NEW CHIMERIC ENZYMES AND THEIR APPLICATIONS
20200165585 · 2020-05-28
Inventors
Cpc classification
C12N2795/00022
CHEMISTRY; METALLURGY
C12Y201/01056
CHEMISTRY; METALLURGY
C12Y306/01
CHEMISTRY; METALLURGY
International classification
C12N9/12
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a chimeric enzyme comprising or consisting of at least one catalytic domain of a capping enzyme and at least one RNA-binding domain of a protein-RNA tethering system as well as its application for the production of an RNA molecule with a 5-terminal cap.
Claims
1. A chimeric enzyme comprising: at least one catalytic domain of a capping enzyme; and at least one RNA-binding domain of a protein-RNA tethering system; wherein said RNA-binding domain binds specifically to a RNA element of said protein-RNA tethering system, said RNA element consisting of a specific RNA sequence and/or structure; and wherein said RNA-binding domain is a bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system.
2. The chimeric enzyme according to claim 1, characterized in that it comprises: at least one catalytic domain of a RNA triphosphatase; at least one catalytic domain of a guanylyltransferase; at least one catalytic domain of a N.sup.7-guanine methyltransferase; and at least one RNA-binding domain of a protein-RNA tethering system; wherein at least one of said catalytic domains is a catalytic domain of a cap-0 canonical capping enzyme.
3. (canceled)
4. The chimeric enzyme according to claim 1, wherein the RNA-binding domain is a bacteriophage RNA-binding domain of a bacteriophage protein selected from the group consisting of the MS2 coat protein, the R17 coat protein and the lambdoid N antitermination proteins.
5. The chimeric enzyme according to claim 1, characterized in that it further comprises at least one catalytic domain of a poly(A) polymerase.
6. The chimeric enzyme according to claim 1, characterized in that it further comprises at least one catalytic domain of a DNA-dependent RNA polymerase.
7. An isolated nucleic acid molecule or a group of isolated nucleic acid molecule(s), said nucleic acid molecule(s) encoding a chimeric enzyme according to claim 1 or an isolated nucleic acid molecule encoding a chimeric enzyme, characterized in that its sequence comprises a nucleic acid sequence encoding a RNA-binding domain of a protein-RNA tethering system fused in frame to: a nucleic acid sequence encoding at least one catalytic domain of a poly(A) polymerase; and a nucleic acid sequence encoding at least one catalytic domain of a capping enzyme; wherein said RNA-binding domain binds specifically to a RNA element of said protein-RNA tethering system, said RNA element consisting of a specific RNA sequence and/or structure; and wherein said RNA-binding domain is a bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system.
8. The isolated nucleic acid molecule according to claim 7, characterized in that its sequence further comprises a nucleic acid sequence encoding at least one catalytic domain of a DNA-dependent RNA polymerase.
9. The isolated nucleic acid molecule according to claim 7, characterized in that its sequence further comprises a nucleic acid sequence encoding a ribosome skipping motif between said nucleic acid sequence encoding a catalytic domain of a poly(A) polymerase and said nucleic acid sequence encoding at least one catalytic domain of a capping enzyme.
10. (canceled)
11. A vector comprising a nucleic acid molecule or a group of nucleic acid molecules according to claim 7.
12. A host cell comprising a nucleic acid molecule or a group of nucleic acid molecules according to claim 7.
13. (canceled)
14. A method for the production of an RNA molecule with a 5-terminal can and optionally comprising at least one chemical modification, wherein said method comprises in vitro or ex vivo use of: i) a chimeric enzyme according to claim 1; and/or ii) an isolated nucleic acid molecule encoding a chimeric enzyme according to claim 1 or characterized in that its sequence comprises a nucleic acid sequence encoding a RNA-binding domain of a protein-RNA tethering system fused in frame to: a nucleic acid sequence encoding at least one catalytic domain of a poly(A) polymerase; and a nucleic acid sequence encoding at least one catalytic domain of a capping enzyme: wherein said RNA-binding domain binds specifically to a RNA element of said protein-RNA tethering system, said RNA element consisting of a specific RNA sequence and/or structure; and wherein said RNA-binding domain is a bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system; and/or iii) a group of isolated nucleic acid molecules encoding a chimeric enzyme according to claim 1.
15. (canceled)
16. An in vitro or ex vivo method for producing a RNA molecule with a 5-terminal cap encoded by a DNA sequence, in a host cell, said method comprising the step of expressing in the host cell a nucleic acid molecule according to claim 7 or a group of isolated nucleic acid molecules according to claim 7, wherein said DNA sequence is covalently linked to at least one sequence encoding the RNA element of said protein-RNA tethering system, which specifically binds to said RNA-binding domain.
17. (canceled)
18. A kit for the production of an RNA molecule with a 5-terminal cap, comprising: i) at least one chimeric enzyme according to claim 1; and/or ii) an isolated nucleic acid molecule encoding a chimeric enzyme according to claim 1, or characterized in that its sequence comprises a nucleic acid sequence encoding a RNA-binding domain of a protein-RNA tethering system fused in frame to: a nucleic acid sequence encoding at least one catalytic domain of a poly(A) polymerase; and a nucleic acid sequence encoding at least one catalytic domain of a capping enzyme; wherein said RNA-binding domain binds specifically to a RNA element of said protein-RNA tethering system, said RNA element consisting of a specific RNA sequence and/or structure; and wherein said RNA-binding domain is a bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system; and/or iii) a group of isolated nucleic acid molecules encoding a chimeric enzyme according claim 1; and optionally a DNA sequence encoding said RNA molecule, which is covalently linked to at least one sequence encoding the RNA element of said protein-RNA tethering system, which specifically binds to said RNA-binding domain.
19-20. (canceled)
21. A pharmaceutical composition comprising: i) at least one chimeric enzyme according to claim 1; and/or ii) an isolated nucleic acid molecule encoding a chimeric enzyme according to claim 1, or characterized in that its sequence comprises a nucleic acid sequence encoding a RNA-binding domain of a protein-RNA tethering system fused in frame to: a nucleic acid sequence encoding at least one catalytic domain of a poly(A) polymerase; and a nucleic acid sequence encoding at least one catalytic domain of a capping enzyme: wherein said RNA-binding domain binds specifically to a RNA element of said protein-RNA tethering system, said RNA element consisting of a specific RNA sequence and/or structure; and wherein said RNA-binding domain is a bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system; and/or iii) a group of isolated nucleic acid molecules encoding a chimeric enzyme according to claim 1.
22. (canceled)
23. The chimeric enzyme according to claim 1, wherein the capping enzyme is selected from the group consisting of cap-0 canonical capping enzymes, cap-0 non-canonical capping enzymes, cap-i capping enzymes and cap-2 capping enzymes.
24. The chimeric enzyme, according to claim 2, wherein at least one of said catalytic domains is a catalytic domain of a virus cap-0 canonical capping enzyme.
25. The chimeric enzyme according to claim 6, wherein said at least one catalytic domain of a DNA-dependent RNA polymerase is a bacteriophage DNA-dependent RNA polymerase.
26. The isolated nucleic molecule according to claim 7, characterized in that its sequence comprises a nucleic acid sequence encoding a RNA-binding domain of a protein-RNA tethering system fused in frame in the order to: a nucleic acid sequence encoding at least one catalytic domain of a poly(A) polymerase; and a nucleic acid sequence encoding at least one catalytic domain of a capping enzyme; wherein said RNA-binding domain binds specifically to a RNA element of said protein-RNA tethering system, said RNA element consisting of a specific RNA sequence and/or structure; and wherein said RNA-binding domain is a bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system.
27. The isolated nucleic acid molecule according to claim 26, characterized in that its sequence further comprises a nucleic acid sequence encoding a ribosome skipping motif between said nucleic acid sequence encoding a catalytic domain of a poly(A) polymerase and said nucleic acid sequence encoding at least one catalytic domain of a capping enzyme.
28. The isolated nucleic acid molecule according to claim 8, wherein said nucleic acid sequence encoding at least one catalytic domain of a DNA-dependent RNA polymerase encodes a bacteriophage DNA-dependent RNA polymerase.
Description
BRIEF DESCRIPTION OF DRAWINGS
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[0403] The present invention will be explained in detail with examples in the following, but the technical scope of the present invention is not limited to these examples.
EXAMPLE 1: D1R/D12L, THE VACCINIA VIRUS CAPPING ENZYME TETHERED TO LUCIFERASE REPORTER MRNA INCREASES ITS EXPRESSION
[0404] 1. Objectives
[0405] The objective of this experiment was to determine if the heterodimeric vaccinia virus capping enzyme appropriately tethered to Firefly Luciferase reporter mRNA synthesized in cellulo increases its expression.
[0406] The vaccinia virus capping enzyme consist of two subunits, which form a heterodimer: (I) a 95 kDa subunit encoded by the vaccinia virus D1R gene (genomic sequence AY243312.1; UniProtKB/Swiss-Prot accession number P04298), designated hereafter as D1R, which has RNA-triphosphatase, RNA guanylyltransferase and RNA N7-guanine methyltransferase enzymatic activities (Cong and Shuman 1993, Niles and Christen 1993, Higman and Niles 1994, Mao and Shuman 1994, Gong and Shuman 2003), (ii) and a 31-kDa subunit encoded by the vaccinia virus D12L gene (genomic sequence AY243312.1; UniProtKB/Swiss-Prot accession number P04318), designated hereafter as D12L, which has no intrinsic enzymatic activity, but enhances the RNA N7-guanine methyltransferase activity of the D1R subunit (Higman, Bourgeois et al. 1992, Higman, Christen et al. 1994, Mao and Shuman 1994). Cotransfection of plasmids encoding these two subunits therefore generate in cellulo the heterodimer D1R/D12L capping enzyme, which can eventually fused to a protein tethering domain.
