Enzymatic synthesis of L-nucleic acids
09850471 · 2017-12-26
Assignee
Inventors
- Andreas Pech (Halle, DE)
- Ralf David (Leipzig, DE)
- Florian Jarosch (Berlin, DE)
- Michael Jahnz (Berlin, DE)
- Sven Klussmann (Berlin, DE)
Cpc classification
C12P19/34
CHEMISTRY; METALLURGY
C12N15/115
CHEMISTRY; METALLURGY
C12Y207/07007
CHEMISTRY; METALLURGY
International classification
C12N9/12
CHEMISTRY; METALLURGY
C12N15/10
CHEMISTRY; METALLURGY
Abstract
The invention relates to a method of reacting one or more L-nucleotides with a first L-nucleic acid in the presence of a D-enzyme that adds the one or more L-nucleotides to the 3′ end of the first L-nucleic acid.
Claims
1. A method for adding one or more L-nucleotides to the 3′ end of a first L-nucleic acid comprising reacting the one or more L-nucleotides with the first L-nucleic acid in the presence of a protein comprising an enzymatic activity of adding one or more L-nucleotides to the 3′ end of an L-nucleic acid, wherein the protein consists of African Swine Fever Virus polymerase X or polymerase DPO4, with the proviso that every amino acid of said polymerase X and polymerase DPO4 is substituted with the corresponding D-amino acid.
2. The method according to claim 1, wherein said African Swine Fever Virus polymerase X, comprises an amino acid sequence at least 85% identical to SEQ ID NO: 1.
3. The method according to claim 1, wherein said polymerase DPO4, comprises an amino acid sequence at least 90% identical to SEQ ID NO: 15.
4. The method according to claim 1, further comprising a second L-nucleic acid, wherein one molecule of the first L-nucleic acid is hybridized to one molecule of the second L-nucleic acid, optionally through Watson-Crick base pairing.
5. A method for amplifying a target L-nucleic acid comprising reacting said target L-nucleic acid with an L-nucleotide and a protein comprising an enzymatic activity of amplifying a target L-nucleic acid, wherein the protein consists of African Swine Fever Virus polymerase X or polymerase DPO4, with the proviso that every amino acid of said polymerase X and polymerase DPO4 is substituted with the corresponding D-amino acid.
6. The method according to claim 5, wherein said African Swine Fever Virus polymerase X, comprises an amino acid sequence at least 85% identical to SEQ ID NO: 1.
7. The method according to claim 5, wherein said polymerase DPO4, comprises an amino acid sequence at least 90% identical to SEQ ID NO: 15.
8. A protein that adds one or more L-nucleotides to the 3′ end of a first L-nucleic acid, wherein the protein consists of African Swine Fever Virus polymerase X or polymerase DPO4, with the proviso that every amino acid of said polymerase X and polymerase DPO4 is substituted with the corresponding D-amino acid.
9. The protein according to claim 8, wherein said African Swine Fever Virus polymerase X, comprises an amino acid sequence at least 85% identical to SEQ ID NO: 1.
10. The protein according to claim 8, wherein said polymerase DPO4, comprises an amino acid sequence at least 90% identical to SEQ ID NO: 15.
11. A polymerase of African Swine Fever Virus polymerase X or polymerase DPO4, with the proviso that every amino acid of said polymerase X and polymerase DPO4 is substituted with the corresponding D-amino acid.
12. The polymerase of claim 11, wherein said African Swine Fever Virus polymerase X, comprises an amino acid sequence at least 85% identical to SEQ ID NO: 1.
13. The polymerase of claim 11, wherein said polymerase DPO4, comprises an amino acid sequence at least 90% identical to SEQ ID NO: 15.
Description
(1) The present invention is further illustrated by the figures, examples and the sequence listing from which further features, embodiments and advantages may be taken, wherein
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EXAMPLES
Abbreviation as Used in the Examples
(27) ACN acetonitrile (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) DCM dichloromethane (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) DIPEA N,N-diisipropylamine (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) EDT (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) Fmoc 9-Fluorenyl-methoxycarbonyl-HATU HATU (2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) (CreoSalus, Louisville Ky., USA) HFIP 1,1,1,3,3,3-hexafluorophosphate (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) HPLC High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography) MeIm methyl imidazole (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) MeOH methanol (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) MSNT 1-(Mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (Merck KGaA, Darmstadt, Germany) NMP N-methyl-pyrrolidone (Iris Biotech GmbH, Marktredwitz, Deutschland) PyBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphat (MERCK KGAA, DARMSTADT, GERMANY) SDS Sodium dodecyl sulfate (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) TBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetralluoroborat (Merck KGaA, Darmstadt, Germany) tBu (tert.-Butyl-) TFA trifluoroacetic acid (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) TFE 1,1,1-trifluoroethanol (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) THF (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) TIS (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) TLC Thin layer chromatography Tris Tris(hydroxymethyl)aminomethane (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) UPLC Ultra-performance liquid chromatography
Example 1—Recombinant Expression and Purification of Wild-Type and Variants of Polymerase X
(28) Polymerase X from African swine fever virus (abbr. ASFV) was described and characterized by Oliveros et al in 1997. The wild-type gene of the polymerase X has an open reading frame (abbr. ORF) of only 525 base pairs including start codon and stop codon (Oliveros et al, 1997). The encoded protein has a length of only 174 amino acids. This example describes how polymerase X as well as variants thereof have been expressed in E. coli and been purified using a His.sub.6-Tag.
(29) 1.1 Expression Constructs
(30) Since the codon usage of ASFV differs from E. coli, an E. coli-codon-optimized synthetic gene for polymerase X was purchased from GeneArt AG (Regensburg, Germany). The synthetic gene sequence was provided in pCR4-Blunt-TOPO vector (originator company: Invitrogen, Karlsruhe, Germany). The codon-optimized open reading frame including start codon and two stop codons had the following sequence:
(31) TABLE-US-00002 ATGCTGACCCTGATTCAGGGCAAAAAAATCGTGAACCATCTGCGTAGCCG TCTGGCCTTTGAATATAACGGCCAGCTGATTAAAATTCTGAGCAAAAACA TTGTGGCGGTGGGCAGCCTGCGTCGTGAAGAAAAAATGCTGAACGATGTG GATCTGCTGATTATTGTGCCGGAAAAAAAACTGCTGAAACATGTGCTGCC GAACATTCGTATTAAAGGCCTGAGCTTTAGCGTGAAAGTGTGCGGCGAAC GTAAATGCGTGCTGTTTATCGAATGGGAAAAAAAAACCTACCAGCTGGAC CTGTTTACCGCGCTGGCCGAAGAAAAACCGTATGCGATCTTTCATTTTAC CGGTCCGGTGAGCTATCTGATTCGTATTCGTGCGGCGCTGAAAAAAAAAA ACTACAAACTGAACCAGTATGGCCTGTTTAAAAACCAGACCCTGGTGCCG CTGAAAATTACCACCGAAAAAGAACTGATTAAAGAACTGGGCTTTACCTA TCGCATTCCGAAAAAACGCCTGTAATAA.
(32) In order to obtain an expression construct for polymerase X, also referred to as all-L polymerase X, the gene of the polymerase X was cut out from pCR4-Blunt-TOPO with BamHI and PstI and subcloned in pRSET-A vector (Invitrogen, Karlsruhe, Germany). Subcloning added a His.sub.6-Tag to the N-terminus, and brought the gene under control of the T7 promoter. The construct was named pMJ14 and was used for expression of all-L polymerase X in E. coli. The protein polymerase X expressed from pMJ14 had the following sequence of 210 amino acids:
(33) TABLE-US-00003 MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSMLTLIQGKKIVNHL RSRLAFEYNGQLIKILSKNIVAVGSLRREEKMLNDVDLLIIVPEKKLLKH VLPNIRIKGLSFSVKVCGERKCVLFIEWEKKTYQLDLFTALAEEKPYAIF HFTGPVSYLIRIRAALKKKNYKLNQYGLFKNQTLVPLKITTEKELIKELG FTYRIPKKRL.
(34) The initial 36 amino acids represented the His.sub.6-Tag including a few spacer amino acids and some other sequence tags (T7 Gene 10 leader, Anti-Express Epitope). The final 174 amino acid part was identical to the polymerase X protein sequence as found in ASFV.
(35) Expression constructs for variants of all-L polymerase X were made using the commercially available QuikChange kit (Stratagene GmbH, Waldbronn, Germany) according to manufacturer's protocol. Plasmid pMJ14 served as template. Oligonucleotides needed for QuikChange were either synthesized at the NOXXON facility (QC10_up, QC10_low) or purchased from Purimex (Grebenstein, Germany) (QC26_up, QC26_low, QC27_up, QC27_low, QC31_up, QC31_low). The following variant expression constructs were made and used for expression of the variants of the all-L polymerase in E. coli:
(36) TABLE-US-00004 oligonucleotides used expression for QuikChange variant construct mutagenesis procedure I124G pMJ130 QC10_up (5′-TCCGGTGAGCTATCTGGGTCGTATTCG TGCGGCG-3′) QC10_low (5′-CGCCGCACGAATACGACCCAGATAGC TCACCGGA-3′) V80G pMJ356 QC26_up (5′-TGAGCTTTAGCGTGAAAGGGTGCGGCG AACG-3′) QC26_low (5′-CGTTCGCCGCACCCTTTCACGCTAAAG CTCA-3′) V80A pMJ357 QC27_up (5′-TGAGCTTTAGCGTGAAAGCGTGCGGCG AACG-3′) QC27_low (5′-CGTTCGCCGCACGCTTTCACGCTAAAG CTCA-3′) C86S pMJ412 QC31_up (5′-TGAAAGTGTGCGGCGAACGTAAAAGCG TGCTGTTTA-3′) QC31_low (5′-TAAACAGCACGCTTTTACGTTCGCCGC ACACTTTCA-3′)
1.2 Protein Expression in E. coli
(37) All-L-polymerase X was expressed in E. coli using expression construct pMJ14. Variants of the all-L-polymerase X were expressed from pMJ130, pMJ356, pMJ357 or pMJ412. For expression, the appropriate expression construct was transformed in competent E. coli strain BL-21 (DE3) pLysS' (Novagen/VWR, Dresden, Germany) and maintained with the antibiotic Ampicillin. The culture was grown at 37° C. in 2YT medium until the optical density at 600 nm reached approx. 0.6. Then protein expression was induced by adding Isopropyl beta-D-1-thiogalactopyranoside (abbr. IPTG) to a final concentration of 0.4 mM. Expression was performed for 4 hours at 30° C. Cells were harvested by centrifugation and either stored at −80° C. or immediately processed.
