
- the present invention provides a method for translating in vitro a ribonucleic acid template into an amino acid sequence of interest, the method comprising:
- a translation reaction mixture comprising: (i) a ribosome-depleted red blood cells lysate, (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes;
- step (pre-a) to form a translation system, contacting the translation reaction mixture as defined in step (pre-a) with:
- RNA ribonucleic acid template
- a transcription reaction mixture comprising (i) a DNA encoding the amino acid sequence of interest, and (ii) the necessary element for transcribing said DNA into the ribonucleic acid template;
- step (b) is conducted during at least 15 minutes, preferably at least 30 minutes, and by order of preference at least 60 minutes, 75 minutes, 90 minutes, 120 minutes, 150 minutes. Further, step (b) is preferably conducted between about 30 ' ⁇ and about 37 °C, and more preferably at 30 ⁇ €.
- RNA template used in step (a) (i) may be readily produced by the one skilled in the art according to known methods.
- the RNA template may be produced by chemical synthesis or by in vitro transcription. It can also be a purified native template.
- the poly (A) tail of the RNA template can be encoded in a DNA coding sequence which can be transcribed to generate an RNA template with a poly (A) tail of defined length.
- An alternative method of generating the poly (A) tail is the use of a poly (A) polymerase in an in vitro reaction to add the tail to the template as a posttranscriptional modification.
- a 5' cap portion may be added co-transcriptionally to the RNA template by the RNA polymerase according to protocols well known to one skilled in the art. If the template is a purified native RNA template, the cap structure will already be in place.
- the ribosomes used to carried out the method are obtained from eukaryotic cells previously transfected, with a DNA (the DNA may be, for instance, a cDNA or a gene) coding for a protein of interest. Since the transcription occurs in cellulo and the translation begins within the cells, the mRNAs are in contact with the ribosomes. Thus, the ribosomes are isolated together with the mRNAs.
- the invention concerns a method for translating in vitro a ribonucleic acid template into an amino acid sequence of interest, the method comprising:
- step (pre-a) after a time sufficient to allow the eukaryotic transfected cells to achieve RNA transcription and to begin translation, isolating ribosomes from the transfected cells of step (pre-a) under conditions that allow the RNA produced from the DNA encoding the amino acid sequence of interest to be isolated together with the ribosomes;
- a translation system comprising (i) a ribosome-depleted red blood cells lysate, (ii) ribosomes isolated from the eukaryotic cells in step (a) for a time sufficient to achieve translation of the ribonucleic acid template into the amino acid sequence of interest.
- the cells are generally cultured during at least between 12h and 48h, preferably 36 hours after transfection.
- RNAse inhibitor To isolate the RNA linked to the ribosomes, it is better to add RNAse inhibitor during cell lysis then after ribosomal fraction resuspension (however if buffer are prepared in RNase free conditions and all ribosomal purification step are carried out at 4°C, RNase inhibitors are not inevitably required). Methods to isolate mRNA linked to the ribosomes are well known to the one skilled in the art.
- step (b) is conducted preferably at about 30 ⁇ , during at least 15 minutes, preferably at least 30 minutes, and by order of preference at least 60 minutes, 75 minutes, 90 minutes, 120 minutes, 150 minutes. Further, step (b) is preferably conducted between about 30 °C and about 37 °C, and more preferably at 30 ⁇ €.
- the different embodiments of the method for translating in vitro a ribonucleic acid template into an amino acid sequence of interest according to the present invention can be performed by using a batch system in a conventional manner. Alternatively, they may be carried out by using various already known or usual methods such as a flow method, wherein materials including the components of the translation reaction mixture are continuously supplied or the reaction product is occasionally withdrawn.
- the invention relates to the use of a translation reaction mixture comprising (i) a ribosome-depleted red blood cell lysate, (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes , for in vitro producing a peptide.
- the invention relates to the use of a ribosome-depleted red blood cell lysate and ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes, for producing translation reaction mixture.
- the invention relates to a cell-free translation reaction system for translating in vitro a RNA into a protein comprising: (i) a ribosome-depleted red blood cell lysate, (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes .
- the invention relates to an in vitro method for analyzing an actively translated transcriptome of eukaryotic cells of interest comprising:
- a translation system comprising (i) a ribosome-depleted red blood cells lysate and (ii) ribosomes and RNAs isolated from the eukaryotic cells of interest in step a), for a time sufficient to achieve translation of the RNAs, isolated with the ribosomes in step a), into the corresponding amino acid sequences,
- step b) identifying and optionally quantifying the amino acid sequences obtained in step b).
- actively translated transcriptome refers to the set of mRNAs in a cell which are associated with ribosomes and are therefore in the process of being translated.
- Step c) of identifying and optionally quantifying the amino acid sequences obtained in step b) may be carried out by any technique well-known from the skilled person such as mass spectrometry, in particular liquid chromatography tandem mass spectrometry (LC/MS/MS); peptide mass fingerprinting; or at least partial amino acid sequencing, in particular Edman degradation.
- mass spectrometry in particular liquid chromatography tandem mass spectrometry (LC/MS/MS); peptide mass fingerprinting; or at least partial amino acid sequencing, in particular Edman degradation.
- the amino acid sequences are identified and optionally quantified in step c) by mass spectrometry.
- the translation system incubated in step b) of the method of analysis preferably comprises all necessary components required for achieving proper translation of an RNA into an amino acid sequence.
- necessary components are well known from the skilled person, and include typically tRNAs and aminoacyl-tRNA synthetases, initiation, elongation and termination factors, chaperones, foldases, amino acids (e.g. natural, unnatural, standard and/or non-standard amino acids), energy sources (e.g. nucleotide triphosphate such as ATP, GTP), energy regenerating systems (e.g. creatine phosphate and creatine phosphokinase, myokinase), and salts (Mg 2+ , K + , etc.).
