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WO2021163559A1 - Variants d'adn polymérases de la famille d - Google Patents

Variants d'adn polymérases de la famille d Download PDF

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Publication number
WO2021163559A1
WO2021163559A1 PCT/US2021/017954 US2021017954W WO2021163559A1 WO 2021163559 A1 WO2021163559 A1 WO 2021163559A1 US 2021017954 W US2021017954 W US 2021017954W WO 2021163559 A1 WO2021163559 A1 WO 2021163559A1
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WIPO (PCT)
Prior art keywords
family
polymerase
polymerases
dna
amino acid
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Ceased
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PCT/US2021/017954
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English (en)
Inventor
Andrew F. Gardner
Kelly M. ZATOPEK
Thomas C. Evans
Ece ALPASLAN
Ludovic Sauguet
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut Pasteur
New England Biolabs Inc
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Institut Pasteur
New England Biolabs Inc
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Priority to EP21710761.4A priority Critical patent/EP4103702A1/fr
Publication of WO2021163559A1 publication Critical patent/WO2021163559A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • DNA polymerases are classified into Families A, B, C, D, X, Y and RT according to their amino acid sequences. DNA polymerases have several properties that contribute to their replicative fidelity. For example, some DNA polymerases of Families A, B, and D have proofreading 3' to 5' (3 '-5') exonuclease activity. When a DNA polymerase incorporates an incorrect or modified nucleotide, for example, in a primer strand, it detects structural perturbations caused by mispairing or nucleotide modification and transfers the primer strand from the polymerase domain to the 3 '-5' exonuclease active site.
  • DNA polymerases restrict access to their active sites to prevent incorporation of ribonucleotides.
  • Families A, B, X, Y, and reverse transcriptases have a steric gate that excludes rNTPs from the active site by a steric clash between a bulky amino acid side chain in the steric gate and the 2’-OH of such rNTPs. Reducing the size of the side chain at the steric gate position allows DNA polymerases so modified to incorporate a single rNTP as efficiently as dNTP.
  • Polymerases with such modified steric gates have been extensively employed in molecular biology applications such as single-molecule sequencing, sequencing by synthesis, and single nucleotide polymorphism (SNP) detection.
  • SNP single nucleotide polymorphism
  • PolD is a heterodimeric enzyme consisting of a large 5 ’-3’ polymerase subunit and a small MRE11-like 3'-5' exonuclease subunit. The activity of each subunit requires the other subunit to be present.
  • Family D DNA polymerases preferentially incorporate dNTPs over rNTPs. The molecular basis for this selectivity has not yet been identified, which may have limited the use of wild type Family D polymerases.
  • a variant Family D polymerase may have an amino acid sequence according to SEQ ID NO:l and (b) includes a substitution at a position corresponding to a position selected from positions 106-161, 243-267, 326-330, 361-365, 385-397, 441-451, 657-667, 822-829, 919-928, and 940-962, 981-997 of SEQ ID NO: 1.
  • FIGURE 1 shows an alignment of Family D polymerase sequences from Pyrococcus abyssi (P.ab) and Euryarchaeota (9°N PolD-L). Based on a cryo-electron microscopy structure of the P.ab sequence bound to DNA, amino acid residues that (a) appear to be within 20 A of the catalytic site of polD are shown under a solid bar, (b) appear to be within 12 A of the 2’-OH group of the substrate nucleotide are underlined, and (c) may contact the substrate nucleotide are shown in bold.
  • compositions, methods and kits are provided here that improve among other things, the synthesis of DNA that contains ribonucleotides using a class of polymerases that do not belong to the DNA polymerase A or B families.
  • Family D polymerases preferentially incorporate deoxyribonucleoside triphosphates relative to larger ribonucleoside triphosphates, which may be attributed to a steric gate motif limiting substrate access to the catalytic site.
  • Variant Family D polymerases with modifications in or modifications impacting this steric gate are provided in some embodiments disclosed herein. For example, variant Family D polymerases may have an enhanced ability to exclude ribonucleotides from the active site.
  • a protein refers to one or more proteins, i.e., a single protein and multiple proteins.
  • claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.
  • Numeric ranges are inclusive of the numbers defining the range. All numbers should be understood to encompass the midpoint of the integer above and below the integer i.e., the number 2 encompasses 1.5-2.5. The number 2.5 encompasses 2.45-2.55 etc. When sample numerical values are provided, each alone may represent an intermediate value in a range of values and together may represent the extremes of a range unless specified.
  • buffering agent refers to an agent that allows a solution to resist changes in pH when acid or alkali is added to the solution.
  • suitable non-naturally occurring buffering agents include, for example, any of Tris, HEPES, TAPS, MOPS, tricine, and MES.
