Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/686—Polymerase chain reaction [PCR]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1241—Nucleotidyltransferases (2.7.7)
  • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase

Definitions

• RNA-dependent RNA polymerase methods and kits for amplification and / or labeling of RNA
• the invention relates to an RNA-dependent RNA polymerase and to methods and kits for labeling and / or for the primer-dependent and -independent amplification of ribonucleic acid (RNA), in particular of viral, eukaryotic and prokaryotic and double-stranded ribonucleic acid (RNA) for use in biotechnology and the medicine.
• RNA ribonucleic acid
• the amplification method according to the invention is particularly suitable for microarray technology, for the production of siRNA and for the diagnosis of viral infections by the detection of viral RNA in patient material.
• the labeling method according to the invention is particularly suitable for the purification of RNA by affinity binding, as well as for the labeling of RNA for use in molecular biological methods that are used in the characterization of the function and / or structure of viral, eukaryotic and prokaryotic and double-stranded ribonucleic acid (RNA). be used.
• RNA ribonucleic acid
• RNA is an important component of the eukaryotic and prokaryotic cells. It is involved in several vital functions: transcription, i. the description of the information of the genetic material (deoxyribonucleic acid, DNA), the transmission of this information from the nucleus to the cytoplasm (mRNA), and the translation, i. the translation of the rewritten information into amino acids or proteins.
• the RNA also forms the so-called transfer RNA, an important building block for translation.
• RNA is also an important component of ribosomes. These are structural units of the cytoplasm, at which the translation of the information into protein takes place.
• RNA Ribonucleic acid
• miRNA miRNA
• siRNA small interfering RNA
• siRNA in vitro has hitherto been carried out by chemical synthesis or by means of DNA-dependent RNA polymerases such as T7 RNA polymerase.
• DNA-dependent RNA polymerases such as T7 RNA polymerase.
• two complementary DNA strands are synthesized by PCR using a primer which also contains the sequence for a T7 promoter.
• the resulting complementary RNA strands are hybridized and digested by RNAse I.
• RNA amplification plays an important role in the detection of viral infections in patient material.
• RNA viruses ie viral pathogens with an RNA-derived genetic material.
• Important pathogens from this group of viruses include the human immunodeficiency virus (HIV), the hepatitis C virus (HCV), the hepatitis A virus, the flu virus (influenza A, B and C) but also the Avian influenza virus, the recently re-identified SARS virus, the polio virus (poliovirus), the measles virus Virus, the mumps virus, the rubella virus and the diarrhea viruses (rotavirus, sapovirus and norovirus).
• HCV human immunodeficiency virus
• HCV hepatitis C virus
• HCV hepatitis A virus
• flu virus influenza A, B and C
• Avian influenza virus the recently re-identified SARS virus
• polio virus poliovirus
• measles virus Virus the measles virus Virus
• RNA in patient material is currently taking place in four steps. After recovering the RNA from the specimen, follow the steps:
• RNA-dependent DNA polymerases the so-called reverse transcriptases. This requires the use of a primer. Primers are oligonucleotides specific for the sequence to be amplified. Primer and the RNA-dependent DNA polymerase allow the targeted amplification of the genome, but in DNA form.
• RNA-dependent RNA polymerases e.g. the T7 polymerase.
• RNA-dependent RNA polymerases e.g. the bacteriophage Q ⁇ RNA polymerase (also called Q ⁇ replicase), poliovirus or hepatitis C virus RNA polymerase.
• the genome of bacteriophage Q ⁇ consists of a single-stranded (+) RNA that is replicated by the RNA-dependent RNA polymerase.
• the Q ⁇ replicase produces a (-) strand RNA copy of the (+) strand, which as well as (+) strands can function as a template. In this way an exponential duplication can be achieved.
• the Q ⁇ system is already used to detect analytes such as nucleic acids (Chu et al (1986), Nucleic Acids Research 14, 5591-5603, Lizardi et al (1988), Biotechnology 6, 1197-1202).
• RNA-dependent RNA polymerases require specific RNA sequences for the initiation of RNA replication. In addition, they are also dependent on certain auxiliary proteins for the amplification of RNA. Therefore, these RNA-dependent RNA polymerases are unable to amplify heterologous RNA.