[0407] 2. Methods
[0408] a. Plasmids
[0409] The coding sequences of the following plasmids were optimized for expression in human cells with respect to codon adaptation index using the GeneOptimizer algorithm (Raab, Graf et al. 2010). All gene sequences were artificially synthesized and assembled from stepwise PCR using oligonucleotides, cloned and fully sequenced.
[0410] For all the following examples, the conditions tested consist of a variable combination of several plasmids. In the present example, the pK1ERNAP/pT3RNAP plasmids together with Firefly luciferase reporter plasmids were used to generate in cellulo the Firefly Luciferase mRNA with or without tethering domain, which then can be specifically modified by enzyme produced by the test plasmid appropriately tethered to the Firefly Luciferase mRNA.
[0411] The expression plasmids consisted of the phage T3 and K1E RNA polymerase open reading-frames (ORFs), which were subcloned in the pCMVScript plasmid backbone (Stratagene, La Jolla, Calif.), following the removal of the T7 10 promoter sequence. These corresponding plasmids, designated as p-followed by the name of the ORF, have the following design (i.e. pK1ERNAP or pT3RNAP;
[0412] The test plasmids contained the coding sequence of the capping enzymes under investigation with (
[0413] The Firefly Luciferase reporter plasmids containing the Firefly Luciferase gene under control of the K1E or T3 RNA polymerase promoters, contained a 5-UTR sequence, Kozak consensus sequence followed by the ORF of Luciferase gene from Photinus pyralis and stop codon, RNA tethering domain consisting of four BoxBr in tandem from A virus (optional, lacking in the untethered version; nucleotides 38312-38298 of genomic sequence of Enterobacteria phage lambda KT232076.1; SEQ ID N.sup.o 7 corresponding to the nucleotide sequence of the BoxBr RNA stem-loops from A bacteriophage), poly(A) track of 40 adenosine residues, followed by a self-cleaving RNA sequence from the genomic ribozyme of the hepatitis D virus, and terminated by the bacteriophage T7 10 transcription stop. These plasmids were designated either pK1Ep-Luciferase/pT3p-Luciferase in their untethered versions (
[0414] b. Cell Culture and Transfection
[0415] For standard experiments, the Human Embryonic Kidney 293 (HEK-293, ATCC CRL 1573) were routinely grown at 37 C. in 5% CO.sub.2 atmosphere at 100% relative humidity. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4 mM L-alanyl-L-glutamine, 10% fetal bovine serum (FBS), 1% non-essential amino-acids, 1% sodium pyruvate, 1% penicillin and streptomycin, and 0.25% fungizone.
[0416] Cells were routinely plated in 24-well plates at 110.sup.5 cells per well the day before transfection and transfected at 80% cell confluence. Transient transfection was performed with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif.) according to manufacturer's recommendations. Except otherwise stated, cells were transfected with 2 l of Lipofectamine 2000 and 0.8 g of total plasmid DNA. For standard luciferase and hSEAP gene reporter expression assays, cells were analyzed 48 hours after transfection, except otherwise stated.
[0417] c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
[0418] Luciferase luminescence was assayed by the Luciferase Assay System (Promega, Madison, Wis.) according to the manufacturer's recommendations. In brief, cells were lysed in Cell Culture Lysis Reagent buffer (CLR), and then centrifuged at 12,000g for two minutes at 4 C. Luciferase Assay Reagent (Promega; 100 l/well) diluted at 1:10 was added to supernatant (20 l/well). Luminescence readout was taken on a Tristar 2 microplate reader (Berthold, Bad Wildbad, Germany) with a read time of one second per well.
[0419] In order to normalize for transfection efficacy, cells were transfected with the pORF-eSEAP plasmid (InvivoGen, San Diego, Calif.), which encodes for the human secreted embryonic alkaline phosphatase (hSEAP) driven by the EF-1/HTLV composite promoter. Enzymatic activity was assayed in cell culture medium using the Quanti-Blue colorimetric enzyme assay kit (InvivoGen). Gene reporter expression was expressed as the ratio of luciferase luminescence (RLU, relative light units) to eSEAP absorbance (OD, optic density).
[0420] d. Statistical Analysis
[0421] Statistical analyses were performed with paired two-tailed Student's t-test. Results are means (n4)standard deviation. P-value<0.05 was considered statistically significant.
[0422] 3. Results
[0423] In this set of experiments, the Firefly Luciferase mRNA was produced by the phage K1E or T3 RNA polymerases by cotransfection of pK1ERNAP/pT3RNAP and pK1Ep-Luciferase-4xBoxBr/pT3p-Luciferase-4xBoxBr for the tethered version of the Firefly Luciferase reporter plasmids or pK1Ep-Luciferase-4xBoxBr/pT3p-Luciferase-4xBoxBr for their untethered version. The test plasmids contain the coding sequence of the D1R/D12L vaccinia virus with or without tethering domain were co-transfected. The translatability of the resulting transcripts, which is expected to increase in case of proficient capping, is measured by the Firefly Luciferase assay. A general depiction of the assay is shown
[0424] Results of the first set of experiments with the K1E-driven system are shown in the table below:
TABLE-US-00001 Plasmids mean SEM (1) pK1ERNAP, pK1Ep-Luciferase 35 329 3 113 (2) pK1ERNAP, pK1Ep-Luciferase-4xBoxBr 34 784 4 388 (3) pK1ERNAP, pD1R, pK1Ep-Luciferase- 315 159 36 188 4xBoxBr (4) pK1ERNAP, pD12L, pK1Ep-Luciferase- 60 212 2 219 4xBoxBr (5) pK1ERNAP, pD1R, pD12L, pK1Ep- 851 056 144 590 Luciferase-4xBoxBr (6) pK1ERNAP, pD1R, pN-pD12L, pK1Ep- 2 237 689 92 709 Luciferase-4xBoxBr (7) Baseline 14 537 3 145
[0425] As expected when capping is lacking, Firefly Luciferase mRNA generated by pK1Ep-Luciferase and pK1Ep-Luciferase-4xBoxBr cotransfected with the K1ERNAP plasmid alone (designated pK1ERNAP) was poorly expressed (row 1 and 2). Cotransfection of the untethered D1R plasmid (designated pD1R) together with pK1ERNAP/pK1Ep-Luciferase-4xBoxBr increased the expression by approximately 9-fold in comparison to cotransfection of pK1ERNAP/pK1Ep-Luciferase-4xBoxBr plasmids only (row 3 vs. 1 or 2, p<0.05, two-way Student t-test), whereas the transfection of the untethered pD12L plasmid with pK1ERNAP/pK1Ep-Luciferase-4xBoxBr had virtually no effect on Firefly Luciferase expression (row 4 vs. 1 or 2, p=NS, two-way Student t-test). The cotransfection of the untethered pD12L and pD1R plasmids together with pK1ERNAP/pK1Ep-Luciferase-4xBoxBr, which result in the vaccinia virus D1R/D12L capping enzyme heterodimer without tethering domain, significantly increased the expression of Firefly Luciferase in comparison to previous conditions, therefore confirming that mRNA capping is requested for mRNA translation (row 5 vs. 1 to 4, p=NS, two-way Student t-test p<0.05, two-way Student t-test). Finally, cotransfection of the tethered D12L plasmid (pN-D12L) and D1R together with pK1ERNAP/pK1Ep-Luciferase-4xBoxBr, which produces the tethering vaccinia virus capping enzyme D1R/N-D12L, increased drastically the expression of the Luciferase mRNA by 63.3 (row 6 vs. 2) and 2.6-fold (row 6 vs. 5) in comparison to no vaccinia virus capping enzyme (pK1ERNAP/pK1Ep-Luciferase-4xBoxBr alone), or untethering vaccinia virus capping enzyme (pK1ERNAP/pK1Ep-Luciferase-4xBoxBr/pD1R/pD12L), respectively.
[0426] In a second set of experiments, the Firefly Luciferase mRNA was produced by the phage T3 RNA polymerase and tethered to the vaccinia virus capping enzyme. Results of the second set of experiments with the T3-driven system are shown in the table below:
TABLE-US-00002 Plasmids mean SEM (1) pT3RNAP, pT3p-Luciferase 40 387 5 041 (2) pT3RNAP, pT3p-Luciferase-4xBoxBr 43 682 2 072 (3) pT3RNAP, pD1R, pT3p-Luciferase-4xBoxBr 82 445 6 913 (4) pT3RNAP, pD12L, pT3p-Luciferase-4xBoxBr 47 167 1 342 (5) pT3RNAP, pD1R, pD12L, pT3p-Luciferase- 110 371 5 390 4xBoxBr (6) pT3RNAP, pD1R, pNA-pD12L, pT3p-Luciferase- 353 096 12 560 4xBoxBr (7) Baseline 7 269 1 901
[0427] This second set of experiments gave very similar results with cotransfection results in the following order: RNA with no 4xBoxBr and no capping enzyme (row 1, pT3RNAP/pT3p-Luciferase) 4xBoxBr-RNA and no capping enzyme (row 2, pT3RNAP/pT3p-Luciferase-4xBoxBr) 4xBoxBr-RNA with D12L subunit alone (row 4, pT3RNAP/pD12L/pT3p-Luciferase-4xBoxBr)<4xBoxBr-RNA with D1R (row 3, pT3RNAP/pD1R/pT3p-Luciferase-4xBoxBr)<4xBoxBr-RNA with untethered D1R/D12L capping enzyme (row 5, pT3RNAP/pD1R/pD12L/UpT3p-Luciferase-4xBoxBr)<<4xBoxBr-RNA with tethered D1R/D12L capping enzyme (row 6, pT3RNAP/pD1R/pN-D12L/UpT3p-Luciferase-4xBoxBr). The expression levels of this latter condition was statistically greater than all other conditions, especially 3.2 fold higher than with the untethered D1R/D12L capping enzyme, therefore demonstrating the importance of guiding the D1R/D12L capping enzyme to the target reporter mRNA by tethering domain (p<0.05, two-way Student t-test).
