Classifications

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  • 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)
  • C12N9/1264—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal nucleotidyl transferase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • 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
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
  • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• • 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)
  • C12Y207/07031—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase

Definitions

• the present invention falls within the field of enzyme enhancement.
• the present invention relates to an improved variant of a polX family DNA polymerase, a nucleic acid encoding this variant, the production of this variant in a host cell, its use for the synthesis of a nucleic acid molecule without a template strand and a kit for synthesizing a nucleic acid molecule without a template strand.
• polymerase DNAs of the polX family are involved in a wide range of biological processes, particularly in the mechanisms of DNA repair or error correction occurring in DNA sequences. These enzymes are capable of introducing nucleotides into the excised nucleic acid strands following the identification of errors in the sequence.
• Polymerase DNAs of the polX family include ⁇ (Pol ⁇ ), ⁇ (Pol ⁇ ), ⁇ (Pol ⁇ ), yeast IV (Pol IV) and terminal deoxyribonucleotidyl transferase (TdT) polymerase DNAs.
• the TdT in particular is widely used in enzymatic synthesis processes of nucleic acid molecules.
• DNA polymerases generally allow only the incorporation of natural nucleotides. In all cases, the natural DNA polymerases lose their catalytic activity in the presence of unnatural nucleotides, and in particular nucleotides modified in 3 'OH, having a greater steric hindrance than the natural nucleotides.
• modified nucleotides may be useful for some specific applications. It has therefore been necessary to develop enzymes capable of catalyzing the synthesis of a nucleic acid strand by incorporating such nucleotides. DNA polymerase variants have thus been developed for the purpose of functioning with nucleotides having important structural modifications.
• the present invention removes certain technological barriers that prevent the use on an industrial scale of DNA polymerases for the enzymatic synthesis of nucleic acids.
• the present invention thus provides variants of DNA polymerases of the polX family capable of synthesizing a nucleic acid in the absence of a template strand and able to use modified nucleotides.
• the variants developed have modified nucleotide incorporation capabilities much higher than those of natural DNA polymerases from which they are derived.
• the variants of DNA polymerases that are the subject of the present invention are particularly effective for the incorporation of nucleotides exhibiting changes in the sugar level.
• the inventors have developed variants having an increased catalytic pocket volume relative to that of the DNA polymerases from which they originate, favoring the incorporation of modified nucleotides which are more cumbersome than the natural nucleotides.
• the variants of DNA polymerases of the polX family that are the subject of the present invention comprise at least one mutation on an amino acid intervening directly at the level of the catalytic cavity of the enzyme, or allowing the deformation of the contours of this cavity to accommodate the steric hindrance due to the changes present at the nucleotide level.
• the introduced mutations allow the broadening of the catalytic cavity of the enzyme in which the 3'-OH end of the modified nucleotides is housed.
• the mutations carried out allow the inflation or increase of the volume of the catalytic cavity, the increase of the access to the catalytic pocket by the nucleotides modified in 3'-OH and / or confer the necessary flexibility to the structure of the enzyme to allow it to accommodate sterically important modifications of nucleotides modified in 3'-OH.
• the modified nucleotide penetrates into the heart of the catalytic pocket whose access is enlarged and adopts an optimal spatial conformation, a phosphodiester bond between the 3'-OH end of the last nucleotide of the nucleic acid strand and the 5'-triphosphate end of the modified nucleotide is created.
• the subject of the invention is therefore a variant of a DNA polymerase of the polX family capable of synthesizing a nucleic acid molecule without a template strand, or a variant of a functional fragment of such a polymerase, said variant comprising at least one a mutation of a residue at at least one position selected from the group consisting of T331, G332, G333, F334, R336, K338, H342, D343, V344, D345, F346, A397, D399 D434, V436, A446, L447, L448, G449, W450, G452, R454, Q455, F456, E457, R458, R461, N474, E491, D501, Y502, 1503, P505, R508, N509 and A510, or a functionally equivalent residue, the positions indicated being determined by alignment with SEQ ID No. 1.
• the variant is capable of synthesizing a strand of DNA or an RNA strand.
• the present invention relates in particular to a variant of a DNA polymerase of the polX family and in particular of a yeast Pol IV, Pol ⁇ or wild-type TdT, and comprising the selected mutation (s).
• the variant according to the present invention is a variant of the TdT of sequence SEQ ID No. 1 or a homologous sequence which has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the sequence of SEQ ID No. 1, and carries the selected mutation (s).
• the invention also relates to a nucleic acid encoding a variant of a DNA polymerase of the polX family according to the present invention, an expression cassette comprising a nucleic acid according to the present invention and a vector comprising a nucleic acid or a cassette of expression according to the present invention.
• the nucleic acid encoding the variant of the present invention may be that of the mature form or the precursor form of the DNA polymerase according to the present invention.
• the present invention also relates to the use of a nucleic acid, an expression cassette or a vector according to the present invention to transform or transfect a host cell. It further relates to a host cell comprising a nucleic acid, an expression cassette or a vector encoding a DNA polymerase of the polX family according to the present invention. It relates to the use of such a nucleic acid, such an expression cassette, such a vector or such a host cell to produce a variant of a DNA polymerase of the polX family according to the present invention. .
• It also relates to a method for producing a variant of a polX family DNA polymerase according to the present invention comprising the transformation or transfection of a host cell with a nucleic acid, an expression cassette or a vector according to the invention. the present invention, culturing the transformed / transfected host cell under culture conditions permitting expression of the nucleic acid encoding said variant, and optionally harvesting the variant of a DNA polymerase of the polX family produced by the host cell.