(38) 1.3 Protein Purification
(39) Fresh or frozen E. coli cells were resuspended on ice in ‘lyse and bind buffer’ (50 mM Na-Phosphate, pH 7.5, 500 mM NaCl, 40 mM Imidazole) and lysed using a ‘French Press’ (G. Heinemann, Schwäbisch Gmand, Germany) cell disrupter. Purification was done at 4° C. using ‘Ni-NTA Superflow’ material (Qiagen, Hilden, Germany). Step elution was done with elution buffer (50 mM Na-Phosphate, pH 7.5, 500 mM NaCl, 200 mM Imidazole). Fractions were analyzed using SDS-PAGE (Invitrogen, Karlsruhe, Germany), pooled and, if required, further purified with anion-ion-exchange chromatography on an ‘AKTA purifier’ system using ‘Q Sepharose fast flow’ material (GE healthcare, Freiburg, Germany). Protein identity was confirmed by MALDI mass spectrometry and correct fractions were pooled, concentrated and re-buffered. Purified protein was stored at −20° C. in a buffer consisting of 25 mM Na-Phosphate, pH 7.5, 250 mM NaCl, 50% glycerol. Protein concentrations were estimated by BCA-protein assay (Pierce/Perbio Science, Bonn, Germany) using a bovine serum albumin (abbr. BSA) standard.
Example 2—Activity Confirmation of Polymerase X and Variants of Polymerase X
(40) The activity assays for the all-L-polymerase X and variants of all-L-polymerase X (see Example 1) were done with different types of substrate complexes formed by oligonucleotides, wherein the substrates and oligonucleotides consists of D-DNA-nucleotides.
(41) 2.1 Activity Assays on Substrates with 1-Nucleotide Gap
(42) List of Oligonucleotides for the 1-Gap Substrates:
(43) TABLE-US-00005 Length, Name nt Sequence (5′.fwdarw.3′) SP-1 15 GATCACAGTGAGTAC D(g1)P 17 Phosphate-GTAAAACGACGGCCAGT MJ_1_140_DD 33 ACTGGCCGTCGTTTTACAGTACTCACTG TGATC MJ_1_141_DD 33 ACTGGCCGTCGTTTTACCGTACTCACTG TGATC MJ_1_142_DD 33 ACTGGCCGTCGTTTTACGGTACTCACTG TGATC SP1c + 18(g1) 33 ACTGGCCGTCGTTTTACTGTACTCACTG TGATC
(44) Substrate complexes were made by annealing a template strand of a DNA oligonucleotide consisting of 33 nucleotides (also referred to as lower strand) with two different DNA oligonucleotides consisting of 15 and 17 nucleotides, respectively, which hybridized to the template strand at its 5′-end and 3′ end, respectively, resulting in a gap of one nucleotide in the upper strand. The complexes contained either A, C, G or T at the template position within the gap. Before annealing, oligonucleotide SP-1 consisting of 15 nucleotides was radioactively labeled at its 5′-end with .sup.32P by a standard kinase reaction employing Gamma-.sup.32P-Adenosine-Triphosphate (ATP) and T4 polynucleotide kinase. Annealing was done in 10 mM Tris-HCl, 5 mM MgCl.sub.2, pH 8.0 by heating 10 min at 65° C. and slowly cooling down. Unincorporated gamma-.sup.32P-ATP was removed by purification over NAP-columns (GE healthcare).
(45) In the activity assay, all-L-polymerase X and the variants thereof were combined with D-configurated 1-gap substrate complexes (see
(46) All-L-polymerase X and the variants I124G, V80A and V80G were active under these conditions and filled the 1 nucleotide gap between the two upper strand DNA oligonucleotides.
(47) 2.2 Activity Assay on a Substrate with 6-Nucleotide Gap
(48) List of Oligonucleotides for the 6-Gap Substrate:
(49) TABLE-US-00006 Length, Name nt Sequence (5′.fwdarw.3′) SP-1 15 GATCACAGTGAGTAC D(g6)P 12 Phosphate-ACGACGGCCAGT SP1c + 18(g6) 33 ACTGGCCGTCGTTCTATTGTACTCACTG TGATC
(50) Substrate complexes were made by annealing a template strand of a DNA oligonucleotide consisting of 33 nucleotides (referred to as lower strand) with two different DNA oligonucleotides consisting of 15 and 12 nucleotides, respectively, which hybridized to the template strand at its 5′-end and 3′ end, respectively, resulting in a gap of six nucleotides in the upper strand. Before annealing, oligonucleotide SP-1 consisting of 15 nucleotides was radioactively labeled at its 5′-end with .sup.32P by a standard kinase reaction employing Gamma-.sup.32P-Adenosine-Triphosphate (ATP) and T4 polynucleotide kinase. Annealing was done in 10 mM Tris-HCl, 5 mM MgCl.sub.2, pH 8.0 by heating 10 min at 65° C. and slowly cooling down. Unincorporated gamma-.sup.32P-ATP was removed by purification over NAP-columns (GE healthcare, Freiburg, Germany).
(51) In the activity assay, all-L-polymerase X and variants thereof were combined with D-configurated 6-gap substrate complex (
(52) All-L-polymerase X and the variants (except C86S) were active under these conditions and filled the 6 nucleotide gap between the two upper strand DNA oligonucleotides.
Example 3—Synthesis of a Variant of Polymerase Pol X Consisting of D-Amino Acids
(53) Within the example the synthesis of the all-D polymerase X variant V80A is described. The amino acid sequence of the all-D polymerase X variant V80A is Ac-MLTLIQGICKIVNHLRSRLAFEYNGQLIKILSKNIVAVGSLRREEKMLNDVDLLIIVPEK KLLKHVLPNIRIKGLSFSVKACGERKCVLFIEWEKKTYQLDLFTALAEEKPYAIFHFTG PVSYLIRIRAALKKKNYKLNQYGLFKNQTLVPLKITTEKELIKELGFTYRIPKKRL-OH.
(54) All amino acids used are protected according to the Solid-phase peptide synthesis Fmoc/tBu-strategy requirements (Eric Atherton et al., 1981). All amino acids used are D-amino acids (Bachem, Bubendorf, Switzerland).
(55) 3.1 Synthesis of HO-Gp(Boc).sub.2
(56) The tert-butyloxycarbonyl-protected 4-guanidinophenol was synthesized in analogy to Sekizaki et al. (Sekizaki et al., 1996). According to this 40 mmole N,N′-Bis-(tert-butyloxycarbonyl)-S-methylisothiourea (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) and 60 mmole 4-aminophenol (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) were dissolved in 250 ml THF in a 500 ml bottom round flask. Following this the solution was argon flushed for 10 min and kept stirring for 120 hours while sealed with a CaCl.sub.2 tube.
(57) After evaporating the solvent the residue was precipitated with ice cold methanol. The precipitate was dried under vacuum over P.sub.4O.sub.10. Finally the product was purified using flash chromatography with DCM. Product containing fractions were combined and the solvent was evaporated under reduced pressure. TLC, reversed phase HPLC, mass spectrometry and NMR were used for analytics. The experimentally determined mass corresponds to the calculated mass of 351 Da.
(58) 3.2 Synthesis of H-
(59) 1 mmole Z-
(60) Z-
3.3 Synthesis of All-D-Peptide AcMLTLIQGKKIVNHLRSRLAFEYNGQLIKILSKNIVAVGSL-OGp (1)
(61) 0.10 mmole TentaGel-R-Trityl resin (Rapp Polymere, Tübingen, Deutschland) was loaded with Fmoc-
(62) The cleavage of the fully protected peptide acid was achieved by incubating the peptidyl resin twice in 10 ml 30% (v/v) HFIP in DCM for 2 hours. After filtering off the peptide the solvent was evaporated and the residue precipitated using ice cold diethyl ether. The precipitated peptide was isolated and dried.
(63) 0.01 mmole fully protected peptide, 4 eq. PyBOP and 5 eq. H-
(64) The final product was characterised by HPLC/UPLC (
(65) 3.4 Synthesis of All-
(66) 0.10 mmole TentaGel-R—NH.sub.2 resin (Rapp Polymere, Tübingen, Deutschland) was loaded with Fmoc-
(67) The final product was characterised by HPLC/UPLC (
(68) 3.5 Synthesis of All-
(69) 0.10 mmole TentaGel-R-PHB resin (Rapp Polymere, Tübingen, Deutschland) was loaded with Fmoc-
(70) The final product was characterised by HPLC/UPLC (
(71) 3.6 Synthesis of All-
(72) Peptide 1 was solved 0.2 mM and peptide 2 was solved 0.6 mM in sodium-phosphate buffer (100 mM, pH 8.5, with 100 mM NaCl) containing 2% Triton X100 (Sigma Aldrich Chemie GmbH, Schnelldorf, Germany). After addition of 20 μM Clostripain (Endoprotease Arg-C, Worthington Biochemical Corporation, Lakewood, N.J., USA) the reaction mixture was shaken overnight at 37° C. The precipitated peptides were centrifuged, solved in H.sub.2O/ACN/Formic Acid 60/40/0.5 and purified by reversed-phase HPLC using a RP-18-column (Phenomenex, Aschaffenburg, Germany) with a gradient of ACN in water of 30% to 60% within 30 min. Product containing fractions were combined and freeze dried.