- energy sources e.g. nucleotide triphosphate such as ATP, GTP
- energy regenerating systems e.g. creatine phosphate and creatine phosphokinase, myokinase
- salts Mg 2+ , K +
- the translation system incubated in step b) further comprises at least one element selected from the group consisting of at least a tRNA, at least an aminoacyl-tRNA synthetase, at least an initiation factor, at least an elongation factor, at least a termination factor, at least a chaperone, at least a foldase, at least an amino acid, at least a labelled amino acid, at least an energy source,at least an energy regenerating system and salts.
- the translation system incubated in step b) further comprises at least a labeled amino acid, such as a radiolabeled amino acid ([ 35 S]- methionine, [ 35 S]-cysteine, [ 3 H]/[ 4 C]/[ 5 N]-amino-acids...), a photoreactive amino acid (for instance diazirine-based amino acid analogs), or a biotinylated amino acid. More preferably, the translation system incubated in step b) further comprises radiolabeled amino acids, in particular [ 4 C]-amino acids and/or [ 5 N]-amino acids.
- a labeled amino acid such as a radiolabeled amino acid ([ 35 S]- methionine, [ 35 S]-cysteine, [ 3 H]/[ 4 C]/[ 5 N]-amino-acids).
- a photoreactive amino acid for instance diazirine-based amino acid analogs
- biotinylated amino acid More
- the method of analysis according to the invention can use puromycin-associated nascent chain proteomics (PUNCH-P), as described in Aviner et al. (2013) Genes & Dev. 27:1834-1844, which is based on incorporation of biotinylated puromycin into newly synthesized proteins under cell-free conditions followed by streptavidin affinity incorporation and liquid chromatography- tandem mass spectrometry analysis.
- PUNCH-P puromycin-associated nascent chain proteomics
- the translation system incubated in step b) further comprises biotinylated puromycin, such as 5' biotin-dC-puromycin 3' as for example described in Starck et al. (2004) Chem. & Biol. 11 :999-1008.
- the method of the invention preferably further comprises a step b') of capturing the puromycin-labelled amino acid sequences obtained in step b) on immobilized streptavidin.
- the amino acid sequences captured in step b') are preferably identified and optionally quantified in step c) by liquid chromatography-tandem mass spectrometry.
- Kits according to the invention are particularly advantageously because they can be implemented in the form of high throughput methods.
- kits that are useful in the above methods and use.
- kits comprise, in separate containers or in the same container, (i) a ribosome-depleted red blood cell lysate, and optionally (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1 ) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes.
- kits comprise, in separate containers or in the same container, at least one element chosen from the group consisting of:
- amino acid e.g. a natural, unnatural, standard and/or non-standard amino acid
- - at least a labelled amino acid, for instance a radiolabeled amino acid
- an energy source e.g. nucleotide triphosphate such as ATP, GTP
- an energy source e.g. nucleotide triphosphate such as ATP, GTP
- an energy regenerating system e.g. creatine phosphate and creatine phosphokinase, myokinase
- a buffer solution (cell lysis buffer, ribosome resuspension buffer, reticulocyte supernatant used as translation buffer).
- kits according to the invention may further comprise, in separate containers or in the same container, at least a biotinylated puromycin and optionally at least an immobilized streptavidin.
- the present invention also concerns a kit for analyzing an actively translated transcriptome of eukaryotic cells of interest comprising, in separate containers or in the same container:
- a ribosome-depleted reticulocyte lysate • at least one element chosen from the group consisting of:
- kits according to the invention may also comprise a control sample comprising a given RNA template.
- the kits according to the invention may further comprise instructions for the use of said kit (i) in translating a RNA into an amino acid sequence, and/or (ii) in producing translation reaction mixture.
- kits may also include means for transcribing a DNA of interest into the corresponding RNA.
- means for transcribing a DNA of interest include in particular a vector or a plasmid (wherein the DNA of interest can be cloned to be under control of a promoter), an appropriate RNA polymerase, rCTP, rATP, rUTP, rGTP and an appropriate buffer system.
- the kits are suitable for performing a coupled transcription / translation reaction.
- the means for transcribing a DNA of interest are not comprised in the container(s) that comprise(s) the ribosome-depleted reticulocyte lysate and the ribosomes isolated from eukaryotic cells.
- the eukaryotic cells from which ribosomes (ii) are isolated are not reticulocytes.
- the ribosome-depleted red blood cell lysate (i) is obtained from rabbit red blood cells, preferably from rabbit reticulocytes.
- the eukaryotic cells from which ribosomes (ii) are isolated are human cells, preferably said human cells are not red blood cells.
- the ribosome-depleted red blood cell lysate (i) is obtained from a red blood cell lysate treated with a nuclease (e.g. a calcium activated S7 microccocale nuclease) to dispose of the endogenous mRNAs that are contained into the reticulocytes.
- a nuclease e.g. a calcium activated S7 microccocale nuclease
- an amino acid sequence is meant any type of natural and unnatural peptide, polypeptide or protein.
- natural means amino acid sequences which comprise "standard” amino acids (i.e. Alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine), and/or "nonstandard” amino acids such as pyrrolysine, selenomethionine and selenocysteine (in specific conditions, selenocysteine is encoded by a UGA codon).
- the natural amino acids may be in the form of dextrogyre or levogyre optical isomers.
- unnatural means amino acid sequences which have at least one modified natural amino acid, for instance a modified non-charged amino acid, a modified acidic amino acid, a modified basic amino acid, a non-alpha-amino acid, an amino acid having functional groups selected from the group consisting of nitro, amidine, hydroxylamine, quinone, aliphatic compounds, an amino acid residue such as p- fluorophenylalanine, p-nitrophenylalanine or homophenylalanine.
- the unnatural amino acids may be in the form of dextrogyre or levogyre optical isomers.
- translation reaction mixture refers to a composition which comprises all necessary components required for achieving proper translation of an RNA into an amino acid sequence.
- the translation reaction mixture preferably comprises a buffer system (about pH 7.3).