[0428] 4. Conclusions
[0429] These experiments show that the vaccinia virus capping enzyme, which contains no known or demonstrated binding domain for a specific RNA sequence, drastically increases gene Firefly
[0430] Luciferase reporter expression when appropriately tethered to uncapped and polyadenylated Firefly Luciferase reporter mRNA by the N-BoxBr tethering system.
EXAMPLE 2: NP868R, THE AFRICAN SWINE FEVER VIRUS CAPPING ENZYME TETHERED TO LUCIFERASE REPORTER MRNA INCREASES ITS EXPRESSION
[0431] 1. Objectives
[0432] The objective of this set of experiments was to demonstrate if NP868R, the African swine fever virus capping enzyme, appropriately tethered to polyadenylated Firefly Luciferase reporter mRNA increases its expression in cellulo. NP868R (also named G4R) is a single-unit 868 amino-acids protein, which all enzymatic activities required for cap-0 formation demonstrated in vitro, i.e. RNA-triphosphatase, RNA guanylyltransferase and RNA N7-guanine methyltransferase (Pena, Yanez et al. 1993, Jais 2011, Dixon, Chapman et al. 2013, Jais, Decroly et al. 2018).
[0433] 2. Methods
[0434] a. Plasmids
[0435] The expression (pK1ERNAP or pT3RNAP), as well as the Firefly Luciferase reporter plasmids in their tethered versions (pK1Ep-Luciferase and pT3p-Luciferase) or untethered versions (pK1Ep-Luciferase-4xBoxBr and pT3p-Luciferase-4xBoxBr) were the same as described above.
[0436] The test plasmid consisted of the coding sequence from the African swine fever virus NP868R capping enzyme (NCBI ASFV genomic sequence strain BA71V NC_001659; UniProtKB/Swiss-Prot accession number P32094; SEQ ID N.sup.o 8 and SEQ ID N.sup.o 9 corresponding to the nucleotide and amino acid sequences of African swine fever virus NP868R capping enzyme, respectively) with (pN-NP868R) or without the NA tethering domain (pNP868R) subcloned in the pCMVScript plasmid backbone as described above.
[0437] b. Cell Culture and Transfection
[0438] Same as described in Example 1.
[0439] c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
[0440] Same as described in Example 1.
[0441] d. Statistical Analysis
[0442] Same as described in Example 1.
[0443] 3. Results
[0444] The design of the assay was very similar to Example 1, except that the single subunit capping enzyme NP868R was used instead of the heterodimeric D1R/D12L capping enzyme. In brief, uncapped but polyadenylated Firefly Luciferase mRNA was synthesized in cellulo by the phage K1E or T3 RNA polymerases and its expression in presence of NP868R was assayed (
[0445] Results of the first set of experiment with the K1E-driven system are shown in the table below:
TABLE-US-00003 Plasmids mean SEM (1) pK1ERNAP, pK1Ep-Luciferase 109 401 7 601 (2) pK1ERNAP, pK1Ep-Luciferase-4xBoxBr 147 494 6 310 (3) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 2 433 576 226 803 4xBoxBr (4) pK1ERNAP, pN-pNP868R, pK1Ep- 4 099 936 465 513 Luciferase-4xBoxBr (5) Baseline 10 393 2 302
[0446] In this first set of experiments, cells were cotransfected with pK1ERNAP/pK1Ep-Luciferase or pK1ERNAP/pK1Ep-Luciferase-4xBoxBr plasmids, showed low levels of Firefly Luciferase reporter expression (row 1 and 2). Cotransfection of the pK1ERNAP/pK1Ep-Luciferase-4xBoxBr with the untethered pNP868R plasmid (pNP868R) increased the expression by approximately 29-fold in comparison to pK1ERNAP/pK1Ep-Luciferase-4xBoxBr alone (row 3 vs. 1 or 2 respectively, p<0.05, two-way Student t-test), therefore confirming that mRNA capping is requested for mRNA translation. Finally, cotransfection of pK1ERNAP/pK1Ep-Luciferase-4xBoxBr with tethered NP868R (pN-NP868R) even increased by 2.1-fold the expression of Firefly Luciferase in comparison to untethered NP868R condition, demonstrating the importance of guiding the enzyme to the target mRNA by tethering domains for proficient mRNA capping (raw 4 vs. 3, p<0.05, two-way Student t-test).
[0447] Results of the second set of experiments with the T3-driven system are shown in the table below:
TABLE-US-00004 Plasmids mean SEM (1) pT3RNAP, pT3p-Luciferase 75 065 7 575 (2) pT3RNAP, pT3p-Luciferase-4xBoxBr 62 220 5 781 (3) pT3RNAP, pNP868R, pT3p-Luciferase- 122 957 24 166 4xBoxBr (4) pT3RNAP, pN-NP868R, pT3p-Luciferase- 352 978 16 813 4xBoxBr (5) Baseline 23 464 6 302
[0448] In this second set of experiments, the Firefly Luciferase mRNA was produced by the phage T3 RNA polymerase and tethered to the African swine fever virus capping enzyme. This second set of experiments gave very similar results with cotransfection results in the following order: RNA with no 4xBoxBr and no capping enzyme (row 1, pT3RNAP/pT3p-Luciferase) 4xBoxBr-RNA without capping enzyme (row 2, pT3RNAP/pT3p-Luciferase-4xBoxBr)<4xBoxBr-RNA with untethered NP868R capping enzyme (row 3, pT3RNAP/pNP868R/pT3p-Luciferase-4xBoxBr)<<4xBoxBr-RNA with tethered NP868R capping enzyme (row 4, pT3RNAP/pN-NP868R/pT3p-Luciferase-4xBoxBr). The expression levels of this last condition was statistically greater than all other conditions, especially 2.9 fold higher than with the untethered NP868R capping enzyme, therefore demonstrating the importance of tethering NP868R to the target mRNA by tethering domains for proficient mRNA capping (row 4 vs.3, p<0.05, two-way Student t-test).
[0449] 4. Conclusions
[0450] These experiments show that another capping enzyme, NP868R from the African swine fever virus, which contains no known or predicted binding domain for a specific RNA sequence, increases Firefly Luciferase reporter expression when appropriately tethered to uncapped and polyadenylated reporter mRNA by the N-BoxBr tethering system.
EXAMPLE 3: VARIOUS PROTEIN:RNA TETHERING SYSTEMS, COUPLED TO AFRICAN SWINE FEVER VIRUS CAPPING ENZYME NP868R CAN INCREASE THE EXPRESSION OF LUCIFERASE REPORTER MRNA PRODUCED BY K1E PHAGE RNA POLYMERASE IN HOST-CELL CYTOPLASM
[0451] 1. Objectives
[0452] The objectives of the present experiments were to investigate if other protein:RNA tethering systems than the N-4xBoxBr system can guide the African swine fever virus capping enzyme NP868R in order to increase the expression of appropriately tethered Luciferase reporter mRNA produced by the phage K1E RNA polymerase.
[0453] The following tethering systems were presently tested: i) MS2 protein and the RNA stem loop tethered sequence from MS2 virus (Valegard, Murray et al. 1994, Valegard, Murray et al. 1997), ii) NA peptide from the lambda virus and its BoxBl RNA tethered sequence (Das 1993, Greenblatt, Nodwell et al. 1993, Friedman and Court 1995), iii) NA peptide from the P22 lamboid virus and its BoxBr RNA tethered sequences (Das 1993, Greenblatt, Nodwell et al. 1993, Friedman and Court 1995), iv) NA peptide from the 21 lamboid virus and its BoxBr RNA tethered sequence (Das 1993, Greenblatt, Nodwell et al. 1993, Friedman and Court 1995), v) TAT binding domain from the Human immunodeficiency virus-1 (HIV-1), which contains a biologically validated nuclear localization signal (Duconge and Toulme 1999), and TAR RNA tethered sequence (Dingwall, Ernberg et al. 1990, Weeks, Ampe et al. 1990, Karn, Dingwall et al. 1991, Puglisi, Tan et al. 1992, Frankel and Young 1998), and vi) the human small nuclear ribonucleoprotein U1 subunit 70 (SNRNP70) protein tethering sequence (Romac, Graff et al. 1994), which contains a biologically validated nuclear localization signal (Keene, Query et al. 1999), and its U1snRNA-stem loop tethered sequence.
[0454] 2. Methods
[0455] a. Plasmids
[0456] The pK1ERNAP expression plasmid was described in Example 1.