(73) The final peptide was analyzed by reversed phase UPLC (
(74) 3.7 Synthesis of All-D Polymerase X Variant V80A by Native Chemical Ligation of Peptide 4 with Peptide 3
(75) Both peptides 3 and 4 were solved 0.2 M in TRIS-buffer (pH 8.6) containing 6 M GuanidinHCl (Sigma Aldrich Chemie GmbH, Schnelldorf, Germany), 200 mM mercaptophenyl-acetic acid (Sigma Aldrich Chemie GmbH, Schnelldorf, Germany) and 5 mm Tris(2-carboxyethyl)phosphine hydrochloride (Sigma Aldrich Chemie GmbH, Schnelldorf, Germany). The reaction mixture was shaking 72 h by room temperature. Afterwards the mixture was purified by reversed phase HPLC using a RP-8-column (Phenomenex, Aschaffenburg, Germany) with a gradient of ACN in water of 30% to 60% within 30 min. Fractions which contained the ligation product were pooled and dried. The dry powder was solved in water and purified by size exclusion chromatography using a SEC3000-column (Phenomenex, Aschaffenburg, Germany) with sodium-buffer phosphate (Sigma Aldrich Chemie GmbH, Schnelldorf, Germany) (50 mM, pH 6.8, 0.5% SDS) as eluent. Product containing fractions were combined and freeze dried.
(76) The final product was analyzed by SDS-PAGE (
Example 4—Activity Confirmation of Synthetic Polymerase X Variant Consisting of D-Amino Acids
(77) The dry all-D polymerase X variant V80A according to example 3 was dissolved in 6 M guanidinium hydrochloride and refolded at 4° C. by step-wise dialysis in commercially available dialysis devices (Pierce/PerBio, Bonn, Germany) with 3,500 molecular weight cut-off Final buffer was 50 mM sodium phosphate, 500 mM sodium chloride, pH 7.5. Protein concentration was estimated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) on pre-cast gels (Invitrogen, Karlsruhe, Germany) using a standard series of known protein concentrations followed by SYPRO-RED staining (Invitrogen, Karlsruhe, Germany) and densiometric band analysis on a BioRad Fx scanner instrument.
(78) The activity assay for the all-D polymerase X variant V80A was done with two different substrate types:
(79) 4.1 Activity Assays on Substrates with 1-Nucleotide Gap
(80) Substrates were made by annealing a 33-mer lower strand DNA oligonucleotide with two 17-mer upper strand DNA oligonucleotides resulting in a gap of 1 nucleotide in the upper strand. Oligonucleotides were synthesized in L-configuration. Before annealing, 17-mer upper strand oligonucleotide MJ_1_58_MD was radioactively labeled at its 5′-end with .sup.32P by a standard kinase reaction employing Gamma-.sup.32P-Adenosine-Triphosphate (Gamma-.sup.32P-ATP) and T4 polynucleotide kinase. To facilitate the kinase reaction of the L-oligonucleotide MJ_1_58_MD, two D-configurated guanosine bases were added at the 5′ end during oligonucleotide synthesis. Annealing was done in 10 mM Tris-HCl, 5 mM MgCl.sub.2, pH 8.0 by heating 10 min at 65° C. and slowly cooling down. Unincorporated gamma-32P-ATP was removed by purification over NAP-columns (GE healthcare, Freiburg, Germany). The complexes contained either A, C, G or T at the template position within the gap. For the setup of the substrate complexes see
(81) In the activity assay, synthetic all-D polymerase X variant V80A was combined with L-configurated 1-gap substrate complexes. As a negative control, each substrate was also incubated without all-D polymerase X variant V80A and L-desoxy-nucleotide-triphosphates (dNTP's). Depending on the template base within the 1-gap complex only the corresponding L-dNTP was added during the assay. A typical 6 μl assay contained 50 nM substrate complex, 1.7 ng/μl all-D polymerase X variant V80A, 8 μM of one L-dNTP and buffer (50 mM Tris-HCl, 10 mM MgCl.sub.2, 4% glycerol, 0.1 mg/ml bovine serum albumin (BSA), pH 7.5). L-dNTP's were purchased as custom synthesis from Rasayan, Inc. (Encinitas, Calif., USA). The incubation time was 30 minutes at 37° C. The whole assay volume was mixed with sample buffer/dye, loaded on a denaturing sequencing gel a separated for 4 hours. The gel was exposed to Kodak K screen overnight at −80° C. and read out using BioRad Fx phosphoimager system.
(82) As can be seen from
(83) 4.2 Activity Assay on Substrates with 6-Nucleotide Gap
(84) Substrates were made by annealing a 33-mer lower strand DNA oligonucleotide with two 17-mer and 12-mer upper strand DNA oligonucleotides resulting in a gap of 6 nucleotides in the upper strand. Oligonucleotides were synthesized in L-configuration. Before annealing, 17-mer upper strand oligonucleotide MJ_1_58_MD (L-configuration) was radioactively labeled at its 5′-end with .sup.32P by a standard kinase reaction employing Gamma-.sup.32P-Adenosine-Triphosphate (ATP) and T4 polynucleotide kinase. To facilitate the kinase reaction of the L-oligonucleotide MJ_1_58_MD, two D-configurated guanosine bases were added at the 5′ end during oligonucleotide synthesis. Annealing was done in 10 mM Tris-HCl, 5 mM MgCl.sub.2, pH 8.0 by heating 10 min at 65° C. and slowly cooling down. Unincorporated Gamma-.sup.32P-ATP was removed by purification over NAP-columns (GE healthcare, Freiburg, Germany). For the setup of the substrate complexes see
(85) In the activity assay synthetic all-D polymerase X variant V80A was combined with L-configurated 6-gap substrate complex. As a negative control, the substrate was also incubated without all-D polymerase X variant V80A and desoxy-nucleotide-triphosphates (L-dNTP's). A typical 6 μl assay contained 50 nM substrate complex, up to 1.3 ng/μl all-D polymerase X variant V80A, 8 μM each of the L-dNTP's and buffer (50 mM Tris-HCl, 10 mM MgCl.sub.2, 4% glycerol, 0.1 mg/ml bovine serum albumin (BSA), pH 7.5). L-dNTP's were purchased as custom synthesis by Rasayan, Inc. (Encinitas, Calif., USA). A typical incubation time was 30 minutes at 37° C., but depending on activity of the batch longer incubations were used. The whole assay volume was mixed with sample buffer/dye, loaded on a denaturing sequencing gel a separated for 4 hours. The gel was exposed to Kodak K screen overnight at −80° C. and read out using BioRad Fx phosphoimager system.
(86) As can be seen from
Example 5—DNA Synthesis by Polymerase X and Variants of Polymerase X
(87) Polymerase X from African Swine Fever Virus (abbr. ASFV) is described in literature (Oliveros, 1997) as a highly distributive enzyme with gap-repair function. As shown in example 2 all-L-polymerase X and variants thereof has been shown to catalyze the incorporation of only very few nucleotides after each initiation on gapped substrates. Here we disclose a method which allows using all-L-polymerase X and variants for synthesizing longer DNA and show complete polymerization of a 83-mer strand.
(88) 5.1 Primer-Template Substrate
(89) A primer-template complex has been used to test activity of all-L-polymerase X and variants thereof. The same complex has also been used to test variants V80G and V80A of the all-L-polymerase X.
(90) List of D-Oligonucleotides for the Primer-Template Complex without Gap:
(91) TABLE-US-00007 Length, Config- Name nt uration Sequence (5′.fwdarw.3′) MJ_1_33_DD 19 D Atto532-GGAGCTCAGACTGG CACGC MJ_1_1_DD 83 D GTGGAACCGACAACTTGTGCTG CGTCCAGCATAAGAAAGGAGCT CCCTCAGAAGAAGCTGCGCAGC GTGCCAGTCTGAGCTCC
(92) The substrate was made by annealing a template strand DNA oligonucleotide consisting of 83 nucleotides (MJ_1_1_DD) with a DNA oligonucleotide consisting of 19 nucleotides. Oligonucleotides were synthesized at NOXXON. The oligonucleotide MJ_1_33_DD carries the fluorescent dye Atto-532 (AttoTec, Siegen, Germany). Annealing was done in 10 mM Tris-HCl, 5 mM MgCl2, pH 8.0 by heating 10 mM at 65° C. and slowly cooling down. The primer-template complex is depicted in
(93) 5.2 Reaction at Constant Temperature
(94) In the activity assay, all-L-polymerase X or variants V80G or V80A of all-L-polymerase X were combined with D-configurated primer-template complex. A typical 6 μl assay contained 50 nM substrate complex, up to 1.3 ng/μl all-L-polymerase X or variants V80G or V80A of all-L-polymerase X, 8 μM each of the D-dNTP's and buffer (50 mM Tris-HCl, 10 mM MgCl.sub.2, 4% glycerol, 0.1 mg/ml bovine serum albumin (BSA), pH 7.5). D-dNTP's were purchased from Rovalab (Teltow, Germany). Incubation time was 30 minutes at 37° C. for Pol-X samples. As a negative control, the substrate was also incubated without any all-L-polymerase X or variants V80G or V80A of all-L-polymerase X and without desoxy-nucleotide-triphosphates (D-dNTP's). A positive control was conducted with Taq polymerase (Invitrogen, Karlsruhe, Germany) used at final concentration of 0.083 U/μl in Taq buffer supplied by manufacturer. Taq samples were incubated 30 minutes at 60° C. The whole assay volume was mixed with sample buffer/dye, and separated on a denaturing gel. The gel was read out using BioRad Fx phosphoimager system.
(95) All-L-polymerase X or variants V80G or V80A all-L-polymerase X were active, but were not able to complete the polymerization of the full 83-mer template. The Taq polymerase positive control shows complete polymerization of the 83-mer template, see
(96) 5.3 Reaction Under Thermal Cycling Conditions
(97) Under the assumption that all-L-polymerase X after initiation catalyzes the incorporation of only one nucleotide and then pauses while staying on the DNA substrate, we performed repeated heat pulses (50° C., 2 minutes) in order to allow all-L-polymerase X for dissociation from and reassociation to the template. Using this repeated thermal cycling procedure we were able to perform full polymerization of the 83-mer with all-L-polymerase X. Reactions and controls were set-up as described above for constant temperature, except that the temperature profile for all-L-polymerase X samples was run as follows:
(98) 5 to 25 cycles of (30 minutes at 20° C./2 minutes at 50° C.)
(99) then a final step of 30 minutes at 20° C.
(100) It was observed that from 15 cycles onwards all-L-polymerase X was able to polymerize the full 83-mer template strand, similar to the positive control, see
Example 6—Primer Elongation with a Synthetic Polymerase X Variant Consisting of D-Amino Acids
(101) The method disclosed in this example uses L-configurated substrates for testing all-D configurated polymerase.