• RNA at the 3'-terminus is possible in the prior art only with ATP by the use of E. coli po) y (A) -transferase.
• Methods for labeling RNA with CTP, UTP and GTP are not known. It is therefore an object of the invention to provide an RNA-dependent RNA polymerase and methods and kits for labeling and / or amplifying viral, eukaryotic and prokaryotic single and double-stranded ribonucleic acid (RNA) that are more efficient and faster.
• RNA-dependent RNA polymerase for the amplification and / or labeling of RNA
• the RNA-dependent RNA polymerase belongs to the viruses of the family Caliciviridae and has a "right hand conformation" and the amino acid sequence of the RNA-dependent RNA polymerase comprises the following sequence sections:
• a so-called "right-hand conformation” is understood to mean that the tertiary structure (conformation) of the protein has a folding according to a right hand with finger (finger), palm (palm) and thumb (thumb).
• the sequence section "XXDYS" is the so-called A motif, the A motif is for the
• GXPSG is the so-called B motif, the B motif is in all
• sequence section "YGDD" is the so-called C motif, the C motif represents the active one
• the sequence section "XXYGL” is the so-called D motif, the D motif is a
• the sequence section "XXXXFLXRXX" is the so-called E-motif
• the E-motif is a feature of RNA-dependent RNA polymerases and does not occur in DNA-dependent RNA polymerases.
• RNA-dependent RNA polymerase has the following functions:
• Terminal transferase activity with a preference for UTP and CTP, resulting in "labeling" of the 3 'end of a single-stranded RNA template.
• the primer-dependent amplification of the RNA takes place only in the presence of a sequence-specific primer.
• the amplification is sequence-dependent and occurs by incorporation of AMP, GMP, CMP or UMP.
• Primer-independent amplification of the RNA occurs in the absence of a sequence-specific primer.
• the amplification is sequence-dependent and occurs by incorporation of AMP, GMP, CMP or UMP.
• RNA strand Due to the terminal transferase activity, several nucleotides of the same kind are attached at the 3 ' end of an RNA strand (eg ATP or UTP or CTP or GTP), regardless of the sequence of the RNA strand.
• RNA strand eg ATP or UTP or CTP or GTP
• the RNA-dependent RNA polymerase according to the invention is able to use in vitro heterologous viral, eukaryotic and prokaryotic RNA as a template for the amplification reaction. Both positive-stranded and negative-stranded single-stranded and double-stranded RNA can be used as a template for amplification.
• the RNA-dependent RNA polymerase according to the invention is an RNA-dependent RNA polymerase of a virus from the family Calicivi ⁇ dae.
• the genome of Caliciviridae consists of a polyadenylated (+) - stranded RNA single strand.
• the RNA-dependent RNA polymerase of the calicivirus in viral replication writes the genomic calicivirus RNA into a (-) - stranded antisense RNA (aRNA) to. This results in an RNA aRNA hybrid.
• the (-) - stranded aRNA then serves as a template for the synthesis of new (+) - stranded genomic virus RNA.
• the RNA-dependent RNA polymerase is an RNA-dependent RNA polymerase of a human and / or non-human pathogenic calicivirus. Most preferably, it is an RNA-dependent RNA polymerase of a norovirus, sapovirus, vesivirus or a lagovirus, e.g. the RNA-dependent RNA polymerase of the norovirus strain HuCV / NL / Dresden174 / 1997 / GE or the sapovirus strain pJG-SapO1 or the Vesivir ⁇ s strain FCV / Dresden / 2006 / GE.
• the RNA-dependent RNA polymerase is a protein having an amino acid sequence according to SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3.
• SEQ ID NO 1 is the amino acid sequence of a norovirus RdRP.
• SEQ ID NO 2 is the amino acid sequence of a sapovirus RdRP.
• SEQ ID NO 3 is the amino acid sequence of a vesivirus RdRP.
• RNA-dependent RNA polymerases of norovirus, sapovirus and vesivirus are produced recombinantly by cloning the nucleic acid encoding the norovirus, sapovirus or vesivirus RdRP into a suitable expression vector, for example pET-28 (Novagen).
• a suitable expression vector for example pET-28 (Novagen).
• the expression vector with the gene sequence coding for the virus RdRP is introduced into a suitable host organism for expression.