[0457] The test plasmids consisted of the coding sequence of the African swine fever virus NP868R capping enzyme fused at its amino-terminal end to: i) bacteriophage N-antitermination protein the N-terminus of the entire MS2 protein (pMS2-NP868R, NCBI accession number NC_001417.2, UniProtKB/Swiss-Prot P03612; SEQ ID N.sup.o 36 and SEQ ID N.sup.o 37 corresponding to the nucleotide and amino-acid sequences, respectively), ii) N-terminal peptide from lambda bacteriophage previously described, iii)N-terminal peptide from P22 bacteriophage N-antitermination protein (pP22N-NP868R, UniProtKB/Swiss-Prot P04891), iv)N-terminal peptide from 21 bacteriophage N-antitermination protein (pN21-NP868R, UniProtKB/Swiss-Prot P07243), v) the TAT protein binding domain from HIV-1 isolate HXB2 (pTAT-NP868R, NCBI reference sequence: AAB50256.1), and vi) human small nuclear ribonucleoprotein U1 subunit 70 (SNRNP70) RNA-binding protein sequence (pSNRNP70-NP868R, amino-acid 92-202, NCBI accession number NM 003089.5).
[0458] The RNA tethering domains of the Firefly Luciferase reporter plasmids substituted by four tandem repeats of: i) MS2 RNA stem-loops (pK1Ep-Luciferase-4xMS2sl plasmid; nucleotides 1748-766 from Enterobacteriophage MS2 isolate DL52, NCBI accession number J0966307.1; SEQ ID N.sup.o 38), ii) BoxBl RNA sequence from A virus (pK1Ep-Luciferase-4xABoxBl; NCBI accession number J02459.1 nucleotides 35518-35534), iii) BoxBr RNA sequence from P22 lamboid virus (pK1Ep-Luciferase-4xP22BoxBr; NCBI accession number NC_002371.2, nucleotides 31,953-31,971), iv) BoxBr RNA sequence from 21 lamboid virus (pK1Ep-Luciferase-4x21BoxBr; NCBI accession number AH007390.1, nucleotides 866-883), v) TAR RNA sequence from Human immunodeficiency virus type 1, isolate HXB2 (pK1Ep-Luciferase-4xTAR; NCBI accession number K03455.1, nucleotides 471-497) and vi) U1snRNA RNA stem-loop (pK1Ep-Luciferase-4xU1snRNA; NCBI accession number M28013.1, nucleotides 123-155).
[0459] b. Cell Culture and Transfection
[0460] Same as described in Example 1.
[0461] c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
[0462] Same as described in Example 1.
[0463] d. Statistical Analysis
[0464] Same as described in Example 1.
[0465] 3. Results
[0466] The design of the assay was very similar to Example 2, except that various protein:RNA tethering systems were tested in replacement to the N:BoxBr system. In brief, uncapped Firefly Luciferase mRNA with a short polyadenylation tail of 40 adenosine residues was synthesized in cellulo by the phage K1E RNA polymerase and its expression in presence of NP868R tethered by various systems was assayed (
[0467] Results of these experiments are shown in the table below:
TABLE-US-00005 Plasmids mean SEM MS2-4xMS2sl tethering system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 143 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 1 033 076 113 402 4xMS2sl (3) pK1ERNAP, pMS2-NP868R, pK1Ep- 1 083 076 232 757 Luciferase (4) pK1ERNAP, pMS2-NP868R, pK1Ep- 4 999 936 232 757 Luciferase-4xMS2sl (5) Baseline 162 150 N-4xBoxBl tetherina system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 113 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 1 433 576 113 402 4xBoxBl (3) pK1ERNAP, pN-NP868R, pK1Ep- 1 430 576 232 757 Luciferase (4) pK1ERNAP, pN-NP868R, pK1Ep- 5 699 936 332 757 Luciferase-4xBoxBl (5) Baseline 162 150 NP22-4xP22BoxBr tethering system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 113 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 913 576 113 402 4xP22BoxBr (3) pK1ERNAP, pNP22-NP868R, pK1Ep- 813 576 122 757 Luciferase (4) pK1ERNAP, pNP22-NP868R, pK1Ep- 4 699 936 232 757 Luciferase-4xP22BoxBr (5) Baseline 162 150 N21-4x21BoxBr tethering system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 113 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 1 313 576 113 402 4x21BoxBr (3) pK1ERNAP, pN21-NP868P, pK1Ep- 1 115 600 232 757 Luciferase (4) pK1ERNAP, pN21-NP868P, pK1Ep- 4 919 936 232 757 Luciferase-4x21BoxBr (5) Baseline 162 150 TAT-4xTAR tethering system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 113 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 1 333 076 113 402 4xTAR (3) pK1ERNAP, pTAT-NP868R, pK1Ep- 1 153 076 232 757 Luciferase (4) pK1ERNAP, pTAT-NP868R, pK1Ep- 1 583 076 232 757 Luciferase-4xTAR (5) Baseline 162 150 SNRNP70-4xU1snRNA tethering system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 113 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 1 331 222 38 402 4xU1snRNA (3) pK1ERNAP, pSNRNP70-NP868R, pK1Ep- 1 153 076 232 757 Luciferase (4) pK1ERNAP, pSNRNP70-NP868R, pK1Ep- 1 423 076 157 757 Luciferase-4xU1snRNA (5) Baseline 162 150
[0468] The cotransfection of pK1ERNAP with plasmids having only one component of the tethering system, i.e. the protein domains fused to the NP868R capping enzyme of the test plasmid or Firefly Luciferase reporter plasmids with four tandem RNA tethered repeats introduced in their 3UTR, had no significant effects on the expression of the Firefly Luciferase reporter mRNA with any system when compared to no tethering system (row 2 or 3 vs. 1; p=NS for all comparisons, two-way Student t-test). The cotransfection of pK1ERNAP with plasmids encoding for the components of the MS2-4xMS2sl (i.e. pMS2-NP868R/pK1Ep-Luciferase-4xMS2sl), N-NP868R-4xABoxBl (i.e. pN-NP868R/pK1Ep-Luciferase-4xBoxBl), NP22-NP868R-4xP22BoxBr (i.e. pNP22-NP868R/pK1Ep-Luciferase-4xP22BoxBr), N21-4x21BoxBr (i.e. pN21-NP868R/pK1Ep-Luciferase-4x21BoxBr), tethering system increased significantly by 3.8- to 4.6-fold the expression levels of firefly luciferase reporter in comparison to conditions with the untethering capping enzyme and/or untethered Firefly Luciferase plasmids (row 4 vs. 1-3; p<0.05 for all comparisons, two-way Student t-test). In contrast, the cotransfection of pK1ERNAP with either the TAT/4xTAR tethering system (i.e. pTAT-NP868R/pK1Ep-Luciferase-4xTAR) or the SNRNP70/4xU1snRNA tethering system (i.e. pSNRNP70-NP868R/pK1Ep-Luciferase-4xU1snRNA) shows very low change of Firefly Luciferase in comparison to conditions with the untethering capping enzyme and/or untethered Firefly Luciferase plasmids (row 4 vs. 1-3; p=NS for all comparisons, two-way Student t-test).
[0469] In conclusion, the best performances were obtained with the N-4xBoxBl tethering expression system (i.e. pN-NP868R/pK1Ep-Luciferase-4xBoxBl), with performances of other tethering systems ranging in the following order (i.e. ratio of condition 4 vs.1): N-4xBoxBl>MS2-4xMS2sl>N21-4x21BoxBr>NP22-4xP22BoxBr>>TAT-4xTAR>SNRNP70-4xU1snRNA.
[0470] 4. Conclusions
[0471] The present experiments show that the African swine fever virus capping enzyme NP868R can increase the expression of Firefly Luciferase mRNA produced by the K1E phage RNA polymerase when appropriately tethered to the mRNA by bacteriophage protein-RNA tethering systems.
EXAMPLE 4: BACTERIOPHAGE PROTEIN:RNA TETHERING SYSTEMS, COUPLED TO THE D12L SUBUNIT OF THE VACCINIA VIRUS CAPPING CAN INCREASE THE EXPRESSION OF LUCIFERASE REPORTER MRNA PRODUCED BY K1E PHAGE RNA POLYMERASE IN HOST-CELL CYTOPLASM
[0472] 1. Objectives
[0473] The objectives of the present experiments were to investigate if other protein:RNA tethering systems than the N-4xBoxBr system can be used to guide the heterodimeric capping enzyme from the vaccinia virus to the a target mRNA, and thereby increase its expression.
[0474] The protein:RNA tethering systems tested hereinafter are the same as described in the previous example.
[0475] 2. Methods
[0476] a. Plasmids
[0477] The pK1ERNAP expression plasmid was described in Example 1.
[0478] The test plasmid consisted of the coding sequence of the D12L subunit from the vaccinia virus capping enzyme fused at its amino-terminal end with the tethering protein sequences described above. The D1R plasmid was the same as previously described.
[0479] The Firefly Luciferase reporter plasmids containing the various tethered RNA sequences were the same as described in the previous example.
[0480] b. Cell Culture and Transfection
[0481] Same as described in Example 1.
[0482] c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
[0483] Same as described in Example 1.
[0484] d. Statistical Analysis
[0485] Same as described in Example 1.