(102) 6.1 Primer-Template Substrate
(103) List of L-Oligonucleotides for the Primer-Template Complex without Gap:
(104) TABLE-US-00008 Length, Configu- Name nt ration Sequence (5′.fwdarw.3′) MJ_1_109_MD 21 first D(GG)-L(GGAGCTCAGACTGG two = D, CACGC) others L MJ_1_105_LD 83 L GTGGAACCGACAACTTGTGCTG CGTCCAGCATAAGAAAGGAGCT CCCTCAGAAGAAGCTGCGCAGC GTGCCAGTCTGAGCTCC
(105) The substrate is made by annealing a 83-mer lower strand DNA oligonucleotide with the 19-mer upper strand DNA oligonucleotide. Oligonucleotides are synthesized at NOXXON's in-house facility in L-configuration. Before annealing, 21-mer upper strand oligonucleotide MJ_1_109_MD is radioactively labeled at its 5′-end with .sup.32P by a standard kinase reaction employing Gamma-.sup.32P-Adenosine-Triphosphate (ATP) and T4 polynucleotide kinase. To facilitate the kinase reaction of the L-oligonucleotide MJ_1_109_MD, two D-configurated guanosine bases are added at the 5′ end during oligonucleotide synthesis. Annealing is done in 10 mM Tris-HCl, 5 mM MgCl.sub.2, pH 8.0 by heating 10 min at 65° C. and slowly cooling down. Unincorporated Gamma-.sup.32P-ATP is removed by purification over NAP-columns (GE healthcare, Freiburg, Germany).
(106) 6.2 Reaction at Constant Temperature
(107) In the activity assay, synthetic all-D polymerase X variant V80A is combined with L-configurated primer-template complex. A typical 6 μl assay contains 50 nM substrate complex, up to 1.3 ng/μl all-D polymerase X variant, 8 μM each of the L-dNTP's and buffer (50 mM Tris-HCl, 10 mM MgCl.sub.2, 4% glycerol, 0.1 mg/ml bovine serum albumin (BSA), pH 7.5). L-dNTP's were purchased from Rasayan, Inc. (Encinitas, Calif., USA). Incubation time is at least 30 minutes at 37° C. As a negative control, the substrate is also incubated without any polymerase and without desoxy-nucleotide-triphosphates (L-dNTP's). The whole assay volume is mixed with sample buffer/dye, and separated on a denaturing gel. The gel is read out using BioRad Fx phosphoimager system.
(108) The synthetic all-D polymerase X variant V80A is active under this condition, but—similar to the all-L polymerase X counterpart—is not able to polymerize the full 83 nucleotide template strand.
(109) 6.3 Reaction Under Thermal Cycling Conditions
(110) Analog to example 5 a repeated thermal cycling procedure is used to allow for full polymerization of the 83-mer L-substrate with all-D Polymerase X variant V80A. Reactions and controls are set-up as described above for constant temperature, except that the temperature profile was run as follows:
(111) 5 to 25 cycles of (30 minutes at 20° C./2 minutes at 50° C.)
(112) then a final step of 30 minutes at 20° C.
(113) It is observed that all-D polymerase X variant V80A—similar to the all-L polymerase X counterpart—is able to polymerize the full 83-mer template strand when using thermal cycle elongation.
Example 7—Recombinant Expression and Purification of Polymerase Dpo4 and Variants of Polymerase Dpo4, all Consisting of L-Amino Acids
(114) The polymerase Dpo4 was originally discovered in Sulfolobus Solfataricus (abbr. Sso) (Boudsocq, 2001). The wild-type gene has an open reading frame (abbr. ORF) of 1,059 base pairs including start codon and stop codon. The encoded protein has a length of 352 amino acids. This example describes how polymerase Dpo4 and variants of polymerase Dpo4 have been expressed in E. coli and been purified using Strep-Tag.
(115) 7.1 Expression Constructs
(116) Since the codon usage of Sso differs from E. coli, an E. coli-codon-optimized synthetic gene for wild-type Sso polymerase Dpo4 was purchased from GeneArt AG (Regensburg, Germany). The synthetic gene sequence was provided in pENTRY-IBA10 vector (originator company: IBA GmbH, Göttingen, Germany). The codon-optimized open reading frame including start codon, but not including stop codon had the following sequence:
(117) TABLE-US-00009 ATGATTGTGCTGTTTGTGGATTTTGATTATTTTTATGCCCAGGTGGAAGA AGTTCTGAATCCGAGCCTGAAAGGTAAACCGGTTGTTGTTTGTGTTTTTA GCGGTCGCTTTGAAGATAGCGGTGCAGTTGCAACCGCCAATTATGAAGCC CGTAAATTTGGTGTTAAAGCCGGTATTCCGATTGTTGAAGCCAAAAAAAT TCTGCCGAATGCAGTTTATCTGCCGATGCGCAAAGAAGTTTATCAGCAGG TTAGCAGCCGTATTATGAATCTGCTGCGCGAATATAGCGAAAAAATTGAA ATTGCCAGCATTGATGAAGCCTATCTGGATATTAGCGATAAAGTGCGCGA TTATCGCGAAGCATATAATCTGGGCCTGGAAATTAAAAATAAAATCCTGG AAAAAGAAAAAATTACCGTGACCGTGGGCATTAGCAAAAATAAAGTGTTT GCCAAAATTGCAGCAGATATGGCAAAACCGAATGGCATTAAAGTGATTGA TGATGAAGAAGTGAAACGTCTGATTCGCGAACTGGATATTGCAGATGTTC CGGGTATTGGCAATATTACCGCAGAAAAACTGAAAAAACTGGGCATTAAT AAACTGGTTGATACCCTGAGCATTGAATTTGATAAACTGAAAGGCATGAT TGGTGAAGCGAAAGCCAAATATCTGATTAGCCTGGCACGTGATGAATATA ATGAACCGATTCGTACCCGTGTTCGTAAAAGCATTGGTCGTATTGTGACC ATGAAACGCAATAGCCGTAATCTGGAAGAAATTAAACCGTACCTGTTTCG TGCAATTGAAGAAAGCTATTATAAACTGGATAAACGCATTCCGAAAGCCA TTCATGTTGTTGCAGTTACCGAAGATCTGGATATTGTTAGCCGTGGTCGT ACCTTTCCGCATGGTATTAGCAAAGAAACCGCCTATAGCGAAAGCGTTAA ACTGCTGCAGAAAATCCTGGAAGAAGATGAACGTAAAATTCGTCGTATTG GTGTGCGCTTTAGCAAATTTATTGAAGCCATTGGCCTGGATAAATTTTTT GATACC.
(118) In order to obtain the expression construct for polymerase Dpo4, also referred to as all-L-polymerase Dpo4, the gene was subcloned from pENTRY-IBA10 into the pASG-IBA5 vector (IBA GmbH, Göttingen, Germany), using a commercially available StarGate cloning kit (IBA GmbH). Subcloning added a Strep-Tag II to the N-terminus and a stop codon to the C-terminus, and brought the gene under control of the tet promoter. The construct was named pMJ343 and was used for expression of all-L-polymerase Dpo4 in E. coli. The all-L-polymerase Dpo4 expressed from pMJ343 had the following sequence of 368 amino acids:
(119) TABLE-US-00010 MASAWSHPQFEKSGMIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGR FEDSGAVATANYEARKFGVKAGIPIVEAKKILPNAVYLPMRKEVYQQVSS RIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKE KITVTVGISKNKVFAKIAADMAKPNGIKVIDDEEVKRLIRELDIADVPGI GNITAEKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEP IRTRVRKSIGRIVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHV VAVTEDLDIVSRGRTFPHGISKETAYSESVKLLQKILEEDERKIRRIGVR FSKFIEAIGLDKFFDTGS.
(120) The initial 14 amino acids represented the Strep-Tag II including a few spacer amino acids, the final 2 amino acids represented spacer amino acids, and the middle 352 amino acid part was identical to the polymerase Dpo4 sequence as found in Sso.
(121) Expression constructs for variants of all-L-polymerase Dpo4 were made using the commercially available QuikChange kit (Stratagene GmbH, Waldbronn, Germany) according to manufacturer's protocol. Plasmid pMJ343 served as template. Oligonucleotides needed for QuikChange were either synthesized at NOXXON (QC_38_up, QC_38_low, QC_39_up, QC_39_low, QC_40_up, QC_40_low) or purchased from Purimex (Grebenstein, Germany) (QC_28_up, QC_28_low, QC_29_up, QC_29_low, QC_30_up, QC_30_low).
(122) The following variant expression constructs were made and used for expression of the variants of all-L-polymerase Dpo4 in E. coli
(123) TABLE-US-00011 oligonucleotides used for variant construct QuikChange mutagenesis procedure A155C pMJ361 QC_28_up. (5′CAAAAATAAAGTGTTTGCCAAAATTGCAT GCGATATGGCAAAACCGAATGGCATTAAAG 3′) QC_28_low (5′CTTTAATGCCATTCGGTTTTGCCATATCG CATGCAATTTTGGCAAACACTTTATTTTTG 3′) V203C pMJ362 QC_29_up (5′TGAAAAAACTGGGCATTAATAAACTGTGT GATACCCTGAGCATTGAATTTG 3′) QC_29_low (5′CAAATTCAATGCTCAGGGTATCACACAG TTTATTAATGCCCAGTTTTTTCA 3′) C31S pMJ363 QC_30_up (5′TGAAAGGTAAACCGGTTGTTGTTTCTGT TTTTAGCGGTC 3′) QC_30_low (5′GACCGCTAAAAACAGAAACAACAACCGG TTTACCTTTCA 3′) A155C + pMJ365 QC_28_up V203C QC_28_low QC_29_up QC_29_low S85C pMJ502 QC_38_up (5′ATGCGCAAAGAAGTTTATCAGCAGGTTTG TAGCCGTATTATGAATC 3′) QC_38_low (5′GATTCATAATACGGCTACAAACCTGCTG ATAAACTTCTTTGCGCAT-3′) S86C pMJ503 QC_39_up (5′AAGTTTATCAGCAGGTTAGCTGTCGTATT ATGAATCTGCTGCG 3′) QC_39_low (5′CGCAGCAGATTCATAATACGACAGCTAAC CTGCTGATAAACTT 3′) S96C pMJ504 QC_40_up (5′ATTATGAATCTGCTGCGCGAATATTGTGA AATAAATGAAATTGCCAGCATT 3′) QC_40_low (5′AATGCTGGCAATTTCAATTTTTTCACAAT ATTCGCGCAGCAGATTCATAAT 3′)
7.2 Protein expression in E. coli
(124) All-L-polymerase Dpo4 was expressed in E. coli using expression construct pMJ343. Mutant variants of all-L-polymerase Dpo4 were expressed from pMJ361, pMJ362, pMJ363, pMJ365, pMJ502, pMJ503 or pMJ504. For expression, the appropriate expression construct was transformed in E. coli strain ‘NEB express’ (New England Biolabs, Frankfurt am Main, Germany) using ‘Transformation and Storage Solution’ (Epicentre/Biozym, Hessisch Oldendorf, Germany) and maintained with the antibiotic Ampicillin. Expression was done in medium ‘EnBase Flo’ or ‘EnPresso’ (BioSilta, Oulu, Finland) for 48 h at 30° C. using 200 ng/ml Anhydrotetracyclin (IBA GmbH, Göttingen, Germany) as inducer. Cells were harvested by centrifugation and either stored at −80° C. or immediately processed.