• the host organism is preferably selected from the prokaryotic, preferably Escherichia coli, or eukaryotic host organisms, preferably Sacharomyces cerevisae or insect cells (preferably Sf9 cells) which have been infected with recombinant baculoviruses, which are commonly used for protein expression.
• This host organism contains at least one expression vector with a gene sequence coding for the virus RdRP.
• the norovirus, sapovirus, or vesivirus calicivirus RdRP is fused at the N- or C-terminus to a protein sequence that facilitates purification of the RdRP fusion protein after expression in a host organism.
• this sequence is a so-called histidine marker consisting of a sequence of at least 6 consecutive histidines (His or H).
• Histidine marker allows in a known manner the purification of the protein by affinity chromatography on a nickel or cobalt column.
• RNA-dependent RNA polymerase fused to a histidine marker are the proteins having an amino acid sequence according to SEQ ID NO 4, SEQ ID NO 5 or SEQ ID NO 6.
• SEQ ID NO 4 is the amino acid sequence of a Norovirus RdRP with a histidine tag (His-tag).
• SEQ ID NO 5 is the amino acid sequence of a sapovirus RdRP with a histidine tag (His-tag).
• SEQ ID NO 6 is the amino acid sequence of a vesivirus RdRP with a histidine tag (His-tag).
• the invention also relates to the use of an RNA-dependent RNA polymerase according to the invention for the amplification and / or labeling of RNA.
• a component of the invention is also a method for the amplification of RNA by means of an RNA-dependent RNA polymerase according to the invention with the steps a. Attachment of the RNA-dependent RNA polymerase to the RNA template in the presence or absence of an oligonucleotide primer, for example an oligo-RNA primer or oligo-DNA primer, b. Rewriting the RNA template into antisense RNA by the RNA-dependent RNA polymerase, c. Separation of the RNA / antisense RNA duplex into individual RNA strands
• RNA-dependent RNA polymerase a RNA-dependent RNA polymerase according to the invention
• a suitable reaction buffer a suitable reaction buffer
• NTPs a suitable reaction buffer
• RNase inhibitor e. if necessary stop solution
• the RNA template is preferably used in amounts of from 1 ⁇ g to 4 ⁇ g per 50 ⁇ l reaction mixture.
• concentration of the ribonucleotides ATP 1 CTP, GTP and UTP (NTPs) used is preferably between 0.1 ⁇ mol / l and 1 ⁇ mol / l, particularly preferably 0.4 ⁇ mol / l.
• NTP analogs can also be used in the RNA amplification method according to the invention, for example fluorescently labeled NTPs, biotin-labeled NTPs or radioactively labeled NTPs such as [P 32 ] -labeled NTPs.
• concentration of the RdRP according to the invention is preferably between 1 ⁇ mol / l and 3 ⁇ mol / l.
• the kit contains a. 150 ⁇ mol / l recombinantly produced norovirus RdRP according to SEQ ID NO 1 or SEQ ID NO 4 and / or sapovirus RdRP according to SEQ ID NO 2 or SEQ ID NO 5 and / or Vesivirus RdRP according to SEQ ID NO 3 or SEQ ID NO 6 b. as buffer: 50 mmol / l HEPES, pH 8.0, 3 mmol / l magnesium acetate or manganese chloride (MnCl 2 ), 4 mM DTT c.
• buffer 50 mmol / l HEPES, pH 8.0, 3 mmol / l magnesium acetate or manganese chloride (MnCl 2 ), 4 mM DTT c.
• the process is preferably carried out at a pH between 6.5 and 8.5 and at a temperature between 20 0 C and 4O 0 C.
• a preferred embodiment of the method for RNA amplification is carried out at 3O 0 C with norovirus RdRP or at 37 ° C with sapovirus RdRP under the following buffer conditions: 50 mmol / l HEPES, pH 8.0, 3 mmol / l magnesium acetate or manganese chloride, 4 mmol / l DTT.
• the RNA / antisense RNA duplex is separated into individual RNA strands by heat denaturation, by chemical denaturation, and / or enzymatically, eg, by an enzyme capable of resolving double-stranded RNA into single-stranded RNA, such as: , a helicase.
• an enzyme capable of resolving double-stranded RNA into single-stranded RNA such as: , a helicase.
• the reaction can be kept isothermal.