[0486] 3. Results
[0487] The design of the assay was very similar to Example 1, except that various protein:RNA tethering systems were tested in replacement to the N:BoxBr system (
[0488] Results of these experiments are shown in the table below:
TABLE-US-00006 Plasmids mean SEM MS2-4xMS2sl tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 573 812 41312 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase-4xMS2sl 564 522 92952 (3) pK1ERNAP, pMS2-D12L, pD1R, pK1Ep-Luciferase 687 767 84793 (4) pK1ERNAP, pMS2-D12L, pD1R, pK1Ep-Luciferase- 1 147 529 181568 4xMS2sl (5) Baseline 187 173 N-4xBoxBl tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 573 812 41312 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase-4xBoxBl 566 750 60984 (3) pK1ERNAP, pN-D12L, pD1R, pK1Ep-Luciferase 521 157 161568 (4) pK1ERNAP, pN-D12L, pD1R, pK1Ep-Luciferase- 1 247 165 141568 4xBoxBl (5) Baseline 187 173 NP22-4xP22BoxBr tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 573 812 41312 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase- 748 833 41312 4xP22BoxBr (3) pK1ERNAP, pNP22-D12L, pD1R, pK1Ep-Luciferase 548 192 221568 (4) pK1ERNAP, pNP22-D12L, pD1R, pK1Ep-Luciferase- 1 306 542 127189 4xP22BoxBr (5) Baseline 187 173 N21-4x21BoxBr tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 573 812 41312 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase- 576 702 123936 4x21BoxBr (3) pK1ERNAP, pN21-D12L, pD1R, pK1Ep-Luciferase 704 808 127189 (4) pK1ERNAP, pN21-D12L, pD1R, pK1Ep-Luciferase- 1 432 734 127189 4x21BoxBr (5) Baseline 187 173 TAT-4xTAR tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 573 812 41312 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase-4xTAR 489 422 81968 (3) pK1ERNAP, pTAT-D12L, pD1R, pK1Ep-Luciferase 420 064 190784 (4) pK1ERNAP, pTAT-D12L, pD1R, pK1Ep-Luciferase- 680 137 83594 4xTAR (5) Baseline 187 173 SNRNP70-4xU1snRNA tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 510 718 61968 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase- 582 331 6995 4xU1snRNA (3) pK1ERNAP, pSNRNP70-D12L, pD1R, pK1Ep-Luciferase 630 096 190784 (4) pK1ERNAP, pSNRNP70-D12L, pD1R, pK1Ep-Luciferase- 618 425 129309 4xU1snRNA (5) Baseline 187 173
[0489] The cotransfection of pK1ERNAP with plasmids having only one out of the two components of the tethering system, i.e. the protein domains fused to the D12L subunit of the vaccinia virus capping of the test plasmid or the Firefly Luciferase reporter plasmids with four tandem RNA tethered repeats introduced in their 3UTR, had no significant effects on the expression of the Firefly Luciferase reporter mRNA with any system when compared to no tethering system (row 2 or 3 vs. 1; p=NS for all comparisons, two-way Student t-test). Similarly to previous findings, the cotransfection of pK1ERNAP with plasmids with all the components of the MS2-4xMS2sl and D1R subunit of the vaccinia virus capping enzyme (i.e. pMS2-D12L/pD1R/pK1Ep-Luciferase-4xMS2sl), N-D12L-4xABoxBl (i.e. pN-D12L/pD1R/pK1Ep-Luciferase-4xABoxBl), NP22-D12L-4xP22BoxBr (i.e. pNP22-D12L/pD1R/pK1Ep-Luciferase-4xP22BoxBr), N21-4x21BoxBr (i.e. pN21-D12L/pD1R/pK1Ep-Luciferase-4x21BoxBr), tethering system increased significantly by 2- to 2.5-fold the expression levels of firefly luciferase reporter in comparison to conditions with the untethering capping enzyme and/or untethered Firefly Luciferase plasmids (row 4 vs. 1-3; p<0.05 for all comparisons, two-way Student t-test). In contrast, the cotransfection of pK1ERNAP with either the TAT/4xTAR tethering system (i.e. pTAT-D12L/pD1R/pK1Ep-Luciferase-4xTAR) or the SNRNP70/4xU1snRNA tethering system (i.e. pSNRNP70-D12L/pD1R/pK1Ep-Luciferase-4xU1snRNA) shows very low change of Firefly Luciferase in comparison to conditions with the untethering capping enzyme and/or untethered Firefly Luciferase plasmids (p=NS for all comparisons, two-way Student t-test).
[0490] Finally, the performances of the tethering systems ranged in a different order than in the previous example (i.e. ratio of condition 4 vs.1): N21-4x21BoxBr>NP22-4xP22BoxBr>N-4xBoxBl>MS2-4xMS2sl>>TAT-4xTAR>SNRNP70-4xU1snRNA.
[0491] 4. Conclusions
[0492] The present experiments show that the heterodimeric D1R/D12L capping enzyme from the vaccinia virus can also increase the expression of Firefly Luciferase mRNA produced by the K1E phage RNA polymerase when appropriately tethered to the mRNA by bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system.
EXAMPLE 5: FUSION BETWEEN POLY(A) POLYMERASES AND AFRICAN SWINE FEVER VIRUS NP868R CAPPING ENZYME INCREASE THE EXPRESSION OF LUCIFERASE REPORTER MRNA PRODUCED BY K1E PHAGE RNA POLYMERASE WHEN APPROPRIATELY TETHERED TO THE TARGET TRANSCRIPT
[0493] 1. Objectives
[0494] The present experiments aimed to determine if poly(A) polymerases fused to NP868R African Swine Fever virus capping enzyme can increase the expression of Firefly Luciferase reporter mRNA produced by the K1E phage RNA polymerase when appropriately tethered to the target transcript by the N-BoxBl the thering system.
[0495] 2. Methods
[0496] a. Plasmids
[0497] The ORFs of the following poly(A) polymerases were synthesized: i) PAP1 poly(A) polymerase from Saccharomyces cerevisiae sorted to the cytoplasm by deletion of the 42 carboxyl-terminal amino-acids that contains a nuclear localization signal (Zhelkovsky, Helmling et al. 1998) (NCBI accession number: P29468); the vaccinia virus VP55 poly(A) polymerase (UniProtKB/Swiss-Prot accession number P23371 corresponding to the nucleotide and amino-acid sequences, respectively), the viral R341 poly(A) polymerase from Acanthamoeba polyphaga mimivirus (UniProtKB/Swiss-Prot accession number: E3VZZ8), iv) the viral MG561 poly(A) polymerase from Megavirus chilensis (NCBI Accession number: YP_004894612), v) the viral C475L poly(A) polymerase from the African swine fever virus (UniProtKB/Swiss-Prot accession number: A0A0A1E081), vi) mutant PAPOLA (K656R-K657R mutation of the human PAPOLA, UniProtKB/Swiss-Prot accession number P51003) mutated at its the nuclear localization signal (Raabe, Murthy et al. 1994, Vethantham, Rao et al. 2008), vii) the wild-type canonical Mus musculus testis specific PAPOLB (UniProtKB/Swiss-Prot Q9WVP6).
[0498] Four types of test plasmids were generated by in-frame subcloning of the poly(A) polymerases ORFs: i) in the pCMV-Script backbone only (e.g. pPAP1), ii) downstream to the NA tethering domain (e.g. pN-PAP1), iii) downstream to the N-NP868R protein through a G4 flexible linker (e.g. pN-NP868R-G4-PAP1) resulting in the expression of monomeric protein, or iv) between the N protein tethering domain from the lambda bacteriophage and NP868R through a G4 flexible linker (e.g. pN-PAP1-G4-NP868R) also resulting in the expression of monomeric protein. The design of these two latter constructions is shown
[0499] The Firefly Luciferase reporter plasmids in their untethered (pK1Ep-Luciferase) or tethered version (pK1Ep-Luciferase-4xBoxBl) were the same as described above.
[0500] b. Cell Culture and Transfection
[0501] Same as described in Example 1.
[0502] c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
[0503] Same as described in Example 1.
[0504] d. Statistical Analysis
[0505] Same as described in Example 1.
