WO2021262013A1 - Polymérase de fusion d'adn bst-nec destinée à être utilisée dans la réplication isotherme de séquences spécifiques du virus du sars cov-2 - Google Patents

Polymérase de fusion d'adn bst-nec destinée à être utilisée dans la réplication isotherme de séquences spécifiques du virus du sars cov-2 Download PDF

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Publication number
WO2021262013A1
WO2021262013A1 PCT/PL2020/000074 PL2020000074W WO2021262013A1 WO 2021262013 A1 WO2021262013 A1 WO 2021262013A1 PL 2020000074 W PL2020000074 W PL 2020000074W WO 2021262013 A1 WO2021262013 A1 WO 2021262013A1
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polymerase
bst
dna
nec
virus
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PCT/PL2020/000074
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English (en)
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Marta SKWARECKA
Kasjan SZEMIAKO
Dawid NIDZWORSKI
Sabina ŻOŁĘDOWSKA
Marcin Olszewski
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Geneme Sp. Z O.O.
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Publication of WO2021262013A1 publication Critical patent/WO2021262013A1/fr

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    • 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)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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
    • 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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the subject of the invention is the use of Bst-Nec DNA fusion polymerase to isothermal replication of specific sequences of SARS CoV-2 virus to detect the RNA of the virus in a test sample. It is also possible to directly detect the virus from the swab without isolating the RNA.
  • DNA polymers are enzymes that play an essential role in the replication and repair of DNA. They are widely used in various fields of science, successfully used in sequencing or different variants of PCR (Polymerase Chain Reaction), where they catalyze the process of DNA synthesis in vitro, which takes place cyclically at strictly defined thermal stages. Another approach and increasingly popular in recent times is the use of DNA polymerases in isothermal techniques for DNA amplification, which are not based on thermocycles and the reaction is carried out at a constant elongation temperature. So far, many such techniques have been developed to duplicate both DNA and RNA. The choice of the right polymerase for a given technique depends mainly on its properties.
  • the basic polymerization properties may have the ability to hydrolyse DNA molecules due to the presence of exonucleolytic domain or reverse transcriptase activity. These features are determined by the presence of appropriate domains in their structure.
  • the basic domains that occur in these enzymes are polymerization and 3'-5' and 5'-3' exonucleolytic.
  • polymerases in which the deletion of the exonucleolytic domain leads to a functional protein with partially changed characteristics in relation to the native enzyme.
  • Taq polymerase isolated from thermophilic bacteria Thermus aquaticus, the discovery of which completely changed the face of molecular biology. The inactivity of 5'-3" exonuclease is reduced.
  • Taq289 polymerase is characterized by increased thermostability, however, it also requires increased demand for Mg2+ ions, and the newly formed DNA thread has fewer errors.
  • the polymerase used in isothermal DNA amplification techniques is Bst polymerase, whose native form has an inactive 3'-5' exonucleolytic domain and an active 5'-3' exonucleytic domain, whose activity can be deactivated by means of point mutation in position 73 (Tyr73 ⁇ Phe73 and Tyr73 ⁇ Ala73).
  • This polymerase like Taq polymerase, belongs to family A and is isolated from Bacillus stearothermophilus.
  • Polymerase shows increased tolerance to clinical or environmental inhibitors compared to other polymerases of this family, but still, taking into account the applications of this polymerase, it is important to look for solutions leading to improve mainly its processability and resistance to inhibitors.
  • the NeqSSB protein belongs to the Single-Stranded DNA Binding protein (SSB) family.
  • SSB proteins show a variety of amino acid sequences and structures. Nevertheless, all of them have a characteristic, strongly preserved, about 100 - amino acid domain Oligonucleotide/Oligosaccharide Binding Fold Domain (OB). It is commonly found in proteins that have the capacity to bind SSB, so it determines this basic and common for all SSB proteins feature - non-specific binding of single stranded DNA and, discovered much later, RNA binding.
  • SSB proteins play an essential role in processes closely related to ssDNA. They play an important role in replication, recombination and repair of DNA. They are responsible for interactions with single- stranded DNA, prevent the formation of secondary structures and protect against the degradation effects of nucleases.
  • SSB proteins The discovery of SSB proteins dates back to the mid 1960s.
  • the first detected proteins were SSB phage T4 and E. coli proteins.
  • the high selectivity of this protein to single stranded DNA was also noted.
  • the fundamental role of SSB proteins in processes related to ssDNA is confirmed by the fact that they occur in all living organisms as well as in viruses.
  • the binding of SSB protein to mamdNA consists in packing the aromatic amino acid residues between the bases in the oligonucleotide chain. Moreover, positively charged amino acid residues interact with the phosphate skeleton in the ssDNA molecule.