• a component of the invention is a corresponding kit for the amplification of RNA which, in addition to the kit according to the invention, comprises an enzyme with the ability to separate double-stranded RNA into single-stranded RNA, such as e.g. a helicase containing.
• One embodiment of the invention is a method for primer-dependent amplification of RNA, wherein at least one primer which hybridizes with a section of the RNA template is used, and after the attachment of the RNA-dependent RNA polymerase according to the invention the primer by the RNA-dependent RNA Polymerase is extended according to the sequence of the RNA template.
• an RNA primer or a DNA primer e.g. with a length of 20 to 25 bases, preferably used in a concentration of 0.1 to 1 ⁇ mol / l.
• an RNA template e.g. viral, prokaryotic and eukaryotic RNA can be used.
• polyadenylated RNA polyurydylated RNA, polyguanylated RNA or polycytidylylated RNA
• the poly-U-RNA primer With the help of the poly-U-RNA primer, a specific amplification of the entire cellular mRNA is possible.
• a poly-U primer of 20 to 24 bases in length is used for this purpose.
• the amplified cellular mRNA can be examined, for example, by means of the so-called microarray method.
• the method according to the invention for sequence-specific RNA amplification is also advantageously used for detecting viral RNA in patient material.
• RNA primer that specifically hybridizes to a particular portion of viral RNA
• CSF CSF
• blood plasma or body fluids are used as patient material.
• kits for carrying out the method for the primer-dependent amplification of RNA at least consisting of a. an RNA-dependent RNA polymerase according to the invention b. a suitable reaction buffer c. NTPs d. optionally RNase inhibitor e. if necessary stop solution f. primer
• the kit contains
• RNase inhibitor e. as stop solution: 4 mol / l ammonium acetate, 100 mmol / l EDTA f. 0.1 to 3 ⁇ M primer (homopolymeric or heteropolymeric RNA or DNA oligonucleotide of 15 to 20 bases in length)
• Also part of the invention is a method for primer-independent amplification of RNA, wherein no primer that hybridizes to a portion of the RNA template is inserted and extended by the RNA-dependent RNA polymerase corresponding to the sequence of the RNA template, and a kit for the primer-independent amplification of RNA, at least consisting of
• RNA-dependent RNA polymerase a. an RNA-dependent RNA polymerase according to the invention b. a suitable reaction buffer c. NTPs d. optionally RNase inhibitor e. if necessary stop solution.
• the attachment of the RNA-dependent RNA polymerase to the RNA template is primer-independent.
• the sequence of the sequence-independent RNA amplification of a single-stranded RNA template without the use of an RNA primer is shown schematically in FIG.
• Another embodiment of the invention is a method for primer-independent amplification of poly (C) RNA, characterized in that no primer hybridizing to a portion of the RNA template is used, using GTP, preferably 50 ⁇ M, as the sole nucleotide is extended by the RNA-dependent RNA polymerase according to the sequence of the RNA template.
• RNA primer-independent amplification of the RNA The possible applications of the method according to the invention for the amplification of RNA are manifold.
• the direct detection of viral genome in patient material is possible.
• the entire cellular RNA is obtained and amplified using an RNA primer, any RNA sequence specifically.
• the detection of the viral nucleic acid is then carried out by hybridization to specific probes.
• Another application concerns the nonspecific (RNA primer-independent) amplification of the RNA, which can make it possible to identify previously unidentified viruses or new viral variants.
• Another application concerns microarray technology. This has the differential detection of cellular expression as the goal. This is done by detecting the so-called cellular transcripts, i. the mRNA.
• the invention makes it possible to specifically amplify the mRNA by the use of a poly (U) -oligonucleotide in order subsequently to detect microarrays by hybridization.
• siRNA in vitro Another field of application of the invention is the production of siRNA in vitro. This is done so far with the help of T7 polymerase. It will be for that
• the invention enables the direct, efficient and simple preparation in 1.5 hours and starting from an RNA sequence double-stranded RNA, without the need for a PCR step, in vitro transcription and hybridization of the RNA (which may possibly be sub-optimal).
• a component of the invention is also a method for labeling RNA by means of an RNA-dependent RNA polymerase according to the invention with the steps a. Attachment of the RNA-dependent RNA polymerase to the RNA to be labeled, b. Attaching at least one nucleotide to the 3 'end of the RNA to be labeled.