- the translation reaction mixture may comprise labelled amino acids, for instance radiolabeled amino acids ([ 35 S]-methionine, [ 35 S]-cysteine, [ 3 H]/[ 4 C]/[ 5 N]-amino- acids...), photoreactive amino acids (for instance diazirine-based amino acid analogs), biotinylated amino acids, to help detection of the translated amino acid sequence.
- radiolabeled amino acids [ 35 S]-methionine, [ 35 S]-cysteine, [ 3 H]/[ 4 C]/[ 5 N]-amino- acids
- photoreactive amino acids for instance diazirine-based amino acid analogs
- biotinylated amino acids for example, to help detection of the translated amino acid sequence.
- the only source of ribosomes of the translation system of the invention is the ribosomal pellet isolated from eukaryotic cells.
- the eukaryotic cells used for obtaining the ribosomes (ii) are mammalian cells, for instance human cells, rabbit cells, rodent (e.g. mouse, hamster, guinea pig and rat) cells, horse cells, cow cells, dog cells, cat cells, goat cells, sheep cells etc...
- the ribosomes are isolated from rodent cells or human cells.
- the mammalian cells may be differentiated (mature) cells or undifferentiated such as adult or embryonic stem cells and progenitor cells.
- the eukaryotic cells used for obtaining the ribosomes (ii) may be derived from eukaryotic cell lines or from organs or tissues.
- the eukaryotic cells used for obtaining the ribosomes (ii) may be derived from vertebrate, in particular mammalian, organs or tissues, for instance brain, heart, liver or lung.
- the eukaryotic cells used for obtaining the ribosomes (ii) may be obtained from healthy or pathogenic eukaryotic cell lines, organs or tissues.
- the eukaryotic cells used for obtaining the ribosomes (ii) may be obtained from tumours, metastasis or biopsy samples.
- Said eukaryotic cells may also be eukaryotic cells infected with a virus, a bacterium or a protozoan.
- Ribosomes can also be obtained from invertebrates such as drosophila and zebra fish cells and yeast.
- the eukaryotic cells will be chosen regarding the wished cellular tropism and/or translational characteristics.
- isolated or “purified” refers to biological molecules that are removed from their natural environment and are isolated or separated and are free from other components with which they are naturally associated.
- the ribosomes isolated from eukaryotic cells according to the invention are isolated from cytoplasmic extracts of these eukaryotic cells, more preferably from S10 supernatant extracts of these eukaryotic cells.
- the ribosomes isolated from said eukaryotic cells are preferably isolated from whole extracts of said vertebrate organs or tissues.
- the ribosomes isolated from eukaryotic cells according to the invention are at least 70-95% pure ribosomes (optionally in association with RNAs and/or ribosome- associated proteins) by weight, preferably at least 75% pure ribosomes (optionally in association with RNAs and/or ribosome-associated proteins) by weight, still preferably at least 80%, 85% or 90% pure ribosomes (optionally in association with RNAs and/or ribosome-associated proteins) by weight, most preferably 98%, 99% or 100% pure ribosomes (optionally in association with RNAs and/or ribosome-associated proteins) by weight.
- a cytoplasmic extract from eukaryotic cells including ribosomes does not correspond to ribosomes isolated from encaryotic cells according to the invention since the extracts contain other eukaryotic proteins, in particular other eukaryotic proteins which are not ribosome-associated proteins, and ribosomes included in these extracts do not constitute at least 70-95% pure ribosomes (optionally in association with RNAs and/or ribosome- associated proteins) by weight, preferably at least 75% pure ribosomes (optionally in association with RNAs and/or ribosome-associated proteins) by weight, still preferably at least 80%, 85% or 90% pure ribosomes (optionally in association with RNAs and/or ribosome-associated proteins) by weight, most preferably 98%, 99% or 100% pure ribosomes (optionally in association with RNAs and/or rib
- the methods commonly used for isolating ribosomes are modified so that the lysis buffer and the sucrose cushion used to carry out differential centrifugation comprise no more than 25 mM to 50 nM of KCI.
- An example of method for isolating ribosomes are disclosed in Example 1 of the description described a typical method for purifying ribosomes. Briefly, a pellet of eukaryotic cells is produced by centrifugation, then it is diluted in a hypotonic buffer (buffer comprising Hepes 10mM, CH 3 C0 2 K 10mM, (CH 3 C0 2 ) 2 Mg 1 mM, DTT 1 mM), homogenized and centrifuged (e.g.
- a hypotonic buffer buffer comprising Hepes 10mM, CH 3 C0 2 K 10mM, (CH 3 C0 2 ) 2 Mg 1 mM, DTT 1 mM
- S10 supernatant extract which contains the ribosomes.
- S10 supernatant is centrifuged through a sucrose cushion (e.g. 1 M sucrose in lysis buffer) for example 2h15 at 240 000 g. Then, sucrose solution is removed and the resulting pellet is resuspended in a suspension buffer (buffer comprising Hepes 20mM, NaCI 10mM, KCI 25mM, MgCI 2 1 ,1 mM, ⁇ -mercaptoethanol 7mM).
- the ribosomes isolated from eukaryotic cells are obtained from eukaryotic cells previously transfected, with a DNA (the DNA may be, for instance, a cDNA or a gene) coding for a protein of interest. Since the transcription occurs in cellulo and the translation begins within the cells, the mRNAs are in contact with the ribosomes. Thus, the ribosomes are isolated together with the mRNAs.
- the eukaryotic cells from which the ribosomes are isolated can be eukaryotic cells previously transfected with i) a miRNA or a siRNA that targets a RNA of interest (e.g. a mRNA or a pre-mRNA), or ii) a DNA expressing said non coding RNA, so that the RNA of interest is not expressed (for instance, when the RNA of interest is a mRNA, protein translation does not occur).