• the host cell may be prokaryotic or eukaryotic.
• the host cell may be a microorganism, preferably a bacterium, a yeast or a fungus.
• the host cell is a bacterium, preferably E. coli.
• the host cell is a yeast, preferably P. pastoris or K. lactis.
• the host cell is a mammalian cell, preferably a COS7 or CHO cell.
• the invention also relates to the use of a variant of a DNA polymerase of the polX family according to the present invention for synthesizing a nucleic acid molecule without strand. matrix, from nucleotides modified in 3'-OH.
• the DNA polymerase variant of the polX family according to the present invention can also be used, in the context of the invention, to synthesize a nucleic acid molecule without a template strand, from unmodified nucleotides or from a mixture of modified and unmodified nucleotides.
• the invention also proposes a process for the enzymatic synthesis of a nucleic acid molecule without a template strand, according to which a primer strand is contacted with at least one nucleotide, preferably a nucleotide modified at 3 '-OH, in the presence of a variant of a DNA polymerase of the polX family according to the invention.
• the implementation of the method may in particular be carried out using a purified variant, a culture medium of a host cell transformed to express said variant, and / or a cell extract of such a host cell.
• the subject of the invention is also a kit for the enzymatic synthesis of a nucleic acid molecule without a template strand comprising at least one variant of a DNA polymerase of the polX family according to the invention, nucleotides, preferably modified nucleotides. at 3'-OH, and optionally at least one primer strand, or nucleotide primer, and / or a reaction buffer.
• Figure 1 SDS-PAGE gel of fractions of a TdT variant according to an exemplary embodiment of the invention
• M Molecular weight marker, 1: Centrifugate before loading, 2: Centrifugate after loading, 3: Wash buffer after loading; 4: Elution fraction 3 mL; 5: Elution fraction 30 mL; 6: Peak elution pool; 7: Concentration);
• FIG. 2 Alignment of the amino acid sequences of DNA polymerases Poi ⁇ of Homo sapiens (UniProtKB Q9NP87), Pol ⁇ of Pan troglodytes (UniProtKB H2QUI0), Pol ⁇ of Mus musculus (Uni ProtKB Q924W4), TdT of Canis lupus familiaris (UniProtKB F1 P657), Mus musculus TdT (UniProtKB Q3UZ80), Galius gailus TdT (UniProtKB P36195) and Homo sapiens TdT (Uni ProtKB P04053) obtained using the Mutalin online alignment software (in uv rni: ii dno ii ... inra. jY s or k ; :;; - m- ÎÎ to in.hh on;
• FIG. 3 Comparison of the activity of a truncated wild-type TdT of sequence SEQ ID No. 3 and of several variants of this truncated TdT comprising various substitutions given by the Table 1, in the presence of a primer previously radiolabeled 5 'and modified nucleotides 3'-O-amino-2', 3'-dideoxyadenosine-5'-triphosphate (gel ONH2) or modified nucleotides 3'-biot- EDA-2 ', 3'-dideoxyadenosine-5'-triphosphate (Biot-EDA gel); on SDS-PAGE gel (No: no enzyme present, wt: truncated wild tdT of sequence SEQ ID No. 3, DSi: Variants i defined in Table 1);
• FIG. 4 Study of the activity of the DS124 variant according to the invention (see Table 1), in the presence of a primer previously radioactively labeled at 5 'and various nucleotides modified with 3'-O-amino-2', 3 ' -dideoxyadenosine-5'-triphosphate on SDS-PAGE gel;
• FIG. 5 Study of the activity of the DS22, DS24, DS124, DS125, DS126, DS127 and DS128 variants in the presence of a primer previously radioactively labeled at 5 'and various nucleotides modified with 3'-O-amino-2', 3'-dideoxyadenosine-5'-triphosphate on SDS-PAGE gel;
• Figure 6 Synthesis of a strand of sequence DNA: 5'-GTACGCTAGT-3 '(SEQ ID NO: 15) following the 5' sequence primer - AAAAAAAAAAGGGG-3 '(SEQ ID NO: 14) ) using a variant of TDT according to the invention having the combination of substitutions R336N - R454A - E457G (DS125).
• amino acids are represented in this document by the one-letter or three-letter code according to the following nomenclature:
• A Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gin (glutamine); E: Glu (glutamic acid);
• F Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).
• percent identity between two nucleic acid or amino acid sequences within the meaning of the present invention, it is meant to designate a percentage of nucleotides or identical amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed over their entire length.
• the best alignment or alignment is the alignment for which the percentage identity between the two sequences to be compared, as calculated below, is the highest.
• sequence comparisons between two nucleic acid or amino acid sequences are traditionally performed by comparing these sequences after optimally aligning them, said comparison being made by segment or comparison window to identify and compare the local regions of the sequence. sequence similarity.
• the optimal alignment of the sequences for comparison can be performed, besides manually, using the local homology algorithm of Smith and Waterman (1981) (Ad App Math 2: 482), by means of the local homology algorithm of Neddleman and Wunsch (1970) (J. Mol Biol 48: 443), using the similarity search method of Pearson and Lipman (1988) (Proc Natl Acad Sci USA). 85: 2444), using computer software using these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), using the alignment software. Mutalin line
• the percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences by comparison window in which the region of the nucleic acid or amino acid sequence to be compared. may include additions or deletions relative to the reference sequence for optimal alignment between these two sequences.