• RNA at least consisting of
• RNA-dependent RNA polymerase a. an RNA-dependent RNA polymerase according to the invention b. a suitable reaction buffer c. CTP or UTP or ATP or GTP d. optionally RNase inhibitor e. if necessary stop solution.
• the kit contains
• FIG. 1 Schematic sequence of the sequence-dependent RNA amplification
• FIG. 2 Schematic representation of the sequence-independent RNA amplification
• FIG. 3 Schematic procedure for labeling the 3 'end of the RNA template
• Figure 4 Schematic sequence of the sequence-independent RNA synthesis starting from a poly (C) RNA in the presence of 50 ⁇ W! GTP
• FIG. 5 Expression and purification of Norovirus 3D pD1 in E. coli.
• FIG. 6 RNA synthesis with Norovirus 3D po1 .
• RNA synthesis was carried out in the presence of a synthetic subgenomic RNA (sG-RNA) as a template and in the presence of wild-type norovirus 3D po1 or a norovirus 3D po 'with a mutation in the active site (YGD 343G D 344 G) as indicated.
• the reaction products were analyzed on a non-denaturing agarose gel and visualized by autoradiography. 17, synthetic subgenomic RNA prepared by in vitro transcription at 17.
• sG-RNA synthetic subgenomic RNA
• reaction products were visualized on nondenaturing (0.5 M, left) and denaturing (1.25 M, right) formaldehyde agarose gels.
• T7 synthetic subgenomic RNA produced by in vitro transcription with T7.
• M RNA molecular weight marker.
• FIG. 7 Northern blot analysis of the Norovirus 3D po1 synthesis products.
• RNA synthesis was carried out in the presence of a synthetic subgenomic RNA (sG-RNA) as a template and in the presence of wild-type norovirus 3D po1 or a norovirus 3D po1 with a mutation in the active site (YGD 343 QD 344G ). performed as specified.
• the reaction products were analyzed on a non-denaturing agarose gel and visualized after ethidium bromide staining by UV transillumination.
• T7 synthetic subgenomic RNA produced by in vitro transcription with T7.
• M RNA molecular weight marker. The remaining band visible in the reaction with m3D po1 instead of wild-type 3D po1 is derived from the template used in the reaction.
• T7 synthetic subgenomic RNA produced by in vitro transcription with T7.
• sG-RNA synthetic subgenomic RNA used as template.
• 3D po1 wild-type norovirus 3D po1 .
• RNA synthesis was 2 h at 30 0 C in the presence of 3 uM norovirus 3D PDL subgenomic RNA and increasing concentrations (0.5 to 3 mM) as template (0.024 uM) of MgAGO, MnCl 2 or FeSC ⁇ . Incorporation of [ ⁇ - 32 P] UMP as indicated. For each concentration, the mean and standard deviation of three independent experiments are shown.
• FIG. 9 Primer-dependent initiation of RNA synthesis at higher temperature.
• RNA synthesis was performed in the presence (black bars) or in the absence (gray bars) of a complementary RNA oligonucleotide primer. Incorporation of [ ⁇ - 32 P] CMP, [ ⁇ - 32 P] AMP, [ ⁇ - 32 P] GMP, or [ ⁇ - 32 P] UMP was measured after TCA precipitation and collection of G / C glass fiber filters. The installation values are given.
• FIG. 10 Terminal transferase activity of Norovirus 3D po1
• synthetic subgenomic polyadenylated RNA was used as template.
• the reaction products were analyzed on formaldehyde agarose gels and visualized by autoradiography.
• RNA oligonucleotide primer concentration mM
• M marker (in vitro transcribed subgenomic polyadenylated RNA).
• FIG. 12 De novo initiation of RNA synthesis on norovirus anti-subgenomic RNA.
• RNA reaction products were analyzed on native agarose gels and visualized after ethidium bromide staining by UV transillumination.
• Lanes 1 to 3 RNA synthesis in the presence of wild-type 3D po1 , mutant 3D po1 or in the absence of 3D po1 .