[0506] 3. Results
[0507] The design of the assay was very similar to Example 2, except that the poly(A) polymerases were fused to African Swine Fever virus NP868R capping enzyme (
[0508] Results of these experiments are shown in the table below:
TABLE-US-00007 Plasmids mean SEM C475L poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 150 567 138 816 (2) pK1ERNAP, C475L, pK1Ep-Luciferase-4xBoxBl 240 907 386 848 (3) pK1ERNAP, pN-C475L, pK1Ep-Luciferase-4xBoxBl 301 134 499 738 (4) pK1ERNAP, pN-NP868R, pK1Ep-Luciferase-4xBoxBl 3 538 575 414 672 (5) pK1ERNAP, pC475L, pN-NP868R, pK1Ep-Luciferase- 4 246 290 456 139 4xBoxBl (6) pK1ERNAP, pN-C475L, pN-NP868R, pK1Ep- 6 794 064 364 911 Luciferase-4xBoxBl (7) pK1ERNAP, pN-C475L-G4-NP868R, pK1Ep-Luciferase- 8 685 660 414 672 4xBoxBl (8) pK1ERNAP, pN-NP868R-G4-C475L, pK1Ep-Luciferase- 8 385 560 393 938 4xBoxBl (9) Baseline 22 217 14 176 MG561 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 150 567 138 816 (2) pK1ERNAP, MG561, pK1Ep-Luciferase-4xBoxBl 316 191 360 922 (3) pK1ERNAP, pN-MG561, pK1Ep-Luciferase-4xBoxBl 421 588 832 896 (4) pK1ERNAP, pN-NP868R, pK1Ep-Luciferase-4xBoxBl 3 538 575 414 672 (5) pK1ERNAP, pMG561, pN-NP868R, pK1Ep-Luciferase- 4 246 290 456 139 4xBoxBl (6) pK1ERNAP, pN-MG561, pN-NP868R, pK1Ep- 6 199 583 364 911 Luciferase-4xBoxBl (7) pK1ERNAP, pN-MG561-G4-NP868R, pK1Ep-Luciferase- 8 638 575 734 672 4xBoxBl (8) pK1ERNAP, pN-NP868R-G4-MG561, pK1Ep-Luciferase- 9 070 504 697 938 4xBoxBl (9) Baseline 22 217 14 176 PAP1 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 150 567 138 816 (2) pK1ERNAP, PAP1, pK1Ep-Luciferase-4xBoxBl 225 851 277 632 (3) pK1ERNAP, pN-PAP1, pK1Ep-Luciferase-4xBoxBl 361 361 694 080 (4) pK1ERNAP, pN-NP868R, pK1Ep-Luciferase-4xBoxBl 3 538 575 414 672 (5) pK1ERNAP, pPAP1, pN-NP868R, pK1Ep-Luciferase- 4 352 447 456 139 4xBoxBl (6) pK1ERNAP, pN-PAP1, pN-NP868R, pK1Ep-Luciferase- 6 659 244 364 911 4xBoxBl (7) pK1ERNAP, pN-PAP1-G4-NP868R, pK1Ep-Luciferase- 10 638 575 734 688 4xBoxBl (8) pK1ERNAP, pN-NP868R-G4-PAP1, pK1Ep-Luciferase- 12 538 575 404 078 4xBoxBl (9) Baseline 22 217 14 176 R341 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 150 567 138 816 (2) pK1ERNAP, R341, pK1Ep-Luciferase-4xBoxBl 271 021 249 869 (3) pK1ERNAP, pN-R341, pK1Ep-Luciferase-4xBoxBl 338 776 430 330 (4) pK1ERNAP, pN-NP868R, pK1Ep-Luciferase-4xBoxBl 3 538 575 414 672 (5) pK1ERNAP, pR341, pN-NP868R, pK1Ep-Luciferase- 4 069 361 456 139 4xBoxBl (6) pK1ERNAP, pN-R341, pN-NP868R, pK1Ep-Luciferase- 6 673 752 364 911 4xBoxBl (7) pK1ERNAP, pN-R341-G4-NP868R, pK1Ep-Luciferase- 8 992 399 734 688 4xBoxBl (8) pK1ERNAP, pN-NP868R-G4-R341, pK1Ep-Luciferase- 8 522 353 881 625 4xBoxBl (9) Baseline 22 217 14 176 VP55 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 150 567 138 816 (2) pK1ERNAP, VP55, pK1Ep-Luciferase-4xBoxBl 271 021 388 685 (3) pK1ERNAP, pN-VP55, pK1Ep-Luciferase-4xBoxBl 331 247 291 514 (4) pK1ERNAP, pN-NP868R, pK1Ep-Luciferase-4xBoxBl 3 538 575 414 672 (5) pK1ERNAP, pVP55, pN-NP868R, pK1Ep-Luciferase- 4 493 990 456 139 4xBoxBl (6) pK1ERNAP, pN-VP55, pN-NP868R, pK1Ep-Luciferase- 6 673 576 364 911 4xBoxBl (7) pK1ERNAP, pN-VP55-G4-NP868R, pK1Ep-Luciferase- 8 638 575 134 687 4xBoxBl (8) pK1ERNAP, pN-NP868R-G4-VP55, pK1Ep-Luciferase- 10 107 133 220 406 4xBoxBl (9) Baseline 22 217 14 176 PAPOLA poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 124 567 138 816 (2) pK1ERNAP, PAPOLA, pK1Ep-Luciferase-4xBoxBl 267 819 388 685 (3) pK1ERNAP, pN-PAPOLA, pK1Ep-Luciferase-4xBoxBl 274 047 291 514 (4) pK1ERNAP, pN-NP868R, pK1Ep-Luciferase-4xBoxBl 3 538 575 414 672 (5) pK1ERNAP, pPAPOLA, pN-NP868R, pK1Ep-Luciferase- 4 635 533 456 139 4xBoxBl (6) pK1ERNAP, pN-PAPOLA, pN-NP868R, pK1Ep- 7 231 432 364 911 Luciferase-4xBoxBl (7) pK1ERNAP, pN-PAPOLA-G4-NP868R, pK1Ep- 12 203 575 734 688 Luciferase-4xBoxBl (8) pK1ERNAP, pN-NP868R-G4-PAPOLA, pK1Ep- 11 262 433 220 406 Luciferase-4xBoxBl (9) Baseline 22 217 14 176 PAPOLB polu(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 150 567 138 816 (2) pK1ERNAP, PAPOLB, pK1Ep-Luciferase-4xBoxBl 251 447 360 922 (3) pK1ERNAP, pN-PAPOLB, pK1Ep-Luciferase-4xBoxBl 337 270 832 896 (4) pK1ERNAP, pN-NP868R, pK1Ep-Luciferase-4xBoxBl 3 538 575 414 672 (5) pK1ERNAP, pPAPOLB, pN-NP868R, pK1Ep-Luciferase- 5 874 035 456 139 4xBoxBl (6) pK1ERNAP, pN-PAPOLB, pN-NP868R, pK1Ep- 7 753 726 364 911 Luciferase-4xBoxBl (7) pK1ERNAP, pN-PAPOLB-G4-NP868R, pK1Ep- 9 338 575 734 688 Luciferase-4xBoxBl (8) pK1ERNAP, pN-NP868R-G4-PAPOLB, pK1Ep- 9 808 575 624 484 Luciferase-4xBoxBl (9) Baseline 22 217 14 176
[0509] In the absence of mRNA capping provided by pN-NP868R, non-statistically significant increase of Firefly Luciferase mRNA expression of 1.5-fold and 2.5-fold was observed when untethered (row 2 vs. 1) or tethered poly(A) polymerase plasmids (row 3 vs. 1) were transfected, respectively (p=NS, two-way Student t-test). When the Firefly Luciferase mRNA was capped by co-transfection of pN-NP868R, a statistically significant increase of expression of 1.5-fold (row 5 vs. 4) and 2-fold (row 6 vs. 4) was observed when the untethered or tethered poly(A) polymerases plasmids were cotransfected, respectively (p<0.05 for all untethered poly(A) polymerases vs. no poly(A) polymerases, two-way Student t-test).
[0510] Poly(A) polymerases were fused to NP868R African Swine Fever virus capping enzyme, together with the N-protein tethering domain. Two types of fusion were tested with poly(A) polymerases subcloned either downstream to N-NP868R through a G4 flexible linker or between the N protein tethering domain from the lambda bacteriophage and NP868R through a Ga flexible linker. All tethered fusions genes of both types increased the expression of Firefly Luciferase mRNA in comparison to non-linked enzymes (row 7 and 8 vs. 6; p<0.05 for all comparisons, two-way Student t-test). Activity of the fusion proteins ranged as follows: N-NP868R-G.sub.4-C475L<N-NP868R-G.sub.4-R341<N-MG561-G.sub.4-NP868R<N-VP55-G.sub.4-NP868R<N-C475L-G.sub.4-NP868R<N-R341-G.sub.4-NP868R<N-NP868R-G.sub.4-MG561<N-PAPOLB-G.sub.4-NP868R<N-NP868R-G.sub.4-PAPOLB<N-NP868R-G.sub.4-VP55<N-PAP1-G.sub.4-NP868R<N-NP868R-G.sub.4-PAPOLA<N-PAPOLA-G.sub.4-NP868R<N-NP868R-G.sub.4-PAP1
[0511] 4. Conclusions
[0512] The present experiments show that various poly(A) polymerases including mammalian, yeast, viral and bacterial enzymes fused to the African Swine Fever virus NP868R capping enzyme increase the expression of transcripts produced by phage RNA polymerase, when appropriately tethered with the N-4xBoxBl system. Surprisingly, the fusion between various poly(A) polymerases and NP868R capping enzymes, which are not physically linked in the nature and contain no RNA-binding domain, can act synergistically and this effect is even greater when these fusion proteins are appropriately tethered (rows 7 and 8 in the above Table). These results are really surprising and one skilled in the art could have expected to obtain the same expression rate since the components are the same.
EXAMPLE 6: FUSION OF POLY(A) POLYMERASES AND THE D12 SUBUNIT OF THE HETERODIMERIC VACCINIA VIRUS CAPPING ENZYME CAN INCREASE THE EXPRESSION OF LUCIFERASE REPORTER MRNA PRODUCED BY K1E PHAGE RNA POLYMERASE WHEN APPROPRIATELY TETHERED TO THE TARGET TRANSCRIPT
[0513] 1. Objectives
[0514] The present experiments aim to determine if fusions of poly(A) polymerases with D12 subunit of the heterodimeric vaccinia virus capping enzyme can increase the expression of transcripts produced by phage RNA polymerase, when appropriately tethered with the N-4xBoxBl system.
[0515] 2. Methods
[0516] a. Plasmids
[0517] The pK1ERNAP expression plasmid was described in Example 1.
[0518] The poly(A) polymerases tested were described in previous example and subcloned in-frame (
[0519] The Firefly Luciferase reporter plasmids in their untethered (pK1Ep-Luciferase) or tethered version (pK1Ep-Luciferase-4xBoxBl) were the same as described above.
[0520] b. Cell Culture and Transfection
[0521] Same as described in Example 1.
[0522] c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
[0523] Same as described in Example 1.
[0524] d. Statistical Analysis
[0525] Same as described in Example 1.