• the percentage of identity is calculated by determining the number of identical positions for which the nucleotide or amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window. and multiplying the result obtained by 100 to obtain the percentage identity between these two sequences.
• the subject variants of the present invention are described according to their mutations on specific residues, whose positions are determined by alignment with, or reference to, the enzymatic sequence SEQ ID No. 1.
• any variant bearing these same mutations on functionally equivalent residues is also targeted.
• functionally equivalent residue is meant a residue in a sequence of a DNA polymerase of the polX family of sequence homologous to SEQ ID No. 1 and having an identical functional role.
• Functionally equivalent residues are identified using sequence alignments, for example using Mutalin online alignment software 10881-10890). After alignment, the functionally equivalent residues are at homologous positions on the various sequences considered.
• sequence alignments and the identification of functionally equivalent residues can be between any of the polX family DNA polymerases and their natural variants, including interspecies variants.
• the residue L40 of the human TdT (UniProtKB P04053) is functionally equivalent to the residue M40 of the chicken TdT (UniProtKB P36195) and to the residue V40 of the tr ⁇ of Pan troglodytes (UniProtKB H2QUI0), said residues being considered after sequence alignment ( Figure 2).
• “functional fragment” is meant a DNA polymerase fragment of the polX family exhibiting DNA polymerase activity.
• the fragment may comprise 100, 200, 300, 310, 320, 330, 340, 350, 360, 370, 380 or more consecutive amino acids of a DNA polymerase of the polX family.
• the fragment comprises 380 consecutive amino acids of a DNA polymerase of the polX family consisting of the catalytic fragment of said enzyme.
• the terms "mutant” and “variant” may be used interchangeably to refer to polypeptides derived from DNA polymerases of the polX family, or derived from functional fragments of such DNA polymerases, and in particular a TdT such as Murine TdT according to the sequence SEQ ID No.
• variants comprising an alteration, namely a substitution, an insertion and / or deletion, at one or more positions and having a DNA polymerase activity.
• the variants can be obtained by various techniques well known in the art.
• exemplary techniques for modifying the DNA sequence encoding the wild-type protein include, but are not limited to, site-directed mutagenesis, random mutagenesis and the construction of synthetic oligonucleotides.
• substitution or “mutation” as used herein with respect to an amino acid position or residue means that the amino acid in the position under consideration has been modified with respect to the amino acid of the amino acid protein. wild type of reference. Such modifications include substitutions, deletions and / or insertions of one or more amino acids, and especially 1 to 5, 1 to 4, 1 to 3, 1 to 2 amino acids, at one or more positions, and especially 1 , 2, 3, 4, 5 or more positions.
• substitution in connection with an amino acid position or residue, means that the amino acid in the particular position has been replaced by another amino acid than that in the wild-type or parent DNA polymerase.
• substitution refers to the replacement of one amino acid residue with another selected from the 20 natural amino acid residues, the naturally occurring rare amino acid residues (e.g. hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylsilyl, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine), and rare unnatural amino acid residues, often made synthetically (eg, norleucine, norvaline and cyclohexyl-alanine).
• rare amino acid residues e.g. hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylsilyl, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methyl
• substitution refers to the replacement of one amino acid residue with another selected from the 20 naturally occurring standard amino acid residues (G, P, A, V, L, I, M , C, F, Y, W, H, K, R, Q, N, E, D, S and T).
• the substitution can be a conservative or non-conservative substitution.
• the conservative substitutions are made within the same group of amino acids, among the basic amines (arginine, lysine and histidine), the acidic amino acids (glutamic acid and aspartic acid), the polar amino acids (glutamine and asparagine) , hydrophobic amino acids (methionine, leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine and threonine).
• the basic amines arginine, lysine and histidine
• the acidic amino acids glutmic acid and aspartic acid
• the polar amino acids glutamine and asparagine
• hydrophobic amino acids methionine, leucine, isoleucine and valine
• aromatic amino acids phenylalanine, tryptophan and tyrosine
• small amino acids glycine, alanine, serine and threon
• R454F indicates that the amino acid residue at position 454 of SEQ ID NO: 1 (arginine, R) is replaced by a phenylalanine (F).
• N474S / T / N / Q means that the amino acid residue at position 474 (Asparagine, N) can be replaced by serine (S), threonine (T), asparagine (N) or glutamine (Q) .
• the sign "+" indicates a combination of substitutions.
• the invention relates to DNA polymerase variants of the polX family (EC 2.7.7.7, Advances in Protein Chemistry, Vol 71, 401-440) capable of synthesizing a nucleic acid molecule without a template strand, and in particular a strand of DNA or RNA.
• the DNA polymerases of the polX family include DNA polymerase ⁇ (UniProt P06746 in humans, Q8K409 in mice), ⁇ , ⁇ (UniProt Q9UGP5 in humans, Q9QUG2 and Q9QXE2 in mice) and ⁇ (UniProt Q9NP87 in humans, Q9JIW4 in mice), Pol4 (UniProt A7TER5 in yeast Vanderwaltozyma polyspora, P25615 in yeast Saccharomyces cerevisiae), and terminal deoxyribonucleotidyl transferase or TdT (EC 2.7.7.31, UniProt P04053 in human, P09838 in mice).
• the invention more particularly relates to a DNA polymerase variant of the polX family capable of synthesizing a nucleic acid molecule without template strand, or a variant of a functional fragment of such a polymerase, said variant comprising at least mutating a residue at at least one position selected from the group consisting of T331, G332, G333, F334, R336, K338, H342, D343, V344, D345, F346, A397, D399, D434, V436, A446, L447 , L448, G449, W450, G452, R454, Q455, F456, E457, R458, R461, N474, E491, D501, Y502, 1503, P505, R508, N509 and A510, or a functionally equivalent residue, the indicated positions being determined by alignment with, or reference to, the sequence SEQ ID No. 1.