  • NeqSSB protein belongs to the family of SSB proteins, it differs in its characteristics from classical SSB proteins and is therefore described as NeqSSB-like protein.
  • This protein comes from the hyperthermophilic archaeon Nanoarchaeum equitans, which parasites in the craenarchaeon Ignicoccus hospitalis. Optimal growth conditions of this microorganism require strictly anaerobic conditions and temperature of 90°C.
  • Nanoarchaeum equitans has the smallest known genome consisting of 490,885 base pairs. Unlike most known organisms with lowered genomes, it has a full set of enzymes involved in replication, repair and recombination of DNA, including SSB protein.
  • This invention uses polymerase, which was modified by adding NeqSSB protein to the native enzyme.
  • This protein belongs to the Single-Stranded DNA Binding protein (SSB) family.
  • SSB proteins show a variety of amino acid sequences and structures. Nevertheless, all of them have a characteristic, strongly conserved, about 100 - Oligonucleotide/Oligosaccharide Binding Fold Domain (OB).
  • OB Oligonucleotide/Oligosaccharide Binding Fold Domain
  • NeqSSB belongs to the family of SSB proteins, it differs in its characteristics from classical SSB proteins and is therefore referred to as NeqSSB-like protein.
  • Nanoarchaeum equitans This protein comes from the hyperthermophilic archaeon Nanoarchaeum equitans, which parasites in the craenarchaeon Ignicoccus hospitalis. Optimal growth conditions of this microorganism require strictly anaerobic conditions and temperature of 90°C. Nanoarchaeum equitans has the smallest known genome consisting of 490.885 base pairs. In contrast to most known organisms with lowered genomes, it has a full set of enzymes involved in replication, repair and recombination of DNA, including SSB protein. Studies indicate that the ability to bind all forms of DNA (ssDNA, dsDNA) and mRNA without structural preferences is unusual for other SSB proteins. Moreover, the protein is characterized by high thermostability.
  • NeqSSB-Bst DNA fusion polymerase was obtained by combining Bst polymerase with NeqSSB protein at the N-end of polymerase using a 6- amino acid linker with amino acid sequence: Gly-Ser-Gly-Val-Asp.
  • a comparative analysis of the properties of the patented enzyme with the commercially available reference DNA polymerase Bst showed that the presence of an additional DNA binding protein has a positive effect on the properties of DNA polymerase. It is characterized by increased thermostability, increased processability, and increased tolerance to inhibitors such as heparin, lactoferrin, and humic acid.
  • innovative, fusional DNA polymerase also shows increased sensitivity and reaction specificity, which is crucial in molecular diagnostics.
  • the purpose of this invention is to use Bst-Nec DNA fusion polymerase to detect a virus without the need for RNA isolation.
  • the subject of the invention is the Bst-Nec DNA fusion polymerase for use in isothermal replication of specific sequences of SARS CoV-2 virus.
  • the fusion polymerase according to the to detect RNA of the virus from a sample of biological material without RNA isolation.
  • Biomaterial - means a sample of material obtained in the form of a nasal, nasopharyngeal swab.
  • Fig.l - shows RNA dilutions of SARS-CoV-2 virus detected in the form of curves - LOD of the proposed solution.
  • Fig.2 - shows the lack of response of the proposed invention to RNAs of other coronavi ruses and the response to RNAs of SARS-CoV-2 - specificity to SARS-CoV-2.
  • Fig.3 - shows the analysis of clinical samples with the proposed solution.
  • Table 1 - shows the serial RNA dilutions of SARS-CoV-2 that were used for the LOD assessment. The answers to the proposed invention are shown in figure 1.
  • Table 2 - shows a tabular overview of analyzed RNAs of other coronaviruses and SARS- CoV-2 for specificity assessment.
  • Table 3 - shows a tabular overview of the clinical samples analysed with the proposed solution.
  • Table 4 - shows a tabular comparison of the results obtained with the proposed solution in comparison with the "gold standard” of the RT-PCR method.
  • the polymerase was modified by adding NeqSSB protein to the native enzyme.
  • This protein belongs to the Single-Stranded DNA Binding protein (SSB) family.
  • a serial 10-fold dilution of the virus RNA was made and the minimum number of copies of the virus RNA that can be detected was determined.
  • a reaction specificity test was also performed. In determining the specificity of the test, the reaction to 4 different viruses from the coronavirus family was performed: HCoV-NL63, HCoV-283E, HCoV-OC43, HCoV-229E. The titer of the examined viruses significantly exceeded physiological concentration and was about 1 x 10 7 PFU. The RNA of SARS-CoV-2 virus with PFU about 1 x 10 4 was used as positive control. The reaction was carried out 30 min.
  • the reaction was also performed using clinical samples. The reaction was carried out without RNA isolation directly using the transport medium as a matrix for the reaction. The study showed 100% convergence of isothermal amplification test results with the gold standard PCR reaction. Moreover, the results obtained with the proposed isothermal method require shorter analysis time than the method recommended by WHO (fig.3; tab.3-4). d)
  • the reaction was carried out in the volume of 20 mI.
  • the following composition of the reaction mixture was applied: 10x Isothermal Buffer [200 mM Tris-HCI pH 8.8, 100 mM (NH 4 ) 2 SO 4 ,50 mM KCI, 2 mM MgSO 4 ], 1% Tween 20], 10 mM dNTPs, 100 mM MgSO 4, Starters: 1.6 uM FIP/BIP, 0.2 uM F3/B3, 0.4 uM LoopF/B, NeqSSB-GssFull polymerase 0.32 U/ul reaction, RT 1 U/ul reaction, Inhibitor RNaz 0.4 U/ul reaction