- a miRNA or a siRNA that targets a RNA of interest e.g. a mRNA or a pre-mRNA
- a DNA expressing said non coding RNA e.g. a DNA expressing said non coding RNA
- the cell-free translation system of the invention can be used for in vitro translation assay using ribosomes isolated from eukaryotic cells in which at least one endogenous protein, in particular a protein associated with the ribosomal fraction which is involved in the translational control which, has been depleted by RNA interference.
- red blood cells refers to all types of erythroid cells, in particular to mature erythrocytes or reticulocytes (immature red blood cells).
- the red blood cells are reticulocytes.
- the red blood cells may be obtained from any mammal, for instance from rabbit, human, rodent, horse, cow, dog, cat, goat, sheep etc...
- the red blood cells are of human or rabbit origin. More preferably, the red blood cells are reticulocytes of rabbit.
- a ribosome-depleted red blood cell lysate refers to a red blood cells lystate treated, for example by ultracentrifugation, so that the ribosomes are pelleted and the supernatant of the lysate is free of ribosomes.
- centrifugation conditions e.g. the centrifugal force necessary to pellet the ribosome are well known to the person skilled in the art.
- the ribosome-depleted red blood cell lysate may be obtained as follows: after lysis of the red blood cells, the lysate is centrifuged at 240 OOOg for 2 hours and the lysate supernatant collected.
- red blood cell lysate Methods for obtaining a red blood cell lysate are described for instance by Hunt and Jackson (Hamatol Bluttransfus, 14: 300-307, 1974) and Pelham and Jackson (Eur J Biochem., 67(1 ) : 247-256, 1976).
- the ribosome-depleted red blood cell lysate is a ribosome- depleted rabbit reticulocyte lysate
- the lysate of reticulocytes used to obtain the ribosome- depleted rabbit reticulocyte lysate may be the rabbit reticulocyte lysate marketed by Life Technologies (Ambion®) or PROMEGA®.
- the red blood cell lysate may be treated with a nuclease (e.g. a calcium activated S7 microccocale nuclease) to dispose of the endogenous mRNAs that are contained into the reticulocytes.
- a nuclease e.g. a calcium activated S7 microccocale nuclease
- the red blood cell lysate used to obtain the ribosome- depleted red blood cell lysate is a rabbit reticulocyte lysate not treated with a nuclease ((e.g. a calcium activated S7 microccocale nuclease) (hereinafter abbreviated as "URRL", stands for "untreated rabbit reticulocyte lysate").
- URRL calcium activated S7 microccocale nuclease
- introducing a DNA into eukarvotic cells refers to the introduction of a DNA (the sequence of a gene or the corresponding cDNA) into the cells so that under suitable conditions the DNA is expressed.
- the cells may be transformed, transfected, transduced or infectected with a plasmid, a vector, especially a vector of expression, or transduced with a virus vector, preferably a retrovirus vector, advantageously a lentivirus vector, comprising the DNA of interest, an AAV ("Adeno associated virus”), or inducible expression systems (systeme inductible TET OF/ON).
- a virus vector preferably a retrovirus vector, advantageously a lentivirus vector, comprising the DNA of interest, an AAV ("Adeno associated virus"), or inducible expression systems (systeme inductible TET OF/ON).
- a transcription reaction mixture refers to a composition which comprises all necessary components required for achieving in vitro transcription of a DNA into the corresponding RNA.
- In vitro transcription requires a DNA template containing a promoter (e.g. a prokaryotic phage promoter, for instance T7, T3 or SP6 promoter, or an eukaryotic virus promoter, for example CMV, SV40 promoter), ribonucleotide triphosphates rCTP, rATP, rUTP, rGTP, a buffer system, and an appropriate RNA polymerase.
- a promoter e.g. a prokaryotic phage promoter, for instance T7, T3 or SP6 promoter, or an eukaryotic virus promoter, for example CMV, SV40 promoter
- ribonucleotide triphosphates rCTP, rATP, rUTP, rGTP a buffer system
- the components necessary to carry out an in vitro transcription are well known to the person skilled in the art, and are described in Ricci et al. (Nucleic Acids Res., 39(12):
- Figure 1 illustrates the translational efficiency of different in vitro system.
- RNA with the 5'UTR of ⁇ -globin (A) and (C) or GAPDH (B) upstream of the renilla luciferase was added to the following in vitro systems: Untreated Rabbit Reticulocyte Lysate (URRL), nuclease-treated Rabbit Reticulocyte Lysate (RRL), Wheat Germ Lysate (WG) and Human cell free system (HL) at the concentrations indicated on each panel. Translation was carried out for 30 minutes at 30°C before determination of renilla activity as described in Example 1 . Results are presented as mean +/- SD of three independent experiments.
- Figure 2 is a schematic representation of the experimental procedure used to fractionate the reticulocyte lysate. Briefly, it consists of isolating the ribosome fraction from the postribosomal supernatant by ultracentrifugation of RRL or URRL as depicted.
- the ribosomal fraction Rurrl (if obtained from URRL) or Rrrl (if obtained from RRL) is resuspended in buffer as described in Materials and Methods and the reconstituted lysate is assembled by mixing Su (if obtained from URRL) or Sr (if obtained from RRL) with the resuspended ribosomal fraction.
- Figure 3 illustrates the translational efficiency of the in vitro translation system of the invention.
- Figure 4 illustrates the translational efficiency of the in vitro translation system of the invention as a function of the ribosome concentration.
- FIG. 5 is a schematic representation of the elaboration of the hybrid reconstituted lysate. The same experimental procedure as described above is applied to a cell extract to isolate the ribosomal fraction. A hybrid reconstituted system is then assembled with the supernatant from the URRL (Su) mixed with resuspended ribosomes from cells.
- Figure 6 illustrates the translational efficiency of the in vitro translation system of the invention using ribosomes isolated from different cells.
- Figure 7 illustrates translation efficiency of different reporter mRNAs with the in vitro translation system of the invention.
- the globin renilla reporter construct was used at different concentrations to program the HeLa cell lysate-based protein expression systems marketed by the company Pierce® (Human In Vitro Protein Expression Kit - RNA (Pierce® reference number 88857)) has been discontinued or the hybrid system as indicated on the figure.