• the variant is capable of one strand of DNA and / or one strand of RNA.
• compositions comprising at least one mutation or “comprising at least one mutation”, it is meant that the variant has one or more mutations as indicated with respect to the polypeptide sequence SEQ ID No. 1, but that it may exhibit other modifications, including substitutions, deletions or additions.
• the mutation of one or more residues at the above positions allows the expansion of the catalytic pocket (targeting, for example, the positions W450, D434, D435, H342, D343, T331, R336, D399, R461, and / or R508), increasing accessibility to the catalytic bag (targeting for example positions R458, E455, R454, A397, K338, and / or N509), and / or providing greater flexibility to the structure of the enzyme allowing it to receive modified nucleotides having a large steric hindrance (targeting for example the positions V436, F346, V344, F334, M330, L448, E491, E457 and / or N474).
• the variant objects of the present invention may be variants of Pol IV, Pol ⁇ , ⁇ , ⁇ or TdT, preferentially variants of Pol IV, Pol ⁇ , or TdT.
• the variants may be chimeric enzyme variants, for example combining portions of different sequences of at least two DNA polymerases of the polX family.
• the variant has at least 60% identity with the sequence according to SEQ ID No. 1, preferably at least 70%, 80%, 85%, 90%, 95%, 96%, 97%. , 98%, 99% and less than 100% identity with the sequence according to SEQ ID No. 1.
• the mutation may consist of a substitution, deletion or addition of one or more amino acid residues.
• the annotation X is used, which indicates that the codon coding for the residue in question is replaced by a STOP codon, therefore all the following amino acids and the residue in question are deleted.
• the D501X mutation means that the enzyme terminates at the residue preceding aspartic acid (D) at position 501, i.e. leucine (L) at position 500, all residues above beyond having been deleted.
• the annotation 0 denotes a simple one-off deletion of the residue under consideration.
• the D5O10 mutation means that aspartic acid (D) at position 501 has been deleted.
• the variant according to the invention comprises at least one mutation of a residue at at least one position selected from the group consisting of T331, G332, G333, F334, R336, D343, L447, L448, G449, W450, G452, R454, Q455, E457 and R508, or a functionally equivalent residue, preferentially at least one mutation of a residue at at least one position selected from the group consisting of R336, R454, E457, or a functionally equivalent residue, the positions indicated being determined by alignment with SEQ ID No. 1.
• said variant comprises at least one mutation of a residue at at least two positions selected from the group consisting of R336, R454 and E457, preferentially a mutation of a residue at said three positions R336, R454 and E457 , or a functionally equivalent residue, the positions indicated being determined by alignment with SEQ ID No. 1.
• the variant further comprises at least one mutation of a residue in at least the semi-conserved region of sequence X1X2GGFR1R2GKX3X4 (SEQ ID NO: 4), wherein
• Xi represents a residue selected from M, I, V, L
• X2 represents a residue selected from T
• A M
• Q X3 represents a residue selected from M, K, E, Q, L, S, P, R, D
• X 4 represents a residue selected from T, I, M, F, K, V, Y, E, Q, H, S, R, D.
• said variant has at least one substitution of a residue at at least one position Ri, R 2 and / or K of the semi-conserved region of sequence SEQ ID No. 4.
• the variant further comprises at least one mutation of a residue in at least one semi-conserved region of XiX 2 sequence LGX 3 X4GSRiX 5 X 6 ER 2 (SEQ ID NO: 5) in which
• Xi represents a residue selected from A, C, G, S
• X 2 represents a residue chosen from L, T, R
• X 3 represents a residue selected from W, Y
• X 4 represents a residue selected from T, S, I
• X5 represents a residue selected from Q, L, H, F, Y, N, E, D or O
• X 6 represents a residue selected from F, Y
• said variant has at least one substitution of a residue at at least one position S, Ri and / or E of the semi-conserved region of sequence SEQ ID No. 5.
• the variant further comprises at least one mutation of a residue in at least one semi-conserved region of LXiYX sequence 2 X 3 PX 4 X5RNA (SEQ ID No. 6) in which
• Xi represents a residue selected from D, E, S, P, A, K
• X 2 represents a residue selected from I, L, M, V, A, T
• X 3 represents a residue selected from E, Q, P, Y, L, K, G, N
• X4 represents a residue selected from W, S, V, E, R, Q, T, C, K, H
• X5 represents a residue selected from E, Q, D, H, L.
• said variant has at least one deletion of the residue at the X1 position and / or at least one substitution at the R and / or N positions of the semi-conserved region of sequence SEQ ID No. 6.
• the variant comprises a substitution of a residue for at least one position selected from the group consisting of R336, K338, H342, A397, S453, R454, E457, N474, D501, Y502, 1503, R508.
• a functionally equivalent residue preferably a substitution of a residue at at least one position selected from the group consisting of R336, A397, R454, E457, N474, D501, Y502 and 1503, or a functionally equivalent residue, plus preferably at least one substitution of a residue at at least one position selected from the group consisting of R336, R454, E457, or a functionally equivalent residue, the positions indicated being determined by alignment with SEQ ID No. 1.
• the invention preferably relates to a variant of a polX family DNA polymerase comprising at least one of the group consisting of R336K / H / G / N / D, K338A / C / G / S / T / N, H342A.