• Lanes 4 to 6 RNA synthesis in the presence of wild-type 3D po1 and an RNA oligonucleotide complementary to the 3 ' terminus, mutated 3D po1 and an RNA oligonucleotide complementary to the 3 ' terminus or with an RNA oligonucleotide complementary to the 3 ' ' Term, but without norovirus 3D po1 .
• T template RNA.
• R replication product.
• M RNA molecular weight marker (Kb).
• B Strand separation analysis of replication products. The reaction products were analyzed on formaldehyde agarose gels and visualized after ethidium bromide staining by UV transillumination. Lanes 1 and 2, template (anti-subgenomic RNA) and norovirus 3D pDl replication product .
• M RNA molecular weight marker (Kb).
• FIG. 13 Primer-independent de novo initiation of RNA synthesis on homopoietmer
• RNA synthesis was performed in the presence (black bars) or in the absence (gray bars) of cold CTP 1 ATP, GTP, and UTP for poly (rG), poly (rU), poly (rC), and poly (rA), respectively. Templates performed. The incorporation of [ ⁇ - 32 P] CMP, [OC- 32 P] AMP, [ ⁇ - 32 P] GMP, or [ ⁇ - 32 P] UMP was measured after TCA precipitation and collection of G / C glass fiber filters. The installation values are given.
• FIG. 14 Ampiification of viral and eukaryotic RNA with the norovirus RNA
• reaction 7 Subgenomic astrovirus RNA (serotype 1); Reaction 8; Subgenomic astrovirus RNA (serotype 2); Reaction 9, eukaryotic RNA of the elF4 gene of Xenopus Laevf, reaction 10, sub-genomic norovirus DNA; Reaction 11; T7 transcribed RNA of the elF4 gene of Xenopus Laevi; Reaction 12, negative control.
• FIG. 15 Expression and purification of sapovirus 3D po1 in E. coli.
• FIG. 16 Concentration dependence, substrate dependency, temperature dependence and metal ion dependency of the sapovirus 3D p0 'activity. In all reactions, subgenomic RNA was used as a template.
• RNA synthesis was carried out in the presence of 3 uM sapovirus 3D po 'with subgenomic RNA as a template (0.024 uM), and increasing concentrations (0.5 to 5 mM) of MgAcO or MnCl 2 at 37 0 C for 2 h. Incorporation of [ ⁇ - 32 P] UMP as indicated.
• FIG. 17 RNA synthesis with sapovirus 3D po1 .
• RNA synthesis in the presence of a synthetic subgenomic polyadenylated RNA (sG-poly (A) RNA) as a template was performed with and without the addition of a 3 ⁇ M oligo (U) 2 o RNA primer and in the presence of wild-type sapovirus 3D po1 (3D po1 ) or, as indicated, a 3D po1 mutated in the acitve site (m3D po1 , YGD 343 G J D 344G ).
• the reaction products were analyzed on non-denaturing agarose gels and visualized after ethidium bromide staining with UV transillumination.
• the reaction product was obtained as indicated by RNA synthesis with sapovirus 3D po1 (3D po1 ) using a subgenomic polyadenylated RNA (sG-Poiy (A) RNA) as template.
• the reaction products were visualized on denaturing formaldehyde agarose gels.
• T7 synthetic subgenomic RNA produced by in vitro transcription with T7.
• M RNA molecular weight markers.
• Figure 18 De novo initiation of RNA synthesis on sapovirus anti-subgenomic RNA.
• RNA synthesis was performed in the presence of a synthetic anti-subgenomic RNA (anti-sG RNA) as a template and wild-type sapovirus 3D POI ⁇ 30 POi) or ejner in the ⁇ actjve sjte mutant 3D poi (Pn 30 POi 1
• Figure 19 Terminal transferase activity of sapovirus 3D po1 .
• the terminal transferase activity was investigated by a reaction of 3 ⁇ M sapovirus 3D po1 or an active site mutated 3D po1 (m3D po1 ), which was used in the presence of subgenomic RNA (sG-RNA) as a template (0.024 ⁇ M) and [ ⁇ - 32 P] ATP, [OC- 32 P] GTP, [(X- 32 P] CTP or [ ⁇ - 32 P] UTP for 2 h at 37 ° C (0.024 ⁇ M) as was incubated.
• the reaction products were analyzed on non-denaturing agarose gels and visualized by autoradiography. 17, synthetic subgenomic RNA produced by in vitro transcription with T7.