(125) 7.3 Protein Purification
(126) Fresh or frozen E. coli cells were resuspended on ice in ‘Buffer W’ (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) and lysed using a ‘French Press’ (G. Heinemann, Schwäbisch Gmünd, Germany) cell disrupter. Purification was done at 4° C. on an ‘AKTA Express’ system equipped with 5 ml StrepTrap HP columns (GE healthcare, Freiburg, Germany). Step elution was done with Buffer W including 2.5 mM Desthiobiotin (IBA GmbH, Göttingen, Germany). Fractions were analyzed using SDS-PAGE (Invitrogen, Karlsruhe, Germany), pooled and, if required, further purified with anion-ion-exchange chromatography on an ‘AKTA purifier’ system equipped with ‘Q HP’ columns (GE healthcare, Freiburg, Germany). Protein identity was confirmed by LC-MS mass spectrometry and correct fractions were pooled, concentrated and re-buffered using VivaSpin 15R concentration devices with 10,000 molecular weight cut-off (MWCO) (VivaSciences/Sartorius Stedim Biotech, Göttingen, Germany). Purified protein was stored at −20° C. in a buffer consisting of 100 mM KCl, 10 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 1 mM DTT, 50% glycerol. Protein concentrations were estimated by gel-densiometry using a bovine serum albumin (abbr. BSA) standard on SDS-PAGE and staining with SYPRO Red (Invitrogen, Karlsruhe, Germany).
Example 8—Production of a Synthetic Polymerase Dpo4 Consisting of Two Fragments
(127) All-L polymerase Dpo4 has a length of 352 amino acids. In order to chemically produce the all-L polymerase Dpo4, such a synthetic all-L polymerase Dpo4 had to be assembled from shorter fragments that can be synthesized by solid-phase peptide synthesis, wherein said shorter fragments had to be ligated by a peptide ligation method such as the native chemical ligation. This example describes how ligation fragments 1-154, 155-352, 155-202 and 203-352 of Dpo4 have been expressed in E. coli, purified and ligated to each other by native chemical ligation to yield the synthetic all-L-polymerase Dpo4.
(128) 8.1 Expression Constructs
(129) As disclosed in example 7 the gene for all-L-polymerase Dpo4 has been obtained as a synthetic gene construct from a commercial source (GeneArt, Regensburg, Germany). All fragments of this example were cloned based on that codon-optimized sequence. The following expression constructs for fragments 1-154, 155-352, 155-202 and 203-352 of the all-L-polymerase Dpo4 variant A155C were made:
(130) TABLE-US-00012 Fragment of Dpo4 (amino acid range) expression construct 1-154-thioester pMJ370 155-352 contained A155C mutation pMJ384 203-352 contained A155C mutation pMJ385 155-202 thioester pMJ388
8.1.1 Fragment 1-154-Thioester of All-L-Polymerase Dpo4 Variant A155C—Expression Construct pMJ370
(131) This construct contains the fragment 1-154 of the all-L-polymerase Dpo4 variant A155C followed by an Mxe GyrA intein, which was used to produce the thioester, and a chitin-binding domain (CBD). The construct was assembled from two PCR products. PCR product 1 was made using pMJ343 as a template and primers MJ_1_90_DD (5′-Phosphate-AGCGGCTCTTCGATGATTGTGCTGTTTGTGGATTTT-3′) and MJ_1_91_DD (5′-Phosphate-AGCGGCTCTTCGGCATGCAATTTTGGCAAACACTTT-3′) to amplify the fragment 1-154 of all-L-polymerase Dpo4 variant A155C. PCR product 2 was made using pTWIN1 (New England Biolabs, Frankfurt am Main, Germany) as a template and primers MJ_1_72_DD (5′-Phosphate-AGCGGCTCTTCGTGCATCACGGGAGAT-3′) and MJ_1_73_DD (5′-Phosphate-AGCGGCTCTTCGCCCTTGAAGCTGCCACAAGGCAGGAACGTT-3′) to amplify the Mxe Gyr A intein and the CBD. Primers MJ_1_90_DD and MJ_1_91_DD were from Purimex (Grebenstein, Germany) while primers MJ_1_72_DD and MJ_1_73_DD were from IBA GmbH (Göttingen, Germany). The two PCR products were gel-purified on a flash-gel system (LONZA, Basel, Switzerland) and cloned together in pENTRY-IBA20 using the StarGate Mutagenesis ENTRY cloning kit (IBA GmbH, Göttingen, Germany) to result in pMJ366. Subcloning from pMJ366 in pASG-IBAwt1 (IBA GmbH, Göttingen, Germany) using the StarGate Transfer cloning kit (IBA GmbH, Göttingen, Germany) yielded pMJ370. The construct pMJ370 encodes the fragment 1-154-thioester of all-L-polymerase Dpo4 variant A155C with the following protein sequence of 154 amino acids length (after intein cleavage/thioester production):
(132) TABLE-US-00013 MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEA RKFGVKAGIPIVEAKKILPNAVYLPMRKEVYQQVSSRIMNLLREYSEKIE IASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVF AKIA-thioester.
8.1.2 Fragment 155-352 of all-L-Polymerase Dpo4 Variant A155C—Expression Construct pMJ384
(133) This construct contains a ‘Profinity eXact’ tag followed by the fragment 155-352 of all-L-polymerase Dpo4 variant A155C. The ‘Profinity eXact’ tag was used for purification and proteolytic cleavage. The construct was assembled from two PCR products. PCR product 1 was made using pPAL7 (Bio-Rad, München, Germany) as a template and primers MJ_1_99_DD (5′-Phosphate-AGCGGCTCTTCGATGGGAGGGAAATCAAACGGGGAA-3′) and MJ_1_100_DD (5′-Phosphate-AGCGGCTCTTCGGCACAAAGCTTTGAAGAGCTTGTC-3′) to amplify the ‘Profinity eXact’ tag. PCR product 2 was made using pMJ361 as a template and primers MJ_1_96_DD (5′-Phosphate-AGCGGCTCTTCGTGCGATATGGCAAAACCGAATGGCATTAAA-3′) and MJ_1_97_DD (5′-Phosphate-AGCGGCTCTTCGCCCTTAGGTATCAAAAAATTTATCCAGG-3′) to amplify the dpo4 fragment 155-352 containing the A155C mutation. Primers MJ_1_96_DD, MJ_1_97_DD, MJ_1_99_DD and MJ_1_100_DD were all from Purimex (Grebenstein, Germany). The 2 PCR products were gel-purified on a flash-gel system (LONZA, Basel, Switzerland) and cloned together in pENTRY-IBA20 using the StarGate Mutagenesis ENTRY cloning kit (IBA GmbH, Göttingen, Germany) to result in pMJ382. Subcloning from pMJ382 in pASG-IBA5 (IBA GmbH, Göttingen, Germany) using the StarGate Transfer cloning kit (IBA GmbH, Göttingen, Germany) yielded pMJ384. The construct pMJ384 encodes the fragment 155-352 of all-L-polymerase Dpo4 containing mutation A155C with the following protein sequence of 198 amino acids length (after proteolytic cleavage):
(134) TABLE-US-00014 DMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKLVD TLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRN SRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRGRTFPH GISKETAYSESVKLLQKILEEDERKIRRIGVRFSKFIEAIGLDKFFDT.