• the variant comprises a substitution of a residue at at least two positions selected from the group consisting of R336, R454, E457, or a functionally equivalent residue, preferably a substitution of a residue at said three positions, or a functionally equivalent residue, the positions indicated being determined by alignment with SEQ ID No. 1.
• the substitutions are chosen from the group consisting of R336K / H / G / N / D, R454F / Y / W / A and E457N / D / G / S / T, preferentially from the group consisting of R336N / G, R454A and E457G / N / S / T.
• the variant comprises at least one substitution according to E457G / N / S / T.
• the variant comprises a combination of substitutions selected from the group mentioned above.
• the combination may consist of 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 substitutions selected from this group.
• the invention more particularly relates to variants of a DNA polymerase of the polX family capable of synthesizing a nucleic acid molecule, such as a strand of DNA or RNA without a template strand, or of a fragment functional group of such a polymerase, said variants comprising at least one combination of mutations described in Table 1, the positions indicated being determined by alignment with SEQ ID No. 1.
• the polX family DNA polymerase variant comprises a combination of R336G-E457N substitutions; R336N - E457N; R336N - R454A - E457N; R336N - R454A - E457G; R336N - E457G; and R336G - R454A - E457N.
• the variant is a chimeric construction of DNA polymerases of the polX family.
• chimeric construction is meant a chimeric enzyme constituted by the addition, and especially the fusion or conjugation, of one or more determined sequences of a member of the polX family enzyme to replace one or more homologous sequences. in the variant DNA polymerase considered.
• the invention provides a TdT variant of sequence SEQ ID No. 1 comprising, besides one or more point mutations at one and / or the other of the above positions, a substitution of residues between the positions. C378 to L406, or functionally equivalent residues, by residues H363 to C390 of the ⁇ polymerase of sequence SEQ ID No. 2, or functionally equivalent residues.
• the subject variants of the present invention may have a deletion of one or more successive amino acid residues at the N-terminal portion. These deletions may in particular target one or more enzymatic domains involved in binding with other proteins and / or involved in cellular localization.
• the polypeptide sequence of the TdT comprises at the N-terminal a BRCT domain of interaction with other proteins such as Ku70 / 80 and a nucleus localization domain (NLS).
• the variant is a TdT variant of sequence SEQ ID No. 1 having, in addition to one or more of the mutations described above, a deletion of residues 1-129 corresponding to the N-terminus of the wild TdT.
• mutagenesis strategies may be guided by known information such as natural variant sequences, sequence comparison with bound proteins, physical properties, study of a three-dimensional structure or computer simulations involving several entities.
• the present invention relates to a nucleic acid encoding a variant of a polX family DNA polymerase capable of synthesizing a template-free nucleic acid molecule according to the present invention.
• the present invention also relates to a cassette for expressing a nucleic acid according to the present invention. It also relates to a vector comprising a nucleic acid or an expression cassette according to the present invention.
• the vector may be selected from a plasmid and a viral vector.
• the nucleic acid encoding the DNA polymerase variant may be DNA (cDNA or gDNA), RNA, a mixture of both. It can be in simple chain or duplex form or a mixture of both. It may comprise modified nucleotides, including for example a modified linkage, a modified purine or pyrimidine base, or a modified sugar. It can be prepared by any method known to those skilled in the art, including chemical synthesis, recombination, mutagenesis, etc.
• the expression cassette comprises all the elements necessary for the expression of the variant of a DNA polymerase of the polX family capable of synthesizing a nucleic acid molecule without a template strand according to the present invention, in particular the elements necessary for the transcription and to translation in the host cell.
• the host cell may be prokaryotic or eukaryotic.
• the expression cassette comprises a promoter and a terminator, optionally an amplifier.
• the promoter may be prokaryotic or eukaryotic.
• prokaryotic promoters are: LacI, LacZ, pLacT, ptac, pARA, pBAD, T3 or T7 bacteriophage RNA polymerase promoters, polyhedrin promoter, phage lambda PR or PL promoter.
• preferred eukaryotic promoters are: early CMV promoter, HSV thymidine kinase promoter, SV40 early or late promoter, mouse L-metallothionein promoter, and LTR regions of some retroviruses.
• the skilled person can advantageously refer to the work of Sambrook et al. (1989) or the techniques described by Fuller et al. (1996) Immunology in Current Protocols in Molecular Biology.
• the present invention relates to a vector carrying a nucleic acid or an expression cassette encoding a variant of a DNA polymerase of the polX family capable of synthesizing a nucleic acid molecule without a template strand according to the present invention.
• the vector is preferably an expression vector, i.e. it comprises the elements necessary for the expression of the variant in the host cell.
• the host cell may be a prokaryote, for example E. coli, or a eukaryotic.
• the eukaryote may be a lower eukaryote such as a yeast (for example, P. Pastoris or K.
• the cell may be a mammalian cell, for example COS (green monkey cell line) (e.g., COS 1 (ATCC CRL-1650), COS7 (ATCC CRL-1651), CHO (US 4,889,803; US 5,047,335, CHO- K1 (ATCC CCL-61)), mouse cells and human cells
• COS green monkey cell line
• the vector may be a plasmid, a phage, a phagemid, a cosmid, a virus, a YAC, a BAC, an Agrobacterium pTi plasmid, etc.
• the vector may preferably comprise one or more elements selected from an origin of replication, a multiple cloning site and a selection gene.
• the vector is a plasmid.
• prokaryotic vectors are: pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pBR322, and pRIT5 (Pharmacia), pET (Novagen).