- Pierce® Human In Vitro Protein Expression Kit - RNA (Pierce® reference number 88857)
- hybrid system as indicated on the figure.
- expression of lucif erase was determined and presented as mean +/- SD of two independent experiments realized in duplicates.
- the bottom panel summarizes the results and is plotted in a log. scale.
- Figure 9 is a schematic representation of the RNAs used in Example 4, the presence of the cap and poly(A) being indicated as +/+.
- Figure 10 illustrates the effects of capping and polyadenylation of the reporter mRNA on the efficiency of translation.
- Figure 11 illustrates the comparison of the level of translation of an IRES-containing RNA with different cell free synthesis systems (“CFPS").
- the dual renilla luciferase bicistronic construct in which the EMCV IRES was inserted in the intercistronic spacer was translated in the Su+Rh hybrid system, the RRL and the WG (wheat germ) lysates. Both firefly and luciferase activities were determined after 30 minutes of incubation and are presented in separate graphs. Data are presented as mean +/- SD of three independent experiments.
- Figure 12 is an autoradiogram showing the production of both firefly and renilla luciferase from translation of the construct depicted above.
- the resuling mRNAs were expressed in the RRL (1 and 2) or the hybrid system (3 and 4) in the presence of L protease as indicated on the figure. Positions of the [ 35 S]-methionine labeled reporter genes are indicated on the left handside of the figure.
- Figure 13 illustrates the comparison of the level of translation of i) an IRES- containing RNA (CrPV-renilla) and ii) a RNA containing the beta-globin 5'UTR with Hela ribosomes isolated at different KCI concentrations.
- Luciferase production from RNA constructs containing the CrPV (left panel) or the globin (right panel) 5'UTRs was measured in the hybrid system that had been assembled with ribosomal pellets isolated under different KCI concentrations ranging from 25 mM to 500 mM as indicated on the figure.
- Figure 14 illustrates the study of the recapitulation of the PV (poliovirus) IRES cell tropism.
- RNAs with the ⁇ -globin or the poliovirus 5'UTRs upstream of the renilla luciferase were translated in the RRL or a hybrid system assembled with HeLa ribosomes (Su+Rh). The results are expressed as a % of control which is represented by the value obtained for the globin-renilla construct in both RRL and Su+Rh and set to 100%.
- B In vitro transcribed RNAs described above were electroporated in BHK cells for 1 h before determination of the luciferase activity. The result is expressed as a % of control (globin), which was set to 100 %.
- Figure 16 illustrates the efficient depletion of an endogenous protein.
- HIV-1 renilla-luciferase mRNAs as indicated. Data are presented as mean +/- SD of three independent experiments.
- Figure 17 illustrates the translation from RNAs produced in cellulo by cells transfected with the corresponding cDNA.
- RNAs with the ⁇ -globin or the c- myc 5'UTRs upstream of the renilla luciferase were added to the RRL. Translation was carried out for 30 minutes at 30 °C before determination of renilla activity as described in Materials and Methods.
- B cDNA coding for ⁇ -globin or the c-myc 5'UTRs upstream of the renilla luciferase was transfected in HeLa cells. Cells were lysed 36 hours post-transfection and renilla activity was determined.
- Hybrid system constituted from Su mixed with ribosomes isolated from HeLa cells that had been prealably transfected with cDNA coding for the renilla luciferase driven by the globin or c-myc 5'UTRs (as indicated). Once assembled on ice, the mixture was incubated for 30 min before measuring luciferase activity.
- Figure 19 illustrates the level of translation achieved using ribosomes obtained from different mice organs and tissues (brain, lung, liver and heart) in comparison with the level of translation achieved using ribosomes obtained from HeLa cells, Jurkat cells or stem cells.
- Figure 20 illustrates the detection, by electrophoresis, of viral proteins translated from mRNAs and ribosomes obtained from A549 cells infected with influenza virus (R A 549- p R8 ) or non-infected A549 cells (R A 54g) using the translation system of the invention.
- the globin, GAPDH, PV, HIV1 , c-myc 5'UTR, EMCV, CrPV and Utrophin 5'UTRs were obtained by PCR using the pO-glo-renilla, pO-GAPDH-renilla pO-EMCV-renilla, pO- PV-renilla, pO-HIV1 -renilla, pO-CrPV-renilla (Soto Rifo et al., Nucleic Acids Res., 35(18): e121 , 2007; Soto-Rifo et al., Nucleic Acids Res., 201 1 ; Soto-Rifo et al., Embo.
- PCR products were digested and cloned in p1 -renilla and pCDNA3.1 -renilla backbone vectors previously digested by Pvull and BamHI or Hpal and BamHI restriction enzymes respectively.
- the pCDNA3.1 vector was modified after the CMV promotor to minimize the number of nucleotides added upstream of the 5'UTR. Position of the +1 transcription site was controlled by rapid amplification of cDNA extremity (RACE) (Ambion kit).
- P1 -bicistronic construction was cloned with the combination of the simple digest ⁇ - ⁇ - globin-firefly vector (Aflll restriction site) and EMCV-renilla insert obtained by PCR using the p1 -EMCV-renilla.
- pRS-shCtrl and pRS-shDDX3 vectors were respectively generated by a 5'-
- RNAs were transcribed using the T7 RNA polymerase from templates linearized either at the Aflll for Polyadenylated RNAs or at the EcoRV sites for non-polyadenylated RNAs. Uncapped RNAs were obtained by using 1 ⁇ g of linear DNA template, 20U of T7 RNA polymerase (Promega), 40U of RNAsin (promega), 1 ,6mM of each ribonucleotide triphosphate, 3mM DTT in transcription buffer (40mM Tris-HCI (pH 7,9), 6mM MgCI 2 , 2mM spermidine and 10mM NaCI).
- RNAs integrity was checked by electrophoresis on non-denaturing agarose gel.