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'objet de la présente invention est une polymérase de fusion Bst-Nec DIMA destinée à être utilisée dans la reproduction isotherme de séquences spécifiques du virus du SARS-CoV-2.
PCT/PL2020/000074 2020-06-26 2020-08-31 Polymérase de fusion d'adn bst-nec destinée à être utilisée dans la réplication isotherme de séquences spécifiques du virus du sars cov-2 WO2021262013A1 (fr)

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PL434484A PL241065B1 (pl) 2020-06-26 2020-06-26 Zastosowanie fuzyjnej polimerazy DNA Bst-Nec do izotermalnego powielania specyficznych sekwencji wirusa SARS CoV-2
PLP.434484 2020-06-26

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4036237A1 (fr) * 2021-01-27 2022-08-03 Instytut Biotechnologii I Medycyny Molekularnej Pwo-neqssb polymérase, méthode pour sa préparation, plasmide recombinant, amorces et l'utilisation de la polymérase

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007029200A2 (fr) * 2005-09-09 2007-03-15 Invitrogen Corporation Proteines hybrides ssb-polymerase
WO2020005084A1 (fr) * 2018-06-27 2020-01-02 Instytut Biotechnologii I Medycyny Molekularnej Adn polymérase monocaténaire de fusion bst, molécule d'acide nucléique codant pour l'adn polymérase de fusion neqssb-bst, procédé de préparation et utilisation associés

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007029200A2 (fr) * 2005-09-09 2007-03-15 Invitrogen Corporation Proteines hybrides ssb-polymerase
WO2020005084A1 (fr) * 2018-06-27 2020-01-02 Instytut Biotechnologii I Medycyny Molekularnej Adn polymérase monocaténaire de fusion bst, molécule d'acide nucléique codant pour l'adn polymérase de fusion neqssb-bst, procédé de préparation et utilisation associés

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MARCIN OLSZEWSKI ET AL: "Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans-Expression and characterization", PLOS ONE, vol. 12, no. 9, 1 September 2017 (2017-09-01), pages e0184162, XP055626163, DOI: 10.1371/journal.pone.0184162 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4036237A1 (fr) * 2021-01-27 2022-08-03 Instytut Biotechnologii I Medycyny Molekularnej Pwo-neqssb polymérase, méthode pour sa préparation, plasmide recombinant, amorces et l'utilisation de la polymérase

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PL434484A1 (pl) 2021-12-27

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