• Non-exhaustive examples of eukaryotic vectors are: pWLNEO, pSV2CAT, pPICZ, pcDNA3.1 (+) Hyg (Invitrogen), pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pCI-neo (Stratagene), pMSG, pSVL (Pharmacia); and pQE-30 (QLAexpress).
• the viral vectors may be non-exhaustively adenoviruses, AAVs, HSVs, lentiviruses, etc.
• the expression vector is a plasmid or a viral vector.
• the sequence coding for the variant according to the present invention may comprise or not comprise a signal peptide.
• a signal peptide In the case where it does not include a methionine may be optionally added to the N-terminus.
• a heterologous signal peptide can be introduced. This heterologous signal peptide can be derived from a prokaryote such as E. coli or from a eukaryotic, especially a mammalian, insect or yeast cell.
• the present invention relates to the use of a polynucleotide, an expression cassette or a vector according to the present invention to transform or transfect a cell.
• the present invention relates to a host cell comprising a nucleic acid, an expression cassette or a vector encoding a variant of a polX DNA polymerase capable of synthesizing a nucleic acid molecule without a template strand and its use to produce a variant of a polX family DNA polymerase capable of synthesizing a nucleic acid molecule without a recombinant template strand according to the present invention.
• the term "host cell” encompasses the daughter cells resulting from the culture or growth of this cell.
• the cell is non-human and non-embryonic. It also relates to a method for producing a variant of a DNA polymerase of the polX family capable of synthesizing a nucleic acid molecule without recombinant template strand according to the present invention comprising the transformation or transfection of a cell with a polynucleotide, an expression cassette or a vector according to the present invention; culturing the transfected / transformed cell; and harvesting the variant of a DNA polymerase of the polX family capable of synthesizing a nucleic acid molecule without template strand produced by the cell.
• the method for producing a variant of a polX family DNA polymerase capable of synthesizing a recombinant template-free nucleic acid molecule according to the present invention comprising providing a cell comprising a polynucleotide, an expression cassette or a vector according to the present invention; culturing the transfected / transformed cell; and harvesting the variant of a polX family DNA polymerase capable of synthesizing a template-free nucleic acid molecule produced by the cell.
• the cell may be transformed / transfected transiently or stably by the nucleic acid encoding the variant.
• This nucleic acid may be contained in the cell as an episome or in a chromosomal form.
• the DNA polymerase variants according to the present invention are particularly interesting for the synthesis of nucleic acids without a template strand. More particularly, the variants according to the invention have an increased catalytic pocket particularly adapted for the synthesis of nucleic acid by means of modified nucleotides having a bigger bulk than the natural nucleotides.
• the variants according to the invention may in particular make it possible to incorporate modified nucleotides, such as those described in application WO2016 / 034807, into a nucleic acid strand.
• the kinetics of incorporation of DNA polymerase variants is greatly improved compared with the kinetics of incorporation of wild-type DNA polymerase.
• variants may advantageously be used in the context of a high performance enzymatic DNA synthesis method.
• the subject of the invention is therefore also a use of a variant of a DNA polymerase of the polX family according to the present invention for synthesizing a nucleic acid molecule. without template strand, from nucleotides modified in 3 '-OH, and in particular those described in application WO2016034807.
• the subject of the invention is also a process for the enzymatic synthesis of a nucleic acid molecule without a template strand, according to which a primer strand is brought into contact with at least one nucleotide, preferably a nucleotide modified with 3'-OH, in presence of a variant of a DNA polymerase of the polX family according to the invention.
• variants according to the invention can be used to implement the synthesis method described in application WO2015 / 159023.
• the subject of the invention is also a kit for the enzymatic synthesis of a nucleic acid molecule without a template strand comprising at least one variant of a DNA polymerase of the polX family according to the invention, nucleotides, preferably modified nucleotides. in 3'-OH, and optionally at least one nucleotide primer.
• the truncated mouse TdT gene was generated from plasmid pET28b whose construction is described in [Boulé et al., 1998, Mol. Biotechnol, 10, 199-208].
• the corresponding sequence SEQ ID No. 3 (corresponding to SEQ ID No. 1 truncated of the first 120 amino acids) was amplified using the following primers:
• T7-pro TAATACGACTCACTATAGGG (SEQ ID NO: 7)
• ⁇ T7-ter GCTAGTTATTGCTCAGCGG (SEQ ID NO: 8) according to standard PCR and molecular biology techniques. It was cloned into a plasmid pET32 to give the vector pET32-SEQ ID No. 3.
• the plasmid pET32-SEQ ID No. 3 was first sequenced and then transformed into commercial E. coli strains BL21 (DE3) (Novagen). Colonies capable of growing on kanamycin / chloramphenicol dishes have been isolated and noted Ec-SEQ ID No. 3.
• the vector pET32-SEQ ID No. 3 was used as the starting vector. Primers with point mutation (or in some cases point mutations, if close enough) were generated from Agilent's online tool:
• the QuickChange II kit (Agilent) was used to generate the plasmids of the variants comprising the desired mutation (s).
• the mutagenesis protocol given by the manufacturer has been scrupulously respected in order to obtain a plasmid pET32-DSi (i is the number of the variant considered given in Table 1).
• plasmid pET32-DSx was first sequenced and then transformed into the commercial E. coli strains BL21 (DE3) (Novagen). Colonies capable of growing on kanamycin / chloramphenicol dishes were isolated and noted Ec-DSx.