  • corresponding to refers to positions that lie across from one another when sequences are aligned, e.g., by the BLAST algorithm.
  • An amino acid position in a functional or structural motif in one polymerase may correspond to a position within a functionally equivalent functional or structural motif in another polymerase.
  • DNA polymerase refers to an enzyme that is capable of replicating DNA and optionally may have exonuclease activity.
  • DNA template refers to the DNA strand read by a DNA polymerase and of which a copy is synthesized.
  • “Family D polymerase” refers to a heterodimeric archaeal DNA polymerase having a small exonuclease subunit (DPI) and a large polymerase subunit (DP2).
  • Family D polymerases may be produced in cells in an immature form and then undergo post-translational processing (e.g ., removal of amino-terminal sequences, inteins, or both). Examples of Family D polymerases may include 9°N PolD (Euryarchaeota), Genbank Accession No. KPV61551.1 (Bathyarchaeota), Accession No.
  • fusion protein refers to protein composed of a plurality of polypeptide components that are un-joined in their native state. Fusion proteins may be a combination of two, three or four or more different proteins.
  • polypeptide is not intended to be limited to a fusion of two heterologous amino acid sequences.
  • a fusion protein may have one or more heterologous domains added to the N-terminus, C-terminus, and or the middle portion of the protein. If two parts of a fusion protein are “heterologous”, they are not part of the same protein in its natural state.
  • fusion proteins include a variant Family D polymerase fused to an SS07 DNA binding peptide (see for example, US Patent 6,627,424), a transcription factor (see for example, US patent 10,041,051), a binding protein suitable for immobilization such as maltose binding domain (MBP), a histidine tag (“His-tag”), chitin binding domain (CBD) or a SNAP-Tag® (New England Biolabs, Ipswich, MA (see for example US patents 7,939,284 and 7,888,090)).
  • MBP maltose binding domain
  • His-tag histidine tag
  • CBD chitin binding domain
  • SNAP-Tag® New England Biolabs, Ipswich, MA (see for example US patents 7,939,284 and 7,888,090)
  • fusion proteins include a heterologous targeting sequence, a linker, an epitope tag, a detectable fusion partner, such as a fluorescent protein, b-galactosidase, luciferase and the functionally similar peptides.
  • NTP refers to a nucleoside triphosphate including, for example, any deoxyribonucleoside triphosphate (“dNTP”) and any ribonucleoside triphosphate (“rNTP”).
  • dNTP deoxyribonucleoside triphosphate
  • rNTP ribonucleoside triphosphate
  • non-naturally occurring refers to a polynucleotide, polypeptide, carbohydrate, lipid, or composition that does not exist in nature.
  • a polynucleotide, polypeptide, carbohydrate, lipid, or composition may differ from naturally occurring polynucleotides polypeptides, carbohydrates, lipids, or compositions in one or more respects.
  • a polymer e.g ., a polynucleotide, polypeptide, or carbohydrate
  • the component building blocks e.g., nucleotide sequence, amino acid sequence, or sugar molecules.
  • a polymer may differ from a naturally occurring polymer with respect to the molecule(s) to which it is linked.
  • a “non-naturally occurring” protein may differ from naturally occurring proteins in its secondary, tertiary, or quaternary structure, by having a chemical bond (e.g., a covalent bond including a peptide bond, a phosphate bond, a disulfide bond, an ester bond, and ether bond, and others) to a polypeptide (e.g., a fusion protein), a lipid, a carbohydrate, or any other molecule.
  • a chemical bond e.g., a covalent bond including a peptide bond, a phosphate bond, a disulfide bond, an ester bond, and ether bond, and others
  • a “non-naturally occurring” polynucleotide or nucleic acid may contain one or more other modifications (e.g., an added label or other moiety) to the 5’- end, the 3’ end, and/or between the 5’- and 3 ’-ends (e.g., methylation) of the nucleic acid.
  • a “non-naturally occurring” composition may differ from naturally occurring compositions in one or more of the following respects: (a) having components that are not combined in nature, (b) having components in concentrations not found in nature, (c) omitting one or components otherwise found in naturally occurring compositions, (d) having a form not found in nature, e.g., dried, freeze dried, crystalline, aqueous, and (e) having one or more additional components beyond those found in nature (e.g., buffering agents, a detergent, a dye, a solvent or a preservative).
  • All publications, patents, and patent applications identified in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
  • polynucleotide copy refers to the product of polymerization activity of a DNA polymerase.
  • a polynucleotide copy may comprise deoxyribonucleotides with or without ribonucleotides.
  • position refers to the place such amino acid occupies in the primary sequence of a peptide or polypeptide numbered from its amino terminus to its carboxy terminus.
  • substitution at a position in a comparator amino acid sequence refers to any difference at that position relative to the corresponding position in a reference sequence, including a deletion, an insertion, and a different amino acid, where the comparator and reference sequences are at least 80% identical to each other.