8.1.3 Fragment 203-352 of all-L-Polymerase Dpo4 Variant A155C—Expression Construct pMJ385
(135) This construct contains a ‘Profinity eXact’ tag followed by the dpo4 fragment 203-352 containing mutation V203C. The ‘Profinity eXact’ tag was used for purification and proteolytic cleavage. The construct was assembled from two PCR products. PCR product 1 was made using pPAL7 (Bio-Rad, München, Germany) as a template and primers MJ_1_99_DD (5′-Phosphate-AGCGGCTCTTCGATGGGAGGGAAATCAAACGGGGAA-3′) and MJ_1_100_DD (5′-Phosphate-AGCGGCTCTTCGGCACAAAGCTTTGAAGAGCTTGTC-3′) to amplify the ‘Profinity eXact’ tag. PCR product 2 was made using pMJ362 as a template and primers MJ_1_98_DD (5′-Phosphate-AGCGGCTCTTCGTGTGATACCCTGAGCATTGAATTT-3′) and MJ_1_97_DD (5′-Phosphate-AGCGGCTCTTCGCCCTTAGGTATCAAAAAATTTATCCAGG-3′) to amplify the dpo4 fragment 203-352 containing the V203C mutation. Primers MJ_1_97_DD, MJ_1_98_DD, MJ_1_99_DD and MJ_1_100_DD were all from Purimex (Grebenstein, Germany). The 2 PCR products were gel-purified on a flash-gel system (LONZA, Basel, Switzerland) and cloned together in pENTRY-IBA20 using the StarGate Mutagenesis ENTRY cloning kit (IBA GmbH, Göttingen, Germany) to result in pMJ383. Subcloning from pMJ383 in pASG-IBA5 (IBA GmbH, Göttingen, Germany) using the StarGate Transfer cloning kit (IBA GmbH, Göttingen, Germany) yielded pMJ385. The construct pMJ385 encodes the dpo4 fragment 203-352 V203C with the following protein sequence of 150 amino acids length (after proteolytic cleavage):
(136) TABLE-US-00015 DTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMK RNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRGRTF PHGISKETAYSESVKLLQKILEEDERKIRRIGVRFSKFIEAIGLDKFFDT
8.1.4 Fragment 155-202 of all-L-Polymerase Dpo4—Expression Construct pMJ388
(137) This construct contains the dpo4 fragment 155-202 followed by an Mxe GyrA intein, which was used to produce the thioester, and a chitin-binding domain (CBD). The construct was assembled from two PCR products. PCR product 1 was made using pMJ343 as a template and primers MJ_1_101_DD (5′-Phosphate-AGCGGCTCTTCGATGGCAGATATGGCAAAACCGAAT-3′) and MJ_1_102_DD (5′-Phosphate-AGCGGCTCTTCGGCACAGTTTATTAATGCCCAGTTT-3′) to amplify the dpo4 155-202 fragment. PCR product 2 was made using pTWIN1 (New England Biolabs, Frankfurt am Main, Germany) as a template and primers MJ_1_72_DD (5′-Phosphate-AGCGGCTCTTCGTGCATCACGGGAGAT-3′) and MJ_1_73_DD (5′-Phosphate-AGCGGCTCTTCGCCCTTGAAGCTGCCACAAGGCAGGAACGTT-3′) to amplify the Mxe Gyr A intein and the CBD. Primers MJ_1_101_DD and MJ_1_102_DD were from Purimex (Grebenstein, Germany) while primers MJ_1_72_DD and MJ_1_73_DD were from IBA GmbH (Göttingen, Germany). The 2 PCR products were gel-purified on a flash-gel system (LONZA, Basel, Switzerland) and cloned together in pENTRY-IBA20 using the StarGate Mutagenesis ENTRY cloning kit (IBA GmbH, Göttingen, Germany) to result in pMJ386. Subcloning from pMJ386 in pASG-IBAwt1 (IBA GmbH, Göttingen, Germany) using the StarGate Transfer cloning kit (IBA GmbH, Göttingen, Germany) yielded pMJ388. The construct pMJ388 encodes the dpo4 fragment 155-202 with the following protein sequence of 48 amino acids length (after E. coli mediated cleavage of the initial Methionine and after intein cleavage/thioester production):
(138) TABLE-US-00016 ADMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKL- thioester
8.2 Protein Expression in E. coli
(139) For expression, the appropriate expression construct was transformed in E. coli strain ‘NEB express’ (New England Biolabs, Frankfurt am Main, Germany) using ‘Transformation and Storage Solution’ (Epicentre/Biozym, Hessisch Oldendorf, Germany) and maintained with the antibiotic Ampicillin. Expression was done in medium ‘EnBase Flo’ or ‘EnPresso’ (BioSilta, Oulu, Finland) at ambient temperature, using 200 ng/ml Anhydrotetracyclin (IBA GmbH, Göttingen, Germany) as inducer during an overnight period. Cells were harvested by centrifugation and either stored at −80° C. or immediately processed.
(140) 8.3 Purification and Generation of a Thioester from Constructs pMJ370 and pMJ388 with Mxe Gyr A Intein
(141) Fresh or frozen E. coli cells were resuspended on ice in ‘column buffer’ (20 mM HEPES, pH 8.5, 500 mM NaCl) and lysed using a ‘French Press’ (G. Heinemann, Schwäbisch Gmünd, Germany) cell disrupter. Purification was done at 4° C. on an ‘AKTA Express’ system (GE healthcare, Freiburg, Germany) equipped with columns containing chitin binding beads (New Englands Biolabs, Frankfurt am Main, Germany). After applying cell lysate and washing with column buffer until baseline, the columns were incubated 20 hours at 4° C. with 50 mM 2-mercaptoethane sulfonate (abbr. MESNA) in column buffer to induce intein-mediated protein cleavage and thioester formation. Cleaved protein carrying thioester was washed out of the column with column buffer, concentrated and subjected to gelfiltration using BioGel P60 medium material (BioRad, München, Germany) in a buffer consisting of 5 mM Bis-Tris, pH 6.5, 250 mM NaCl. Protein concentrations were estimated by gel-densiometry using a bovine serum albumin (BSA) standard on SDS-PAGE and staining with SYPRO Red (Invitrogen, Karlsruhe, Germany). Protein identity and the presence of the thioester were confirmed by LC-MS mass spectrometry.
(142) 8.4 Purification and Proteolytic Cleavage from Construct pMJ384 with ‘Profinity eXact’ Tag
(143) Purification of fragment 155-352 of the all-L-polymerase Dpo4 variant A155C from pMJ384 was done as follows: Fresh or frozen E. coli cells were resuspended on ice in ‘Buffer W’ (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) and lysed using a ‘French Press’ (G. Heinemann, Schwäbisch Gmünd, Germany) cell disrupter. Purification was done at 4° C. on an ‘ÄKTA Express’ system equipped with 5 ml StrepTrap HP columns (GE healthcare, Freiburg, Germany). Step elution was done with Buffer W including 2.5 mM Desthiobiotin (IBA GmbH, Göttingen, Germany). Eluted protein was subjected to buffer exchange in ‘Profinity eXact elution buffer’ (0.1 M Na-phosphate, pH 7.2, 0.1 M NaF) using a HiPrep 26/10 desalting column (GE healthcare, Freiburg, Germany) and then slowly pumped through a Profinity eXact column. The sample was concentrated, supplemented with Tris(2-carboxyethyl)phosphine) (TCEP) to 1 mM final concentration and applied to gelfiltration using a HiLoad 16/60 Superdex 75 prep grade column (GE healthcare, Freiburg, Germany) developed in a buffer consisting of 5 mM Bis-Tris, pH 6.5, 250 mM NaCl. Protein was concentrated and stored at −80° C. Protein concentrations were estimated by gel-densiometry using a bovine serum albumin (BSA) standard on SDS-PAGE and staining with SYPRO Red (Invitrogen, Karlsruhe, Germany). Protein identity was confirmed by LC-MS mass spectrometry.
(144) 8.5 Purification and Proteolytic Cleavage from Construct pMJ385 with ‘Profinity eXact’ Tag
(145) Purification of dpo4 fragment 203-352 V203C from pMJ385 was done as follows: Inclusion bodies were prepared and denatured as described in the ‘i-FOLD Protein refolding system’ manual (Novagen/Merck-Millipore, Darmstadt, Germany). Solubilized protein was subjected to buffer exchange into ‘Buffer W’ (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) using Sephadex G-25 fine material (GE healthcare, Freiburg, Germany) and purified at 4° C. on an ‘AKTA Express’ system equipped with StrepTrap HP columns (GE healthcare, Freiburg, Germany). Step elution was done with Buffer W including 2.5 mM Desthiobiotin (IBA GmbH, Göttingen, Germany). Eluted protein solution was supplemented with NaF to a final concentration of 0.1 M and Tris(2-carboxyethyl)phosphine) (TCEP) to a final concentration of 1 mM and then slowly pumped through a Profinity eXact column. Flowthrough was diluted 1:3 with deionized water and pH adjusted to 7.2 using HCl. The sample was further purified by cation-exchange-chromatography using HiTrap SP HP columns (GE healthcare, Freiburg, Germany) equilibrated in ‘Buffer A’ (50 mM Na-Phosphate, pH 7.2, 1 mM 2-mercaptoethanol). Step elution was done using 17%, 25% and 100% of ‘Buffer B’ (50 mM Na-Phosphate, pH 7.2, 1 M NaCl, 1 mM 2-mercaptoethanol). Fractions were pooled, concentrated and applied to gelfiltration using a HiLoad 16/60 Superdex 75 prep grade column (GE healthcare, Freiburg, Germany) developed in deionized water. Protein was shock frozen in liquid nitrogen and lyophilized. Protein concentrations were estimated by gel-densiometry using a bovine serum albumin (BSA) standard on SDS-PAGE and staining with SYPRO Red (Invitrogen, Karlsruhe, Germany). Protein identity was confirmed by LC-MS mass spectrometry.
(146) 8.6 Synthesis of Synthetic All-L-Polymerase Dpo4 Variant A155C by Native Chemical Ligation of the Fragments 1-154-Thioester and 155-352
(147) The fragments 1-154-thioester and 155-352 of all-L-polymerase Dpo4 variant A155C were solved 50 μM in TRIS-buffer (pH 8.6) containing 2% Triton X100, 1% thiophenol and 5 mM tris(2-carboxyethyl)phosphine hydrochloride. The reaction mixture was shaken 72 h by room temperature. Afterwards the ligation success was analyzed by SDS-PAGE (
(148) 8.7 Synthesis of Fragment 155-352 by Native Chemical Ligation of the Fragments 155-202-Thioester and 203-352
(149) The fragments 155-202-thioester and 203-352 V203C of all-L-polymerase Dpo4 were solved 0.2 M in TRIS-buffer (pH 8.6) containing 2% SDS, 1% thiophenol and 5 mM tris(2-carboxyethyl)phosphine hydrochloride. The reaction mixture was shaken 72 h by room temperature. Analysis of the ligation success was performed by SDS-PAGE (
Example 9—Activity Confirmation of Polymerase Dpo4 and Variants of Polymerase Dpo4 Consisting of L-Amino Acids
(150) This example describes a PCR activity test for all-L-polymerase Dpo4 and variants of all-L-polymerase Dpo4 according to example 7 and for the synthetic all-L-polymerase Dpo4 according to example 8.
(151) 9.1 Templates for PCR Activity Assay
(152) Template for the PCR reaction is a 83-mer single-stranded D-DNA oligonucleotide (MJ_1_1_DD) from which, in the first thermal cycle, the opposite strand is made. Thereafter both strands serve as template for exponential amplification. Template DNA oligonucleotide and DNA primers are synthesized at NOXXON in D-configuration.