• Ec-SEQ ID NO: 3 and Ec-DSx cells were precultured in a 250 mL Erlenmeyer flask containing 50 mL of LB medium to which appropriate amounts of kanamycin and chloramphenicol were added. The culture was incubated at 37 ° C with stirring overnight. The preculture was then used to seed a 5 L Erlenmeyer flask containing 2 L of LB medium supplemented with appropriate amounts of kanamycin and chloramphenicol. The optical density (OD) of departure was 0.01. The culture was incubated at 37 ° C with shaking. OD was regularly measured to a value between 0.6 and 0.9.
• the cell pellet frozen in the previous step was thawed in a water bath heated to 25-37 ° C. Once completely thawed it was resuspended in about 100 mL of lysis buffer. Particular attention has been paid to the re-suspension which should lead to a very homogeneous solution and in particular to the total absence of aggregates.
• the cells were lysed using a French press under a pressure of 14,000 psi.
• the lysate collected was centrifuged at high speed, 10,000 g for 1 h to 1:30.
• the centrifugate was filtered through a 0.2 ⁇ filter and collected in a tube of sufficient volume.
• TdT was purified on an affinity column. 5 mL His-Trap Crude columns (GE Life Sciences) were used with peristaltic pumps (Peristaltic Pump - MINIPULS® Evolution, Gilson). In a first step the column was equilibrated using 2 to 3 CV (column volume) of lysis buffer. The centrifugate from the previous step was then loaded onto the column at a rate of between about 0.5 and 5 mL / min. After the entire centrifugate was loaded, the column was washed with 3 CV of lysis buffer and then 3 CV of wash buffer (20 mM Tris-HCl, pH 8.3, 0.5 M NaCl, 60 mM imidazol).
• the elution buffer (20 mM Tris-HCl, pH 8.3, 0.5 M NaCl, 1 M imidazol) was injected into the column at about 0.5 to 1 ml / min for a total volume of 3 HP. Throughout the elution phase the column outlet was collected in 1 mL fractions. These fractions were analyzed by SDS-PAGE to determine which fractions contain the elution peak. Once determined, these were pooled in a single fraction and dialyzed against dialysis buffer (20 mM Tris-HCl, pH 6.8, 200 mM NaCl, 50 mM MgOAc, 100 mM [NH 4 ] 2 SO 4 .
• the activity of different variants according to the invention was determined by the following test. The results were compared with those obtained with the natural enzyme from which each of the variants is derived. Activity test
• the primer used of sequence 5 '-AAAAAAAAAAGGGG-3' (SEQ ID No. 14), was previously radioactively labeled at 5 'by means of a standard labeling protocol involving the enzyme PNK (NEB) and the use of radioactive ATP (PerkinElmer).
• Buffer 10x of 250 mM Tris-HCl pH 7.2, 80 mM MgC, 3.3 mM ZnSO 4 was used.
• the modified nucleotides used are 3'-O-amino-2 ', 3'-dideoxynucleotide-5'-triphosphate (ONH2, Firebird Biosciences) or 3'-biot-EDA-2', 3'-dideoxynucleotide-5 ' -triphosphate (Biot-EDA, Jena Biosciences), such as 3'-O-amino-2 ', 3'-dideoxyadenosine-5'-triphosphate or 3'-biot-EDA-2', 3'-dideoxyadenosine-5 ' -triphosphate for example.
• the 3'-O-amino group is a larger group attached to the 3'-OH end.
• the 3'-biot-EDA group is an extremely bulky and non-flexible group attached to the 3'-OH end.
• the performance of incorporation of a modified nucleotide given by the variants listed in Table 1, relative to the natural TdT (SEQ ID No. 3) were evaluated by carrying out simultaneous activity tests, for which only the enzyme varies.
• a denaturing 16% polyacrylamide gel (Biorad) was used for the analysis of the previous activity test.
• the gel was previously cast and allowed to polymerize. It was then mounted on an appropriately sized electrophoresis tank filled with TBE buffer (Sigma). The different samples were directly loaded onto the gel without pre-treatment.
• the gel was then subjected to a potential difference of 500 to 2000 V for 3 to 6 hours. Once the migration is satisfactory, the gel was disarmed and then transferred to an incubation cassette.
• a phosphor screen (Amersham) was used for 10 to 60 min for revelation using a Typhoon instrument (GE Life Sciences) previously set with a suitable detection mode.
• the natural TdT (wt column) is incapable of incorporating the modified nucleotides 3'-O-amino-2 ', 3'-dideoxyadenosine-5'-triphosphate as shown in FIG. comparison with the negative control (column No).
• a first group of variants (DS7 to DS34 columns) is capable of about 50% incorporation.
• a second group of variants (columns DS46 to DS73) is capable of more than 95% incorporation, sometimes more than 98%.
• a third group of variants (columns DS83 to DSI 06) is capable of incorporation between 60 and 80%.
• a first group of variants (columns DS7 to DS34) is capable of incorporation between 5 and 10% approximately.
• a second group of variants (columns DS46 to DS73) is capable of incorporation at more than 30%, sometimes more than 40%.
• a third group of variants (columns DS83 to DSI 06) is capable of incorporation between 10 and 25%.
• TdT variants according to the invention are all capable of using modified nucleotides, in particular 3'-OH, as substrate, unlike the wild-type enzyme.
• certain variants have very high levels of incorporation, even in the presence of nucleotides carrying modifications tending to increase very significantly the steric hindrance of said nucleotide.
• the enzymes are brought into contact with modified nucleotides ONH2 and incubated at 37 ° C. according to different times. The reactions are stopped in order to observe the kinetics of incorporation of DS124 and to compare it with the kinetics of the natural enzyme WT (SEQ ID No. 3).