- Hela, C2C12, BHK and Jurkat cells were obtained originally from American Tissue Type Culture Collection. Mouse stem cells were kindly donated by D.Aubert (IGF-Lyon, France).
- Hela, BHK and C2C12 cells were typically grown in DMEM containing 10% fetal calf serum (FSC) supplemented with 50U/ml of penicillin, 50 ⁇ g/ml of streptomycin (PS) under a humidified atmosphere containing 5% C02 at 37°C.
- FSC fetal calf serum
- PS streptomycin
- C2C12 differentiation is induced by DMEM containing 2% horse serum.
- Jurkat cells were grown in (RPMI) containing 10% FSC supplemented with 50U/ml of penicillin, 50 ⁇ g/ml of streptomycin, 10mM Hepes (pH 7,2-7,5), 2mM L-Glutamine and 1 mM pyruvate under a humidified atmosphere containing 5% C02 at 37°C.
- Mouse stem cells were grown in GMEM containing 10% FSC supplemented with 50U/ml of penicillin, 50 ⁇ g/ml of streptomycin, 1 % MEM unessential amino acids (Gibco), 1 mM sodium pyruvate, 2mM L-Glutamine, 40 ⁇ ⁇ -mercaptoethanol and 400 ⁇ _ Leukemia inhibitory factor (LIF) (Chemicon) under a humidified atmosphere containing 7,5% C02 at 37 °C.
- LIF Leukemia inhibitory factor
- DDX3 knock-down stable clone of Hela cells were obtained by transfecting the pRS-shCtrl or pRS-shDDX3 vectors that were maintained and selected in DMEM growth media supplemented with ⁇ g/mL puromycin, 10% FSC and 1 % PS.
- Hela cells were transfected with ⁇ g of total DNA containing 500ng, 6 ⁇ g or ⁇ g of interest plasmid DNA per 175cm 2 containing 1 ,5.10 7 cells using cationic polymers (JetPEI from Polyplus) as specified by the manufacturer. Cells were lysed 24h to 48 h after transfection either to determine luciferase activity or to pellet and isolate the ribosomes as indicated in the manuscript (see below).
- Hela, C2C12 and BHK cells were electroporated with 100ng of in vitro synthesized mRNAs (see below) for 10 5 cells with the NeonTM system (life technology) following supplier's indications. Cells were lysed 1 h after transfection and luciferase activity was determined. Ribosome purification
- S10 preparation The pellet of 10 8 cells was diluted in an isovolume of lysis buffer (buffer R: Hepes 10mMM, CH 3 C0 2 K 10mM, (CH 3 C0 2 ) 2 Mg 1 mM, DTT 1 mM). The cell suspension was homogenized by potter and centrifuged at 16 000 g for 10mn to yield the S10 supernatant extract.
- buffer R Hepes 10mMM, CH 3 C0 2 K 10mM, (CH 3 C0 2 ) 2 Mg 1 mM, DTT 1 mM.
- Ribosomal fraction 300 ⁇ of S10 preparation was centrifuged through a sucrose cushion (1 M sucrose in buffer R) for 2h15 at 240 000 g. After removal of the sucrose solution, the resulting pellet is gently rinsed in buffer R2 and resuspended in buffer R2 (buffer R2: Hepes 20mM, NaCI 10mM, KCI 25mM, MgCI 2 1 ,1 mM, ⁇ -mercaptoethanol 7mM) and stored at -S0°C.
- Fractionation of the reticulocyte lysate After centrifugation of 1 ml of URRL/RRL, 950 ⁇ of post-ribosomal supernatant is collected, frozen and stored at -80° C. The ribosomal pellet is rinsed in buffer R2 and resuspended in buffer R2 as above.
- RNAs were translated at 2,7 nM unless specified in the figure legend in a final volume of 10 ⁇ of lysate (either crude or reconstituted as indicated on figures) supplemented with 75mM KCI, 0,75mM MgCI2, 20 ⁇ amino acids mix.
- the translation reaction is left incubated for 30mn at 30 °C before the reaction is stopped by addition of renilla lysis buffer (Promega).
- renilla lysis buffer Promega
- [ 35 S]-methionine labeled radioactive proteins were translated in presence of 20 ⁇ of amino acids mix minus methionine and 5 ⁇ & ⁇ of [ 35 S]-methionine (Perkin Elmer) for 30 mn before the reaction was stopped by addition of SDS loading buffer.
- the L-protease from the foot and mouth disease virus (FMDV) or GFP for control was produced by in vitro translation using RRL as previously described (Ohlmann et al., RNA, 5(6): 764-778, 1999) and 0,1 ⁇ (per 10 ⁇ final volume of lysate) was added and incubated for 10 mn at 30 °C prior to the start of the translation reaction.
- FMDV foot and mouth disease virus
- Renilla activity was measured using the renilla luciferase Assay System (Promega Co, Madison, Wl, USA) in a Mithras (Berthold technologies) with 50 ⁇ substrate injection and 10 secondes of signal integration program.
- Cytoplasmic RNA extraction and RT-qPCR was performed as previously described (Ricci et al., Nucleic Acids Res., 39(12): 5215-5231 , 201 1 ).
- the inventors wanted to compare the most commonly used ones such as the rabbit reticulocyte (treated or not with the S7 nuclease), the wheat germ and the newly available Human lysates from Pierce which is prepared from HeLa cell extracts. For each of these lysates, their ability to translate in vitro transcribed mRNAs has been monitored.
- the renilla luciferase whose expression was driven either by the ⁇ -globin (50 nts) or the GAPDH (102 nts) 5' untranslated region (5'UTR) was used.
- RNA constructs harbor a m 7 GTP cap moiety together with a 50 adenylate residue poly(A) and were translated in either the crude rabbit reticulocyte lysate (URRL), the micrococcal treated rabbit reticulocyte lysate (RRL), the wheat germ lysate (WG) and the human in vitro protein expression system (HL pierce). Results obtained are summarized in Fig. 1 A for the mRNA that is driven by the ⁇ -globin 5'UTR and 1 B for the one driven by the GAPDH leader. For both mRNAs, the best translational efficiency was obtained in the rabbit reticulocyte derived systems, namely URRL and RRL.