(153) List of Oligonucleotides for the PCR Activity Assay:
(154) TABLE-US-00017 Length, Name nt Sequence (5′.fwdarw.3′) MJ_1_1_DD 83 GTGGAACCGACAACTTGTGCTGCGTCCAGCAT AAGAAAGGAGCTCCCTCAGAAGAAGCTGCGCA GCGTGCCAGTCTGAGCTCC DE4.40T7 38 TCTAATACGACTCACTATAGGAGCTCAGACTG GCACGC DE4.40R 20 GTGGAACCGACAACTTGTGC
9.2 PCR Reactions
(155) 15 μl PCR reactions contained 0.2 mM each of the four D-dNTP's, 10 nM 83-mer ssDNA (MJ_1_1_DD) template, 1 μM of forward and reverse primer, 1× ThermoPol buffer (Invitrogen, 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 2 mM MgSO.sub.4, 0.1% Triton X-100, pH 8.8@25° C.) and at least 0.67 ng/μl all-L-polymerase Dpo4 or a variant of all-L-polymerase Dpo4 or the synthetic all-L-polymerase Dpo4. The forward primer is DE4.40T7, the reverse primer is DE4.40R, yielding a PCR product of 102 base pairs length. The D-dNTP's were purchased from Rovalab (Teltow, Germany). Negative controls were conducted by omitting all-L-polymerase Dpo4 or a variant of all-L-polymerase Dpo4 or the synthetic all-L-polymerase Dpo4. Positive controls were conducted using commercially available all-L-dpo4 (New England Biolabs, Frankfurt am Main, Germany).
(156) The thermal cycling program consisted of 1 cycle (85° C., 3 min) then at least 7 cycles (85° C., 30 sec/56° C., 1 min/60° C., 4 min) then hold at 4° C. 4 μl aliquots of the PCR reactions were mixed with sample loading buffer and analyzed on TBE-PAGE or on agarose gels (LONZA, Cologne, Germany). A DNA standard ladder containing, among others, a 100 bp band was applied on the gel.
(157) 9.3 Activity Confirmation
(158) All-L-polymerase Dpo4, the variants A155C, V203C, C31S, A155C/V203C, S85C, S86C, S96C of all-L-polymerase Dpo4 and the synthetic all-L-polymerase Dpo4 were tested. All tested polymerases were able to amplify the template strand in the PCR reaction.
Example 10—Synthesis of a Variant of Polymerase Dpo4 Consisting of D-Amino Acids
(159) Within the example the synthesis of the all-D polymerase Dpo variant A155C/V203C is described. The amino acid sequence of the all-D polymerase Dpo variant A155C/V203C is Ac-MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFG VKAGIPIVEAKKILPNAVYLPMRKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDK VRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAKIACDMAKPNGIKVIDDEEVK RLIRELDIADVPGIGNITAEKLKKLGINKLCDTLSIEFDKLKGMIGEAKAKYLISLARDE YNEPIRTRVRICSIGRIVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTED LDIVSRGRTFPHGISKETAYSESVKLLQKILEEDERKIRRIGVRFSKFIEAIGLDKFFDT-OH.
(160) All amino acids used are protected according to the Solid-phase peptide synthesis Fmoc/tBu-strategy requirements (Eric Atherton et al, 1981). All amino acids used are D-amino acids (Bachem, Bubendorf, Switzerland).
(161) 10.1 Synthesis of H-
(162) 1 mmole Z-
(163) Z-
(164) 10.2 Synthesis of Fully Protected all-D-Peptide H-RTFPHGISKETAYSESVKLLOKILEEDERKIRRIGVRFSKFIEAIGLDKFFDT-NH.sub.2 (1)
(165) 0.1 mmole Fmoc-Sieber rink amide NovaSynTG resin was loaded after Fmoc-deprotection with Fmoc-
(166) Automated synthesis was done using an ABI 433 with the FASTmoc protocol. 10 eq. amino acid were activated using 9 eq. HATU and 20 eq. DIPEA in NMP. Coupling time was 45 min and Fmoc-deprotection was performed three times for 7 min with 20% (v/v) piperidine in NMP. Double coupling was performed after coupling of 42 amino acids.
(167) The cleavage of the fully protected peptide acid was achieved by incubating the peptidyl resin twice in 10 ml 1% (v/v) TFA in DCM for 2 hours. After filtering off the peptide the solvent was evaporated and the residue precipitated using ice cold diethyl ether. The precipitated peptide was isolated and dried.
(168) The final product was characterised by HPLC and mass spectrometry (
(169) 10.3 Synthesis of Fully Protected all-D-Peptide Boc-VDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSRNLEEIK PYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRG-OH (2)
(170) 0.10 mmole TentaGel-R-Trityl resin was loaded with Fmoc-
(171) The cleavage of the fully protected peptide acid was achieved by incubating the peptidyl resin twice in 10 ml 30% (v/v) HFIP in DCM for 2 hours. After filtering off the peptide the solvent was evaporated and the residue precipitated using ice cold diethyl ether. The precipitated peptide was isolated and dried. The final product was characterised by HPLC and mass spectrometry (
(172) 10.4 Synthesis of All-D-Peptide H-VDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSRNLEE IKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRGRTFPHGISKETAYSESVKLLQ KILEEDERKIRRIGVRFSKFIEAIGLDKFFDT-NH.sub.2 (3) by Fragment Condensation of Peptide 1 with Peptide 2.
(173) 5 μmole (2) and 1 eq. (1) were dissolved in 25% (v/v) TFE in DCM. After addition of 5 eq. PyBOP and 10 eq. DIPEA the mixture was stirred overnight. After evaporating the solvent the peptide was precipitated using ice cold diethyl ether and filtered off.
(174) The cleavage of the side chain protection groups of the peptide was performed with 2.5% EDT, 2.5% water, 2.5% TIS in TFA for 2 hours. Following the evaporation of TFA the peptide was precipitated with ice cold diethyl ether. Reversed phase HPLC purification of the N-terminal Fmoc-protected peptide was performed on a C18 column using an ACN/water gradient. Fractions that contain product were combined and freeze dried.
(175) The final product was characterised by HPLC and mass spectrometry (
(176) 10.5 Synthesis of All-D-Peptide Z-CDMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKL-benzyl-thioester (4)
(177) 0.10 mmole TentaGel-R-Trityl resin was loaded with Fmoc-D-Leu-OH as described in Barlos et al. (Barlos et al., 1989). Therefore 0.10 mmol resin was incubated twice for 30 min with 0.6 mmole thionylchloride and subsequently washed with DCM. Following this the resin was incubated 90 min with 0.6 mmole Fmoc-
(178) The cleavage of the fully protected peptide acid was achieved by incubating the peptidyl resin twice in 10 ml 30% (v/v) HFIP in DCM for 2 hours. After filtering off the peptide the solvent was evaporated and the residue precipitated using ice cold diethyl ether. The precipitated peptide was isolated and dried.
(179) The N-terminal Z— and completely side chain protected peptide 4 was solved 1 mM in DMF. After addition of 5 eq. PyBOP, 10 eq. DIPEA and 30 eq. benzyl mercaptan the mixture was stirred for 4 h. Then the DMF was evaporated, the peptide was precipitated and washed with ice-cold diethyl ether. The side chain protecting groups were removed by treatment with 2.5% EDT, 2.5% water, 2.5% TIS in TFA for 2 hours. After the evaporation of TFA the peptide was precipitated and washed with ice cold diethyl ether. The peptide-benzyl-thioester was then purified by reversed-phase HPLC and analyzed by reversed-phase HPLC (
(180) 10.6 Synthesis of All-D-Peptide H-RKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILE KEKITVTVGISKNKVFAKIA-SMe (7)
(181) 0.10 mmole TentaGel-R—NH.sub.2 resin was loaded with Fmoc-
(182) Following this the corresponding thioester was generated by incubation with methyl iodide in DMF overnight according to Sharma et al. (Sharma et al, 2011). After filtering of the resin the peptide thioester containing solvent was evaporated and the residue precipitated using ice cold diethyl ether. The cleavage of side chain protection groups was performed with 2.5% EDT, 2.5% water, 2.5% TIS in TFA for 2 hours. Following the evaporation of TFA the peptide was precipitated with ice cold diethyl ether. Reversed phase HPLC purification of the peptide thioester was performed on a C18 column using an ACN/water gradient. Fractions that contain product were combined and freeze dried.
(183) The final product was characterised by HPLC (
(184) 10.7 Synthesis of Ac-MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFG VKAGIPIVEAKKILPNAVYLPM-OGp (6)
(185) 0.10 mmole TentaGel-R-Trityl resin was loaded with Fmoc-
(186) The cleavage of the fully protected peptide acid was achieved by incubating the peptidyl resin twice in 10 ml 30% (v/v) HFIP in DCM for 2 hours. After filtering off the peptide the solvent was evaporated and the residue precipitated using ice cold diethyl ether. The precipitated peptide was isolated and dried.
(187) 0.01 mmole fully protected peptide, 4 eq. PyBOP and 5 eq. H-
(188) The final product was characterised by HPLC (
(189) 10.8 Synthesis of All-D-Peptide H-CDMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKLCDTLSIEFDK LKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSRNLEEIKPYLFRAIEES YYKLDKRIPKAIHVVAVTEDLDIVSRGRTFPHGISKETAYSESVKLLQKILEEDERKIRR IG VRFSKFIEAIGLDKFFDT-OH (5) by native chemical ligation of peptide 4 with peptide 3 Both peptides 3 and 4 are solved 0.2 M in TRIS-buffer (pH 8.6) containing 2% SDS, 1% thiophenol and 5 mM tris(2-carboxyethyl)phosphine hydrochloride. The reaction mixture shaked 72 h by room temperature. Afterwards the mixture is purified is by reversed-phase HPLC. For removal of the N-terminal Z-protecting group the peptide was solved in 270 eq. TFA and 50 eq. thioanisol and shakes for 6 h at room temperature (Yoshiaki Kiso et al, 1980). After evaporation of TFA the peptide was precipitated and washed by ice-cold diethyl ether and purified again by reversed-phase HPLC (Phenomenex, Aschaffenburg, Germany). Analysis of the free peptide 5 is performed by SDS-PAGE, reversed phase UPLC and ESI-mass spectrometry. The correct mass of the product is found.