• Figure 20 De novo initiation of RNA synthesis by Sapo virus 3D po> on homopolymer templates.
• RNA synthesis was performed using poly (G) RNA, poly (U) RNA, poly (C) RNA, and poly (A) RNA templates with [ ⁇ - 32 P] CTP, [ ⁇ - 32 P ] ATP, [ ⁇ - 32 P] GTP, or [ ⁇ - 32 P] UTP and with or without 3 ⁇ M of an oligo (C) 2 o, oligo (A) 20l oligo (G) 20 or oligo (U) 20 RNA primers were incubated.
• RNA synthesis was performed in the absence of an oligo (RNA) primer but in the presence of 50 ⁇ M cold CTP, ATP, GTP, and UTP, respectively, for poly (G) RNA, poly (U) RNA, poly (C ) RNA or poly (A) RNA
• FIG. 21 Expression and purification of the vesivirus 3D po 'in E. coli.
• FIG. 22 pol
• RNA synthesis in the presence of a synthetic subgenomic polyadenylated RNA (sG-poly (A) RNA) as template was performed with and without the addition of a 3 ⁇ M oligo (U) 20 RNA primer and in the presence of wild-type vesivirus 3D po1 (3D po1 ) examined.
• the reaction products were analyzed on non-denaturing agarose gels and visualized by autoradiography.
• T7 synthetic subgenomic RNA produced by in vitro transcription with T7.
• M RNA molecular weight markers.
• the norovirus RdRP cDNA was obtained by PCR from the norovirus clone pUS-NorII (Genbank accession number: AY741811). It was cloned into the pET-28b (+) vector (Novagen), the expression vector was sequenced and transformed into E. coli CL21 (DE3) pLysS. The cells were at 37 0 C in Luria-Bertani medium with kanamycin (50 mg / L). At an optical density of 0.6 (OD600), protein expression was induced by the addition of isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. The cultures were then incubated at 25 0 C overnight.
• IPTG isopropyl- ⁇ -D-thiogalactopyranoside
• the cell pellets obtained from 250 ml of culture were washed once in 4 ml of phosphate buffered saline (PBS) and 1% Triton X 100 (Sigma).
• the cells were treated at 37 0 C for 15 minutes with DNase (10 U / ml), sonicated on ice, and resuspended in 40 ml binding buffer (20 mM Tris / HCl, pH 7.9, 500 mM NaCl, 5 mM imidazole). After centrifugation at 4300 rpm at 4 0 C for 40 minutes was obtained, the clarified lysate.
• His6-tagged norovirus RdRP was bound to a Ni-nitrilotriacetacetic acid (NTA) -sepharose matrix (Novagen) preequilibrated with the binding buffer.
• NTA Ni-nitrilotriacetacetic acid
• the bound protein was washed with the binding buffer containing 60 mM imidazole and eluted with binding buffer containing 1 M imidazole.
• the eluted protein was dialyzed against buffer A (25 mM Tris-hCI, pH 8.0, 1 mM ⁇ -mercaptoethanol, 100 mM NaCl, 5 mM MgCl 2, 10% glycerol, 0.1% Triton X).
• the protein concentration was determined with the BCA protein assay kit (Pierce) based on the biuret reaction.
• the enzyme was resuspended in a final volume of 50% glycerol and stored at -20 0 C.
• the reaction mixture (50 ⁇ l) consists of 0.5 to 1 ⁇ g RNA template, 10 ⁇ l of the reaction buffer (250 mM HEPES, pH 8.0, 15 mM magnesium acetate, 20 mM DTT), 50 U RNase inhibitor (RNAsin, Promega), 0.4 each mM of ATP, CTP, GTP, UTP, 3 ⁇ M of norovirus RdRP prepared according to Example 1.
• the reaction is carried out at 30 ° C. for 2 hours.
• the reaction is stopped by the addition of 50 ⁇ l of stop solution (4 M ammonium acetate, 100 mM EDTA). Purification is by phenol-chloroform extraction or by MEGAclear kit (Ambion) according to the manufacturer's instructions.
• the transcription products are visualized after ethidium bromide staining by UV transillumination on TBE-buffered agarose gels. Formamide agarose gels can also be used.