- EXAMPLE 3 Design of a novel hybrid in vitro translation system
- the aim of the invention was to design a novel in vitro cell free system that can combine translational efficiency and characteristic features of living cells. To do that, the creation of a hybrid translational system between components of the rabbit reticulocyte lysate with those derived from cultured cells has been considered.
- the renilla construct was translated in both parental lysates (URRL and RRL) and efficiency was compared with the homologous reconstituted systems (Su+Rurrl and Sr+Rrrl). Although translational activity was of similar magnitude for both URRL and RRL, it was not the case once the systems were reconstituted with the Su+Rurrl being about twice as efficient as the reconstituted Sr+Rrrl. Interestingly, the inventors also observed that the Su+Rrrl was the best combination for heterologous systems with a translational yield comparable to that obtained with the parental lysates (Fig. 3). These data suggest that the S100 supernatant from the untreated lysate (Su) is the most efficient fraction and will be selected to serve for the basis of the hybrid lysate (see later).
- the post-ribosomal supernatant of the URRL was used to generate the cytosolic fraction (Su for supernatant of the URRL).
- Hybrid reconstituted systems were assembled by mixing the ribosome fractions (R) with the S100 supernatant from the URRL (Su) in all possible combinations.
- 2.7 nM of the globin-renilla reporter gene was translated for 30 mn before analysis of renilla activity.
- Data showed that all combinations with mammalian ribosomes resulted in efficient protein synthesis to a level comparable to that observed with the reconstituted RRL or the parental lysate (Fig. 6A, compare Rrrl and URRL with Rurrl/Rh/Rj).
- EXAMPLE 4 The hybrid system recapitulates cap/poly(A) synergy and support IRES-driven translation
- nuclease treated rabbit reticulocyte lysate fails to recreate the selective advantage conferred by the addition of the poly(A) tail on transcripts and does not recapitulate the cap/poly(A) synergistic effect on translation (Borman et al., Nucleic Acids Res., 25: 925-932, 2000). Such a property can be found in the crude RRL but use of the latter is restricted by the synthesis of endogenous globin and lipoxygenase which can interfere with ectopic gene translation both at the level of RNA and protein production (Soto Rifo et al., Nucleic Acids Res., 35(18) : e121 , 2007).
- RNAs were added to the hybrid SuRh (Supernatant of URRL with ribosomes from HeLa) or the nuclease treated lysate (RRL) as control. As it could be seen on Fig.
- the inventors used a bicistronic construct coding for the firefly (first gene) and the renilla (second gene) in which the Encephalomyocarditis virus IRES (EMCV) was inserted in the intercistronic spacer.
- EMCV Encephalomyocarditis virus IRES
- the addition of the L protease in the RRL results in a quite attenuated effect of the viral enzyme in comparison to what happens in living cells; as such, in the RRL, cap-dependent translation is decreased and a mild stimulation of the picornaviral IRES is generally observed (Ohlmann et al., Nucleic Acids Res., 23(3): 334-340, 1995; Soto Rifo et al., Nucleic Acids Res., 35(18) : e121 , 2007). Therefore, the in vitro translated L protease to both the RRL and the hybrid system prior to translation of the firefly-EMCV- renilla bicistronic construct used above was added.
- the present data indicate that the addition of ribosomes isolated from HeLa cells confer cap/poly(A) synergy to the lysate, restitute the ability to internal initiation and conserve the good translational efficiency of the reticulocyte lysate. Therefore, it was investigated whether the hybrid system could restitute cellular tropism. It has been shown that some IRES-containing mRNAs, notably those driven by the poliovirus IRES, were poorly expressed in the reticulocyte lysate and this can be partially rescued by the exogenous addition of HeLa S10 cell extracts to the reticulocyte lysate (Borman et al., Nucleic Acids Res., 25: 925-932, 1995).
- the next step was to conduct the reverse experiment which consists of adding ribosomes isolated from cells that do not support PV IRES translation such as Baby Hamster Kidney cells (BHK) (Borman et al., Nucleic Acids Res., 25(5) : 925-932, 1997). Indeed, these cells are thought to lack one, or several, factors that are needed for PV IRES translation (Borman et al., Nucleic Acids Res., 25: 925-932, 1995; Borman et al., Nucleic Acids Res., 25(5): 925-932, 1997). Thus, this property was used to test the limits of the in vitro translation system of the invention.
- BHK Baby Hamster Kidney cells
- a hybrid reconstituted translation system was prepared with ribosomes isolated from BHK cells that were mixed to the S100 URRL supernatant.
- Translation of the reporter mRNAs in the in vitro hybrid system showed that expression from the PV containing construct remained extremely low indicating that BHK ribosomes are unable to support PV-driven translation (Fig. 14C). This contrasts with the globin-renilla construct which was correctly expressed in the hybrid system indicating that the BHK ribosomes are not deficient to support global protein synthesis.
- FIG. 15A shows the translation of capped and polyadenylated in vitro transcribed mRNAs that were electroporated in C2C12 mouse myoblastic cells. Luciferase activity was measured before (undifferentiated) and 15h after (differentiated) horse serum addition as indicated (Fig. 15A). It can be noticed that translation from all three mRNAs was strongly stimulated in C2C12 cells that are engaged in the differentiation process. As expression from the utrophin driven construct was quite low, a blow up picture is presented below.
- EXAMPLE 7 Design of a factor-depleted hybrid system by the use of RNA silencing
- CFPS In contrast with living cells, CFPS offer the possibility to manipulate the level of endogenous components by relatively simple and highly standardized biochemical protocols. As such, removal can be carried out by affinity column chromatography or immunodepletion whereas inactivation can be achieved by enzymatic cleavage, chemicals or antagonist peptides.