  • a substitution in a comparator sequence, in addition to being different than the reference sequence, may differ from all corresponding positions in naturally occurring sequences that are at least 80% identical to the comparator sequence.
  • variant Family D polymerase refers to a non-naturally occurring archaeal Family D DNA polymerase that has an amino acid sequence that is less than 100% identical to the amino acid sequence of a naturally occurring DNA polymerase from archaea or has a non-naturally occurring chemical modification (e.g ., a polypeptide fused to its amino terminal or carboxy terminal end or other chemical modification).
  • a variant amino acid sequence may have at least 95%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of a naturally occurring Family D polymerase without being 100% identical to any known naturally occurring polymerase.
  • a variant Family D polymerase may have a small exonuclease subunit (DPI) and a large polymerase subunit (DP2), with the DPI subunit having or lacking exonuclease activity.
  • DPI small exonuclease subunit
  • DP2 large polymerase subunit
  • a variant Family D polymerase lacking exonuclease activity may comprise only such portion of the DPI subunit as necessary to support catalytic activity of the polymerase (DP2) subunit.
  • a steric gate amino acid blocks ribonucleotide incorporation at low ribonucleotide concentrations and altering the steric gate amino acid has been observed to reduce discrimination against ribonucleotides at low concentration.
  • family D DNA polymerases the position of the rNTP 2'-OH in the active site had not been identified.
  • the present disclosure relates to a low resolution cryo-electron microscopy structure of the Pyrococcus abyssi (P.ab) sequence bound to DNA that provides insight into the possible structure of the active site and surrounding domains.
  • amino acid positions potentially close enough to the catalytic site of the polymerase domain (DP2) to interact with a substrate NTP were identified from structural data using PyMol software (Schrodinger, LLC) and are shown in Tables 1-3.
  • Variant Family D polymerases are provided here that differ from wild type Family D DNA polymerases in their abilities to incorporate rNTPs.
  • the incorporation ratio of adenosine triphosphate to deoxyadenosine triphosphate (rA:dA) may be more than wild type.
  • Use of wild type Family D polymerases may have been limited in the past, in part, by their rNTP/dNTP selectivity.
  • Variant Family D polymerases are not naturally occurring and have at least one substitution relative to wild type in their amino acid sequences.
  • a variant Family D polymerase may have an amino acid sequence comprising a substitution in one or more of the domains shown in Table 1 (optionally, in order, from amino- to carboxy terminal ends).
  • Each of these domains corresponds to the indicated portion of wild type Family D polymerase (e.g ., Pyrococcus abyssi; SEQ ID NO: 1). Amino acid residues in each of these domains may be located in the active protein near the active site (e.g., within about 20 A).
  • a variant Family D polymerase may have an amino acid sequence comprising a substitution at one or more positions that may contact the incoming substrate nucleoside triphosphate, for example, positions shown in Table 2.
  • a variant Family D polymerase may have an amino acid sequence comprising a substitution at one or more positions that may be located in the protein near the active site ( e.g ., within about 12 A), for example, positions shown in Table 3.
  • a variant Family D polymerase may comprise one or more substitutions at one or more positions selected from positions corresponding to positions 106-161, 243-267, 326-330, 361-365, 385-397, 441-451, 657-667, 822-829, 919-928, 940-962, and 981- 997 of SEQ ID NO:l.
  • a variant Family D polymerase may have an amino acid sequence comprising one or more sequences having (a) at least 98% identity to domain I, at least 96% identity to domain II, (c) at least 80% identity to domains III and IV, (d) at least 92% identity to domain V, (e) at least 90% identity to domains VI, VII, and IX, (f) at least 87% identity to domain VIII, (g) at least 95% identity to domain X, and (h) at least 94% identity to domain XI.
  • variant Family D polymerases may have a substitution at a position corresponding to position 923 of SEQ ID NO:l or one or more substitutions in the triplet Pro-His-Thr corresponding to positions 922-924 of SEQ ID NO:l.

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Abstract

La présente invention concerne des polymérases (par exemple, des variants d'ADN polymérases de la famille D) de synthèse de polynucléotides, d'amplification de polynucléotides, de séquençage de polynucléotides, de clonage d'un polynucléotide, ou leurs combinaisons. Par exemple, un variant de polymérase de la famille D peut avoir une séquence d'acides aminés qui (a) est d'au moins 99 % identique à SEQ ID NO:1 et (b) a une substitution au niveau d'une position choisie parmi des positions correspondant aux positions 106-161, 243-267, 326-330, 361-365, 385-397, 441-451, 657-667, 822-829, 919-928, et 940-962, 981-997 de SEQ ID NO:1.
PCT/US2021/017954 2020-02-13 2021-02-12 Variants d'adn polymérases de la famille d Ceased WO2021163559A1 (fr)

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EP1196583B1 (fr) * 1999-04-21 2006-07-12 Centre National De La Recherche Scientifique (Cnrs) Adn polymerase de type ii de pyrococcus abyssi
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