• the reaction mixture (50 ⁇ l) consists of 0.5 to 1 ⁇ g RNA template, 10 ⁇ l of the reaction buffer (250 mM HEPES, pH 8.0, 15 mM magnesium acetate, 20 mM DTT), 50 U RNase inhibitor (RNAsin, Promega), 0.4 each mM of ATP, CTP, GTP, UTP, 0.1 to 1 ⁇ M gene-specific RNA primer, 3 ⁇ M of norovirus RdRP prepared according to Example 1.
• the reaction is carried out at 30 ° C. for 2 hours.
• the reaction is stopped by the addition of 50 ⁇ l of the stop solution (4 M ammonium acetate, 100 mM EDTA).
• the reaction mixture (50 ⁇ l) consists of 0.5 to 1 ⁇ g RNA template, 10 ⁇ l of the reaction buffer (250 mM HEPES, pH 8.0, 15 mM magnesium acetate, 20 mM DTT), 50 U RNase inhibitor (RNAsin, Promega), 0.4 each mM of ATP, CTP 1 GTP 1 UTP, 0.1 to 1 ⁇ M poly (U) 2 o primer, 3 ⁇ M of Norovirus RdRP prepared according to Example 1.
• the reaction is carried out at 30 ° C. for 2 hours.
• the reaction is stopped by the addition of 50 ⁇ l of stop solution (4 M ammonium acetate, 100 mM EDTA).
• the reaction mixture (50 ⁇ l) consists of 0.5 to 1 ⁇ g RNA template, 10 ⁇ l of the reaction buffer (250 mM HEPES, pH 8.0, 15 mM magnesium acetate, 20 mM DTT), 50 U RNase inhibitor (RNAsin, Promega), 0.4 each mM of ATP, CTP, GTP 1 UTP 1 3 ⁇ M of Norovirus RdRP prepared according to Example 1.
• the reaction is carried out at 30 ° C. for 2 hours.
• the reaction is stopped by the addition of 50 ⁇ l of stop solution (4 M ammonium acetate, 100 mM EDTA). Purification is by phenol-chloroform extraction or by MEGAclear kit (Ambion) according to the manufacturer's instructions.
• the transcription products are visualized after ethidium bromide staining by UV transillumination on TBE-buffered 10% polyacrylamide gels. Formamide agarose gels can also be used. Alternatively, armored RNA can be used
• the reaction mixture (50 ⁇ l) consists of 0.5 to 1 ⁇ g RNA template, 10 ⁇ l of the reaction buffer (250 mM HEPES 1 pH 8.0, 15 mM magnesium acetate, 20 mM DTT), 50 U RNase inhibitor (RNAsin, Promega), 0.4 each mM of ATP 1 or CTP 1 or GTP 1 or UTP, 3 ⁇ M of the embodiment 1 Norovirus RdRP.
• the reaction is carried out at 30 ° C for 2 hours.
• the reaction is stopped by the addition of 50 ⁇ l of stop solution (4 M ammonium acetate, 100 mM EDTA). Purification is by phenol-chloroform extraction or by MEGAclear kit (Ambion) according to the manufacturer's instructions.
• the transcription products are visualized after ethidium bromide staining by UV transillumination on TBE-buffered 10% polyacrylamide gels. Formamide agarose gels can also be used. Alternatively, armored RNA can be
• the reaction mixture (50 ⁇ l) consists of 0.5 to 1 ⁇ g poly (C) RNA template, or an RNA with poly (C) RNA at the 3 'end, 10 ⁇ l of the reaction buffer (250 mM HEPES, pH 8.0, 15 mM magnesium acetate, 20 mM DTT), 50 U RNase inhibitor (RNAsin, Promega), 50 ⁇ M of GTP, and 3 ⁇ M of Norovirus RdRP prepared according to Example 1.
• the reaction is carried out at 30 ° C. for 2 hours.
• the reaction is stopped by the addition of 50 ⁇ l of stop solution (4 M ammonium acetate, 100 mM EDTA).
• the cleaning is done by phenol-chloroform extraction or with the MEGAclear Kit (Ambion) according to the manufacturer's instructions.
• the transcription products are visualized after ethidium bromide staining by UV transillumination on TBE-buffered 10% polyacrylamide gels.
• Formamide agarose gels can also be used.
• armored RNA can be used.

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