- affinity column chromatography or immunodepletion
- inactivation can be achieved by enzymatic cleavage, chemicals or antagonist peptides.
- RNA interference in cultured cells prior to ribosome purification.
- DDX3 was exclusively found on the ribosomal fraction with no detectable protein left in the post-ribosomal supernatant (Fig. 16A compare S100 with C100).
- Transfection of RNA constructs harboring the globin or the HIV-1 5' UTR was then realized in HeLa cells that were treated, or not, with shRNAs against DDX3 (Fig. 16B).
- Fig. 16B As previously shown (Soto-Rifo et al., Embo. J., 31 (18) : 3745-3756, 2012), translation from the globin-renilla RNA construct was not affected by the lack of DDX3 whereas the construct driven by the HIV-1 5' UTR exhibited a significant drop in translational efficiency upon DDX3 knockdown (Fig. 16B).
- EXAMPLE 8 Use of the hybrid system from a cDNA transfection
- the hybrid system relies on the isolation of crude ribosomes from cells, it was proposed to take advantage of this situation to isolate ribosome associated RNAs from an ectopically expressed cDNA plasmid.
- the ectopic gene would be synthesized, processed and exported by the cellular machinery; it would undergo transcription, splicing, capping/polyadenylation, nucleus export and translation (see cartoon Fig. 17A). Once translated in the cytoplasm, the ectopic RNA must be associated to polysomes and should be co-purified with the ribosomal fraction.
- cytoplasmic RNA concentration was measured by quantitative RT-PCR and data are summarized in Fig. 17B. This shows a correlation between the amount of plasmid transfected (from 0.5 ; 6 and 12 ⁇ g of input cDNA per 1 ,5.10 7 cells) and the concentration of neo-synthesized renilla-globin RNA found in the cytoplasm (Fig. 17B; from 0.010 picogrammes to 0.190 pg of RNA).
- the hybrid system was assembled by mixing these ribosome containing RNAs to the supernatant obtained from fractionation of the URRL. Upon reconstitution, the hybrid system was incubated for 30 minutes at 30 °C before quantification of luciferase activity (Fig. 17C). It can be observed that the production of luciferase correlated with the amount of RNAs quantified by quantitative RT-PCR. In order to rule out any possible interference with some luciferase protein that could have been co-purified with the HeLa ribosomes, cycloheximide was added to the reconstituted system and this gave virtually no enzymatic activity under these experimental conditions (data not shown). This shows that the RNAs trapped into cellular polysomes are fully functional for translation upon their transfer to the hybrid reconstituted system.
- the c-myc 5'UTR was a good tool to test the in vitro translation system of the invention.
- a capped and polydenylated c-myc driven renilla reporter RNA together with the globin-renilla control was first generated. Both were translated in the RRL (Fig. 18A) and it can be observed that production of luciferase under the control of the c-myc 5'UTR was about 10-fold lower than that from globin in agreement with the work of Willis and colleagues (Stoneley et al., Mol. Cell. Biol. 20(4): 1 162-1 169, 2000a).
- mRNAs that are associated to translating ribosomes can then be extracted from the ribosomal pellet and subjected to high throughput RNA sequencing as described for example in Roberts et al. (201 1 ) Genome Med. 3:74 or Wilhelm et al. (2008) Nature 453:1239-1243. These mRNAs are indeed representative of the whole transcriptome that is being translated under the experimental conditions used.
- cellular endogenous mRNAs can also be transferred in the hybrid system of the invention, thereby producing detectable amounts of proteins that can then be detected, identified and quantified in particular by mass spectrometry.
- mass spectrometry To facilitate this detection, identification and quantification by mass spectrometry, the amino acid mix added in the in vitro hybrid system is replaced by "heavy" amino acids to detect specifically newly synthetized proteins, as described for example in Bantscheff et al. (2007) Anal. Bioanal. Chem. 389:1017-1031 .
- the in vitro hybrid system is supplied with biotinylated puromycin molecules that are incorporated into neo-synthesized peptides and enable purification of these peptides with streptavidin, as described for example in Aviner et al. (2013) Genes Dev. 27:1834-1844.
- EXAMPLE 9 Use of the hybrid system to label all the newly svnthetized protein (cellular and viral) in the case of viral infection
- Example 8 isolation of ribosomes from cells that were previously infected with viruses also contained viral mRNAs. Once transferred in the hybrid system described in Example 8, these viral mRNAs were translated to yield viral proteins that could be detected by radiolabeled methionine incorporation and could be further quantified and purified.
- Figure 20 shows such ribosomes isolated from A549 cells infected with influenza virus.
- the neo-synthetized viral proteins can clearly be observed and are labeled on the right side of the picture.
Abstract
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WO2015085209A1 (en) | 2013-12-06 | 2015-06-11 | President And Fellows Of Harvard College | Paper-based synthetic gene networks |
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BR112019000195A2 (en) * | 2016-07-07 | 2019-04-24 | Rubius Therapeutics, Inc. | compositions and methods related to therapeutic cellular systems expressing exogenous rna |
EP3579845A4 (en) * | 2017-02-09 | 2020-12-30 | Dow Agrosciences LLC | Novel eukaryotic cell-free expression system that does not require an artificial energy regeneration system |
CN113409887A (en) * | 2021-07-07 | 2021-09-17 | 中国科学院生物物理研究所 | Cell-free translation system, method and product |
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2014
- 2014-02-06 JP JP2015556498A patent/JP2016506740A/en active Pending
- 2014-02-06 US US14/766,636 patent/US20150376673A1/en not_active Abandoned
- 2014-02-06 WO PCT/EP2014/052369 patent/WO2014122231A1/en active Application Filing
- 2014-02-06 EP EP14702891.4A patent/EP2954066A1/en not_active Withdrawn
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2015085209A1 (en) | 2013-12-06 | 2015-06-11 | President And Fellows Of Harvard College | Paper-based synthetic gene networks |
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