[0526] 3. Results
[0527] The design of the experiment was similar to previous example, except that the capping enzyme consisted of the vaccinia virus heterodimer D1R/D12 (
[0528] Results of these experiments are shown in the table below:
TABLE-US-00008 Plasmids mean SEM C475L poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 145 279 116 201 (2) pK1ERNAP, C475L, pK1Ep-Luciferase-4xBoxBl 201 661 325 912 (3) pK1ERNAP, pN-C475L, pK1Ep-Luciferase-4xBoxBl 253 700 466 225 (4) pK1ERNAP, pN-D12L, pD1R, pK1Ep-Luciferase-4xBoxBl 3 301 279 429 341 (5) pK1ERNAP, pC475L, pN-D12L, pD1R, pK1Ep-Luciferase- 4 196 498 353 954 4xBoxBl (6) pK1ERNAP, pN-C475L, pN-D12L, pD1R, pK1Ep- 5 272 046 95 796 Luciferase-4xBoxBl (7) pK1ERNAP, pN-C475L-G4-D12L, pD1R, pK1Ep-Luciferase- 7 201 395 453 620 4xBoxBl (8) pK1ERNAP, pN-D1R, pD12L-G4-C475L, pK1Ep-Luciferase- 7 173 174 267 557 4xBoxBl (9) Baseline 23 887 9 510 MG561 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 121 133 125 248 (2) pK1ERNAP, MG561, pK1Ep-Luciferase-4xBoxBl 285 286 334 854 (3) pK1ERNAP, pN-MG561, pK1Ep-Luciferase-4xBoxBl 391 138 866 711 (4) pK1ERNAP, pN-D12L, pD1R, pK1Ep-Luciferase-4xBoxBl 2 240 943 218 840 (5) pK1ERNAP, pMG561, pN-D12L, pD1R, pK1Ep-Luciferase- 3 682 240 466 962 4xBoxBl (6) pK1ERNAP, pN-MG561, pN-D12L, pD1R, pK1Ep- 5 346 676 374 202 Luciferase-4xBoxBl (7) pK1ERNAP, pN-MG561-G4-D12L, pD1R, pK1Ep- 8 858 505 603 377 Luciferase-4xBoxBl (8) pK1ERNAP, pN-D1R, pD12L-G4-MG561, pK1Ep- 7 449 488 527 949 Luciferase-4xBoxBl (9) Baseline 23 887 9 510 PAP1 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 120 765 132 669 (2) pK1ERNAP, PAP1, pK1Ep-Luciferase-4xBoxBl 215 849 176 186 (3) pK1ERNAP, pN-PAP1, pK1Ep-Luciferase-4xBoxBl 229 321 646 674 (4) pK1ERNAP, pN-D12L, pD1R, pK1Ep-Luciferase-4xBoxBl 3 296 890 441 173 (5) pK1ERNAP, pPAP1, pN-D12L, pD1R, pK1Ep-Luciferase- 4 630 608 427 360 4xBoxBl (6) pK1ERNAP, pN-PAP1, pN-D12L, pD1R, pK1Ep-Luciferase- 6 239 087 194 450 4xBoxBl (7) pK1ERNAP, pN-PAP1-G4-D12L, pD1R, pK1Ep-Luciferase- 10 668 967 620 415 4xBoxBl (8) pK1ERNAP, pN-D1R, pD12L-G4-PAP1, pK1Ep-Luciferase- 9 534 665 223 391 4xBoxBl (9) Baseline 23 887 9 510 R341 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 92 989 144 738 (2) pK1ERNAP, R341, pK1Ep-Luciferase-4xBoxBl 282 582 190 160 (3) pK1ERNAP, pN-R341, pK1Ep-Luciferase-4xBoxBl 257 822 449 340 (4) pK1ERNAP, pN-D12L, pD1R, pK1Ep-Luciferase-4xBoxBl 2 684 956 273 600 (5) pK1ERNAP, pR341, pN-D12L, pD1R, pK1Ep-Luciferase- 3 694 895 372 489 4xBoxBl (6) pK1ERNAP, pN-R341, pN-D12L, pD1R, pK1Ep-Luciferase- 5 449 865 413 128 4xBoxBl (7) pK1ERNAP, pN-R341-G4-D12L, pD1R, pK1Ep-Luciferase- 10 180 543 694 655 4xBoxBl (8) pK1ERNAP, pN-D1R, pD12L-G4-R341, pK1Ep-Luciferase- 8 057 982 766 195 4xBoxBl (9) Baseline 23 887 9 510 VP55 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 111 045 96 804 (2) pK1ERNAP, VP55, pK1Ep-Luciferase-4xBoxBl 188 997 328 927 (3) pK1ERNAP, pN-VP55, pK1Ep-Luciferase-4xBoxBl 280 320 285 568 (4) pK1ERNAP, pN-D12L, pD1R, pK1Ep-Luciferase-4xBoxBl 3 466 398 436 289 (5) pK1ERNAP, pVP55, pN-D12L, pD1R, pK1Ep-Luciferase- 4 728 261 461 933 4xBoxBl (6) pK1ERNAP, pN-VP55, pN-D12L, pD1R, pK1Ep-Luciferase- 5 758 344 375 637 4xBoxBl (7) pK1ERNAP, pN-VP55-G4-D12L, pD1R, pK1Ep-Luciferase- 7 863 085 29 635 4xBoxBl (8) pK1ERNAP, pN-D1R, pD12L-G4-VP55, pK1Ep-Luciferase- 8 601 926 252 202 4xBoxBl (9) Baseline 23 887 9 510 PAPOLA poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 174 916 96 128 (2) pK1ERNAP, PAPOLA, pK1Ep-Luciferase-4xBoxBl 185 461 347 658 (3) pK1ERNAP, pN-PAPOLA, pK1Ep-Luciferase-4xBoxBl 245 121 282 829 (4) pK1ERNAP, pN-D12L, pD1R, pK1Ep-Luciferase-4xBoxBl 3 433 158 309 045 (5) pK1ERNAP, pPAPOLA, pN-D12L, pD1R, pK1Ep-Luciferase- 4 454 749 385 659 4xBoxBl (6) pK1ERNAP, pN-PAPOLA, pN-D12L, pD1R, pK1Ep- 6 114 076 325 113 Luciferase-4xBoxBl (7) pK1ERNAP, pN-PAPOLA-G4-D12L, pD1R, pK1Ep- 9 230 982 731 066 Luciferase-4xBoxBl (8) pK1ERNAP, pN-D1R, pD12L-G4-PAPOLA, pK1Ep- 10 284 960 221 627 Luciferase-4xBoxBl (9) Baseline 23 887 9 510 PAPOLB poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xBoxBl 134 279 130 482 (2) pK1ERNAP, PAPOLB, pK1Ep-Luciferase-4xBoxBl 236 351 321 719 (3) pK1ERNAP, pN-PAPOLB, pK1Ep-Luciferase-4xBoxBl 300 637 861 464 (4) pK1ERNAP, pN-D12L, pD1R, pK1Ep-Luciferase-4xBoxBl 3 659 946 363 888 (5) pK1ERNAP, pPAPOLB, pN-D12L, pD1R, pK1Ep-Luciferase- 4 154 652 410 206 4xBoxBl (6) pK1ERNAP, pN-PAPOLB, pN-D12L, pD1R, pK1Ep- 5 972 923 350 054 Luciferase-4xBoxBl (7) pK1ERNAP, pN-PAPOLB-G4-D12L, pD1R, pK1Ep- 7 677 493 552 983 Luciferase-4xBoxBl (8) pK1ERNAP, pN-D1R, pD12L-G4-PAPOLB, pK1Ep- 8 958 357 480 636 Luciferase-4xBoxBl (9) Baseline 23 887 9 510
[0529] In the absence of mRNA capping provided by pN-D12L/D1R, non-statistically significant change of Firefly Luciferase mRNA expression of 1.5-fold and 2-fold was observed when untethered (row 2 vs. 1) or tethered poly(A) polymerase plasmids (row 3 vs. 1) were transfected, respectively (p=NS, two-way Student t-test). Similarly to previous findings, when the Firefly Luciferase mRNA was capped by co-transfection of pN-D12L/D1R, a statistically significant increase of expression of 1.5-fold (row 5 vs. 4) and 2-fold (row 6 vs. 4) was observed when the untethered or tethered poly(A) polymerases plasmids were cotransfected, respectively (p<0.05 for all untethered poly(A) polymerases vs. no poly(A) polymerases, two-way Student t-test).
[0530] Poly(A) polymerases were fused to the D12 subunit of the heterodimeric vaccinia virus capping enzyme, together with the N-protein domain as described above. All tethered fusions genes of both types increased the expression of Firefly Luciferase mRNA in comparison to non-linked enzymes (row 7 and 8 vs. 6; p<0.05 for all comparisons, two-way Student t-test). Activity of the fusion complexes ranged as follows: N-D12L/D1R-G.sub.4-C475L<N-C475L-G.sub.4-D12L/D1R<N-D12L/D1R-G.sub.4-MG561<N-PAPOLB-G.sub.4-D12L/D1R<N-VP55-G.sub.4-D12L/D1R<N-D12L/D1R-Ga-R341<N-D12L/D1R-G.sub.4-VP55<N-MG561-G.sub.4-D12L/D1R<N-D12L/D1R-G.sub.4-PAPOLB<N-PAPOLA-G.sub.4-D12L/D1R<N-D12L/D1R-G.sub.4-PAP1<N-R341-G.sub.4-D12L/D1R<N-D12L/D1R-G.sub.4-PAPOLA<N-PAP1-G.sub.4-D12L/D1R.
[0531] 4. Conclusions
[0532] The present experiments that various poly(A) polymerases fused to the D12 subunit of the heterodimeric vaccinia virus capping enzyme together with D1R subunit, which are not physically linked in the nature and contain no RNA-binding domain, can act synergistically and this effect is even greater when these fusion proteins appropriately tethered.