10.9 Synthesis of All-D-Peptide Ac-MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFG VKAGIPIVEAKKILPNAVYLPMRKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDK VRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAKIA-SMe (8) by Protease-Catalyzed Ligation of Peptide 6 with Peptide 7
(190) Peptide 6 was solved 0.2 mM and peptide 7 was solved 0.6 mM in sodium-phosphate buffer (100 mM, pH 8.5, with 100 mM NaCl) containing 4 M Urea. After addition of 20 μM Clostripain (Endoprotease Arg-C, Worthington Biochemical Corporation, Lakewood, N.J., USA) the reaction mixture was shaken overnight at 37° C. The precipitated peptides were centrifuged, solved in H.sub.2O/Formic Acid 80/20 and will be purified by reversed phase HPLC using a RP-18-column (Phenomenex, Aschaffenburg, Germany) with a gradient of ACN in water of 5% to 95% within 30 min. The final peptide was analyzed by SDS-PAGE (
(191) 10.10 Synthesis of the All-D Polymerase Dpo Variant A115C/V203C by Native Chemical Ligation of Peptide 8 with Peptide 5
(192) Both peptides 5 and 8 are solved 0.2 M in TRIS-buffer (pH 8.6) containing 2% Triton X100, 1% thiophenol and 5 mM tris(2-carboxyethyl)phosphine hydrochloride. The reaction mixture shakes 72 h by room temperature. Afterwards the mixture is purified by reversed-phase HPLC and analyzed by SDS-PAGE, reversed phase UPLC and ESI-mass spectrometry. The correct mass of the ligation product is found.
Example 11—Activity Confirmation of Synthetic Polymerase Dpo4 Consisting of D-Amino Acids
(193) This example describes a PCR activity test for all-D polymerase Dpo4 variant A155C/V203C according to example 10.
(194) Surprisingly, the synthetic all-D polymerase Dpo4 variant A155C/V203C is active without extra refolding efforts and thus is used without further refolding procedure. Protein concentration is estimated by sodium dodecyl sulphate (abbr. SDS) polyacrylamide gel electrophoresis (abbr. PAGE) on pre-cast gels (Invitrogen, Karlsruhe, Germany) using a standard series of known protein concentrations followed by SYPRO-RED staining (Invitrogen, Karlsruhe, Germany) and densiometric band analysis on a BioRad Fx scanner instrument.
(195) 11.1 Templates for PCR Activity Assay
(196) Template for the PCR reaction is a 83-mer single-stranded L-DNA oligonucleotide (MJ_1_105_LD) from which, in the first thermal cycle, the opposite strand is made. Thereafter both strands serve as template for exponential amplification. Template DNA oligonucleotide and DNA primers are synthesized at NOXXON in L-configuration.
(197) List of Oligonucleotides for PCR Activity Assay:
(198) TABLE-US-00018 Length, Configu- Name nt ration Sequence (5′.fwdarw.3′) MJ_1_105_LD 83 L GTGGAACCGACAACTTG TGCTGCGTCCAGCATAA GAAAGGAGCTCCCTCAGA AGAAGCTGCGCAGCGTG CCAGTCTGAGCTCC MJ_oligo_187_LD 38 L TCTAATACGACTCACTAT AGGAGCTCAGACTGGCAC GC MJ_oligo_189_LD 20 L GTGGAACCGACAACTT GTGC
11.2 PCR Reactions
(199) 15 μl PCR reactions contain 0.2 mM each of the four L-dNTP's, 10 nM 83-mer ssDNA (MJ_1_105_LD) template, 1 μM of forward and reverse primer, lx ThermoPol buffer (Invitrogen, 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 2 mM MgSO.sub.4, 0.1% Triton X-100, pH 8.8@25° C.) and at least 0.67 ng/μl all-D polymerase Dpo4 variant A155C/V203C. The forward primer is MJ_oligo_187_LD yielding a PCR product of 102 bp, which is distinguishable from the 83-mer template. The reverse primer is MJ_oligo_189_LD. The L-dNTP's are purchased as custom synthesis by Rasayan, Inc. (Encinitas, Calif., USA).
(200) Negative controls are conducted by omitting the all-D polymerase Dpo4 variant A155C/V203C.
(201) The thermal cycling program consists of 1 cycle (85° C., 3 min) then at least 7 cycles (85° C., 30 sec/56° C., 1 min/60° C., 4 min) then hold at 4° C. 4 μl aliquots of the PCR reactions are mixed with sample loading buffer and analyzed on TBE gels. A DNA standard ladder containing, among others, a 100 bp band is applied on the gel.
(202) 11.3 Activity Confirmation
(203) The PCR reaction with all-D polymerase Dpo4 variant A155C/V203C and L-DNA substrate and L-nucleotides yields a band in the range of about 100 bp as compared with the DNA standard ladder. Said band does not appear in the negative controls, where polymerase is omitted. Also said 102 bp band migrates higher than the 83-mer template. The all-D polymerase Dpo4 variant A155C/V203C dependent appearance of an L-DNA amplification product thus confirms the activity of the synthetic all-D polymerase Dpo4 variant A155C/V203C in a thermal amplification process.
Example 12—Synthesis of D- or L-Nucleic Acids
(204) L-DNA nucleic acids or D-DNA nucleic acids were produced by solid-phase synthesis with an ABI 394 synthesizer (Applied Biosystems, Foster City, Calif., USA) using 2′TBDMS DNA phosphoramidite chemistry with standard exocyclic amine protecting groups (Damha and Ogilvie, 1993). For the DNA synthesis dA(N-Bz)-, dC(N—Ac)—, dG(N-ibu)-, and dT in the D- and L-configuration were applied. All phosphoramidites were purchased from ChemGenes, Wilmington, Mass. After synthesis and deprotection L-DNA nucleic acids or D-DNA nucleic acids were purified by gel electrophoresis.
REFERENCES
(205) The complete bibliographic data of the documents recited herein are, if not indicated to the contrary, as follows, whereby the disclosure of said references is incorporated herein by reference. Altschul S. F., Gish W., et al. (1990) Basic local alignment search tool. J Mol Biol. 215(3):403-10. Altschul S. F., Madden T. L., et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25(17):3389-402. Atherton E., Logan C. J. and Sheppard R. C. (1981) Peptide synthesis. Part 2. Procedures for solid-phase synthesis using Na-fluorenylmethoxycarbonylamino-acids on polyamide supports. Synthesis of substance P and of acyl carrier protein 65-74 decapeptide J. Chem. Soc., Perkin Trans. 1, 538-546 Barbs, K., Gatos, D., Kallitsis, J., Papaphotiu, G., Sotiriu, P., Yao, W. Q. and Schäfer, W. (1989) Darstellung geschützter Peptid-Fragmente unter Einsatz substituierter Triphenylmethyl-Harze. Tetrahedron Letters. 30(30):3943-3946 Bock L. C., Griffin L. C., Latham J. A., Vermaas E. H. and Toole J. J. (1992) Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature. 355(6360):564-6. Boudsocq, F., S. Iwai, et al. (2001). “Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic poleta.” Nucleic Acids Res 29(22): 4607-16. Burmeister P. E. et al (2006) 2′-Deoxy purine, 2′-O-methyl pyrimidine (dRmY) aptamers as candidate therapeutics. Oligonucleotides. 16(4):337-51. Damha M J, Ogilvie K K. (1993) Oligoribonucleotide synthesis. The silyl-phosphoramidite method. Methods Mol Biol. 20:81-114 Ellington A. D. and Szostak J. W. (1990) In vitro selection of RNA molecules that bind specific ligands. Nature. 346(6287):818-22. Freier S. M. and Altmann K. H. (1997) The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. 25(22):4429-43. Klussmann S. (2006). The Aptamer Handbook—Functional Oligonucleotides and their Applications. Edited by S. Klussmann. WILEY-VCH, Weinheim, Germany, ISBN 3-527-31059-2 Klussmann S., Nolte A., Bald R., Erdmann V. A. and Fürste J. P. (1996) Mirror-image RNA that binds D-adenosine. Nat Biotechnol. 14(9):1112-5. Kusser W. (2000) Chemically modified nucleic acid aptamers for in vitro selections: evolving evolution. J Biotechnol 74(1): 27-38. Mairal T., Ozalp V. C., Lozano Sánchez P., et al. (2008) Aptamers: molecular tools for analytical applications. Anal Bioanal Chem. 390(4):989-1007 Mandal K., Uppalapati M., Ault-Riché D., Kenney J., Lowitz J., Sidhu S. S. and Kent S. B. H. (2012) Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography. PNAS. 109(37) McGinnis S., Madden T. L. et al. (2004) BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res. 32(Web Server issue):W20-5. Needleman and Wunsch (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 48(3):443-53. Nolte A., Klussmann S., Bald R., Erdmann V. A. and Fürste J. P. (1996) Mirror-design of L-oligonucleotide ligands binding to L-arginine. Nat Biotechnol. 14(9):1116-9. Oliveros, M., R. J. Yanez, et al. (1997). “Characterization of an African swine fever virus 20-kDa DNA polymerase involved in DNA repair.” J Biol Chem 272(49): 30899-910. Pearson and Lipman (1988) Improved tools for biological sequence comparison. Proc. Nat'l. Acad. Sci. USA 85: 2444 Sambrook et al. (ed.), (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Sekizaki, H., Itoh, K., Toyota, E. and Tanizawa, K. (1996) Synthesis and tryptic hydrolysis of p-guanidinophenyl esters derived from amino acids and peptides. Chemical & Pharmaceutical Bulletin. 44(8):1577-9 Sekizaki, H., Itoh, K., Toyota, E. and Tanizawa, K. (1996) Trypsin-catalyzed peptide synthesis and various p-guanidinophenyl esters as acyl donors. Chemical & Pharmaceutical Bulletin. 44(8):1585-7 Sharma, I. and Crich, D. (2011) Direct Fmoc-Chemistry-Based Solid-Phase Synthesis of Peptidyl Thioesters. Journal of Organic Chemistry. 76(16):6518-24 Smith and Waterman (1981), Adv. Appl. Math. 2: 482 Tuerk C. and Gold L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 249(4968):505-10. Usman N. and Cedergren R. (1992) Exploiting the chemical synthesis of RNA. Trends Biochem Sci. 17(9):334-9 Venkatesan N., Kim S. J., et al. (2003) Novel phosphoramidite building blocks in synthesis and applications toward modified oligonucleotides. Curr Med Chem 10(19): 1973-91 Wincott F, DiRenzo A, et al. (1995). Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res.; 23(14):2677-84.
(206) The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.