EXAMPLE 7: NON-COVALENT TETHERED COUPLING BETWEEN ACANTHAMOEBA POLYPHAGA MIMIVIRUS R341 POLY(A) POLYMERASE AND AFRICAN SWINE FEVER VIRUS NP868R CAPPING ENZYME, CAN INCREASE THE EXPRESSION OF LUCIFERASE REPORTER MRNA PRODUCED BY PHAGE RNA POLYMERASES
[0533] 1. Objectives
[0534] The objectives of this set of experiments were to determine if non-covalent coupling between the R341 poly(A) polymerase and NP868R capping enzyme also results in an active expression system able to enhance the expression of uncapped Firefly Luciferase reporter mRNA when appropriately tethered. In the present experiments, the non-covalent coupling was generated using complementary leucine zippers that form heterodimers.
[0535] 2. Methods
[0536] a. Plasmids
[0537] The pK1ERNAP expression and Firefly Luciferase reporter plasmids in their untethered (pK1Ep-Luciferase) or tethered version (pK1Ep-Luciferase-4xBoxBr) were the same as described above.
[0538] Non-covalent coupling between the Acanthamoeba polyphaga mimivirus R341 poly(A) polymerase and the African swine fever virus NP868R capping enzyme was induced by the EE.sub.1234L and RR.sub.1234L complementary leucine-zippers (SEQ ID N.sup.o 28 and SEQ ID N.sup.o 29 corresponding to the nucleotide and amino-acid sequences of G.sub.4-EE.sub.1234L leucine-zipper, respectively; SEQ ID N.sup.o 30 and SEQ ID N.sup.o 31 corresponding to the nucleotide and amino-acid sequences of RR.sub.1234L-G.sub.4 leucine-zipper, respectively). These amphipathic -helices form an antiparallel heterodimer with dissociation affinity of 10.sup.15M (Moll, Ruvinov et al. 2001). Two non-covalent heterodimeric complexes were generated between NP868R capping enzyme and R341 RNA polymerase (
[0539] b. Cell Culture and Transfection
[0540] Same as described in Example 1.
[0541] c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
[0542] Same as described in Example 1.
[0543] d. Statistical Analysis
[0544] Same as described in Example 1.
[0545] 3. Results
[0546] Uncapped Firefly Luciferase reporter mRNA was generated by the K1E bacteriophage RNA polymerase, using the pK1Ep-Luciferase and pK1Ep-Luciferase-4xBoxBr plasmids as previously described (
[0547] Results of these experiments are shown in the table below:
TABLE-US-00009 Plasmids mean SEM (1) pK1ERNAP, pN-R341 497 650 182 520 (2) pK1ERNAP, pEE1234L-R341 257 650 109 512 (3) pK1ERNAP, pN-R341-EE1234L 522 533 146 016 (4) pK1ERNAP, pN-NP868R 2 377 957 237 063 (5) pK1ERNAP, pRR1234L-NP868R 1 049 099 104 587 (6) pK1ERNAP, pN-NP868R-RR1234L 2 331 330 232 415 (7) pK1ERNAP, pEE1234L-R341, pRR1234L- 4 539 654 325 828 NP868R (8) pK1ERNAP, pN-R341-EE1234L, 12 924 395 325 828 pRR1234L-NP868R (9) pK1ERNAP, pN-NP868R-RR1234L, 10 191 523 270 437 pEE1234L-R341 (10) Baseline 671 257
[0548] The N-tethering of the Acanthamoeba polyphaga mimivirus R341 poly(A) polymerase with or without leucine zipper increased modestly the expression of uncapped 4xBoxBr-Firefly Luciferase mRNA generated by the phage K1E RNA polymerase in comparison to untethered Acanthamoeba polyphaga mimivirus R341 poly(A) polymerase (row 1 or 3 vs. 2). The N-tethering of the African Swine Fever Virus NP868R capping enzyme with or without leucine zipper increased frankly the expression of uncapped Firefly Luciferase 4xBoxBr-mRNA generated by the phage K1E RNA polymerase in comparison to untethered NP868R capping enzyme or no capping enzyme (row 4 or 6 vs. 5).
[0549] The non-covalent coupling between the Acanthamoeba polyphaga mimivirus R341 poly(A) polymerase and the African swine fever virus NP868R capping enzyme was generated using the EE.sub.1234L and RR.sub.1234L complementary high-affinity leucine-zippers. This heterodimeric complex without the N-tethering domain resulted in active complex that significantly increased the expression of uncapped Firefly Luciferase mRNA generated by the phage K1E RNA polymerase in comparison to conditions to either untethered R341 or NP868R with leucine zippers alone (row 7 vs. 2 or 5). Noticeably, the addition of N-tethering at amino-terminal end of the Acanthamoeba polyphaga mimivirus R341 poly(A) polymerase or the African swine fever virus NP868R capping enzyme of this heterodimeric complex increased by 2.80- and 2.24-fold the expression of uncapped Firefly Luciferase mRNA in comparison to the untethered complex (row 8 or 9 vs. 7; p<0.05 for both comparisons, two-way Student t-test).
[0550] 4. Conclusions
[0551] The present experiments show that the artificial coupling between the R341 poly(A) polymerase and the African swine fever virus NP868R capping enzyme, i.e. non-covalent through leucine zippers, also results in synergistically active heterodimers and this effect is even greater when these fusion proteins appropriately tethered.
EXAMPLE 8: ASSEMBLIES BETWEEN THE ACANTHAMOEBA POLYPHAGA MIMIVIRUS R341 POLY(A) POLYMERASE, AFRICAN SWINE FEVER VIRUS NP868R CAPPING ENZYME AND PHAGE K1E RNA POLYMERASE RESULTS IN ACTIVE EXPRESSION COMPLEXES
[0552] 1. Objectives
[0553] The objective of the following experiment was to determine if active complexes could be generated by assembling the Acanthamoeba polyphaga mimivirus poly(A) polymerase R341, the African swine fever virus NP868R capping enzyme and the phage K1E RNA polymerase when appropriately N-tethered.
[0554] The assemblies tested hereinafter are designed according to the common N-R341-[X1]-NP868R-[X2]-K1ERNAP protein scaffold, where [X1] and [X2] are variable.
[0555] The following open-reading-frames were generated to test this hypothesis (
[0560] 2. Methods
[0561] a. Plasmids
[0562] The Firefly Luciferase reporter plasmids in their untethered (pK1Ep-Luciferase) or tethered version (pK1Ep-Luciferase-4xBoxBl) were the same as described above.
[0563] The ORFs previously described were subcloned in the pCMVScript backbone, therefore resulting in the pN-R341-G.sub.4-NP868R-G.sub.4-K1ERNAP, pN-R341-G.sub.4-NP868R-F2A-K1ERNAP, pN-R341-F2A-NP868R-G.sub.4-K1ERNAP, and pN-R341-T2A-NP868R-G.sub.4-K1ERNAP plasmids.
[0564] b. Cell Culture and Transfection
[0565] Same as described in Example 1.
[0566] c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
[0567] Same as described in Example 1.
[0568] d. Statistical Analysis
[0569] Same as described in Example 1.
[0570] 3. Results
[0571] A depiction of the assay is shown
[0572] Results of these experiments are shown in the table below:
TABLE-US-00010 Plasmids mean SEM (1) pN-R341-G4-NP868R, pK1ERNAP, 2 989 500 321 841 pK1Ep-Luciferase-4xBoxBl (2) pN-R341-G4-NP868R-G4-K1ERNAP, 3 882 614 649 466 pK1Ep-Luciferase-4xBoxBl (3) pN-R341-G4-NP868R-F2A-K1ERNAP, 4 393 507 928 702 pK1Ep-Luciferase-4xBoxBl (4) pN-R341-F2A-NP868R-G4-K1ERNAP, 7 778 512 219 138 pK1Ep-Luciferase-4xBoxBl (5) pN-R341-T2A-NP868R-G4-K1ERNAP, 7 861 869 1 120 056 pK1Ep-Luciferase-4xBoxBl (6) Baseline 162 088 56 504
[0573] The various fusion of K1ERNAP coding sequence with N-R341-F2A-NP868R were compared with non-linked N-R341-F2A-NP868R and K1ERNAP. All fusions constructions gave significantly greater expression levels than non-linked N-R341-F2A-NP868R and K1ERNAP (row 2-to-5 vs. 1; p<0.05, two-way Student t-test for all comparisons). Best results were obtained with N-R341-T2A-NP868R-G.sub.4-K1ERNAP and N-R341-F2A-NP868R-G.sub.4-K1ERNAP fusions, with other conditions ranging in the following order: N-R341-T2A-NP868R-G.sub.4-K1ERNAP N-R341-F2A-NP868R-G.sub.4-K1ERNAP>>N-R341-G.sub.4-NP868R-F2A-K1ERNAP>N-R341-G.sub.4-NP868R-G.sub.4-K1ERNAP>pN-R341-G.sub.4-NP868R, pK1ERNAP.
[0574] 4. Conclusions
[0575] The present experiments show that active tethered expression systems can be generated by assembling the poly(A) polymerase R341, African swine fever virus NP868R capping enzyme and phage K1E RNA polymerase under a N-R341-[X1]-NP868R-[X2]-K1ERNAP scaffold, preferably where [X1]=T2A or F2A, and [X2]=G.sub.4. Unexpectedly, the construction N-R341-F2A-NP868R-G.sub.4-K1ERNAP and N-R341-T2A-NP868R-G.sub.4-K1ERNAP allow higher expression rate than the association of the constructions N-R341 with NP868R-G.sub.4-K1ERNAP (row 4 and 5 vs. 1). These results are really surprising and one skilled in the art could have expected to obtain the same expression rate since the components are the same and are not physically linked in the nature and nor contain any RNA-binding domain.
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