• the primer and the buffer used are in accordance with Example 3.
• the modified nucleotides used are 3'-O-amino-2 ', 3'-dideoxynucleotides-5'-triphosphate (ONH2, Firebird Biosciences): 3'-O-amino-2', 3'-dideoxyguanosine-5'- triphosphate, 3'-O-amino-2 ', 3'-dideoxycytidine-5'-triphosphate and 3'-O-amino-2', 3'-dideoxythymidine-5'-triphosphate.
• the 3'-O-amino group is a larger group attached to the 3'-OH end.
• the negative control (column No) gives the expected size of the primer used when it has not been lengthened, that is to say when there has been no incorporation of nucleotides.
• the natural TdT (WT column) is not able to incorporate modified nucleotides (here ONH2-dGTP): we observe a band at the same level as that of column No.
• the DS124 variant is able to incorporate the modified nucleotides with an apparent efficiency of 100%.
• the different variants were put in the presence of a mixture of natural nucleotides and highly concentrated modified nucleotides.
• the concentration of the enzyme is also increased in order to shorten the incubation time and to obtain quantitative addition (see Example 4).
• the primer and the buffer used are identical to Example 3.
• the nucleotide mixture consists of natural nucleotides 2'-deoxynucleotide 5'-triphosphate (Naked, Sigma-Al drich) such as 2'-deoxyguanosine 5'-triphosphate (dGTP) and modified nucleotides 3 -O-amino-2 ', 3'-dideoxynucleotide-5'-triphosphate (ONH2, Firebird Biosciences) such as 3'-O-amino-2', 3'-dideoxyguanosine-5'-triphosphate for example.
• the 3'-O-amino group is a larger group attached to the 3'-OH end.
• the mixture consists of 90% ONH2-dGTP modified nucleotides and 10% dGTP natural nucleotides.
• the negative control (column No) gives the expected size of the primer used when it has not been elongated, that is to say when there has been no incorporation of nucleotides.
• the following samples go in pairs, each pair corresponding to the same enzyme variant tested under both conditions: in the absence and in the presence of nucleotides (in the form of a mixture when they are present).
• the first group is the DS128 variant, constituting a negative control.
• This variant has extremely low levels of nucleotide incorporation: between 5% and 10% incorporation is observed when the nucleotide mixture is present; which corresponds to the proportion of natural nucleotides present in the mixture.
• the second group consists of DS127 and DS22 variants. These variants have high levels of nucleotide incorporation: between 50% and 60% incorporation is observed when the nucleotide mixture is present. Still in this case, an over-addition band corresponding to the successive incorporation of two nucleotides is observed for these two variants. The intensity of this band corresponds to the proportion of natural nucleotides present in the nucleotide mixture.
• the last group consists of DS124, DS24, DS125 and DS126 variants. These variants have extremely high incorporation rates of nucleotides: between 80%> and 100%, for DS 124 and DS 125, when the nucleotide mixture is present. In this case, no over-addition band is present. In the case of the DS24 and DS126 variants, the proportion of non-incorporation is similar to the proportion of natural nucleotides present in the mixture.
• variants of the TdT according to the invention are capable of preferentially using the modified nucleotides among a mixture of modified nucleotides and natural nucleotides.
• these variants have extremely high levels of incorporation of the modified nucleotides and are capable of discriminating the natural nucleotides so as not to incorporate them and thus greatly improve the quality of the DNA synthesize by avoiding over-additions.
• TdT variant having the combination of R336N - R454A - E457G substitutions was generated and produced according to Example 1.
• the DS125 variant is used to synthesize the sequence: 5 '- GTACGCTAGT-3' (SEQ ID NO: 15) following the 5 '-AAAAAAAAAAGGGG-3' sequence primer (SEQ ID NO: 14).
• the primer was radioactively labeled in 5 'by means of a standard labeling protocol involving the enzyme PNK (NEB) and the use of radioactive ATP (PerkinElmer).
• the primer is attached to a solid support by interaction with a sequence capture fragment: 5'-CCTTTTTTTTTT -3 'complementary (SEQ ID No. 16).
• the capture moiety has on its 3 'end a group allowing it to react covalently with a reaction group attached to a surface.
• this group may be NH 2, the reaction group N-hydroxysuccinimide and the surface a magnetic ball (Dynabeads, Thermo Feher).
• the interaction of the primer with the capture fragment is performed under standard DNA fragment hybridization conditions.
• the modified nucleotides used are 3'-O-amino-2 ', 3'-dideoxynucleotides-5'-triphosphate (ONH 2, Firebird Biosciences) such as 3'-O-amino-2', 3'-dideoxyguanosine-5 ' -triphosphate, 3'-O-amino-2 ', 3'-dideoxycytidine-5'-triphosphate, 3'-O-amino-2', 3'-dideoxythymidine-5'-triphosphate or 3'-O-amino- 2 ', 3'-5'-triphosphate dideoxytadénosine.
• the 3'-O-amino group is a larger group attached to the 3'-OH end.
• the 10x buffer consisting of 250 mM Tris-HCl pH 7.2, 80 mM MgCl2, 3.3 mM ZnSO4 was used.
• Wash buffer L used was 25 mM Tris-HCl pH 7.2.
• the deprotection buffer D used consists of 50 mM sodium acetate pH 5.5 in the presence of 10 mM MgCl 2.
• the beads constituting the solid support on which the primers were hybridized for an equivalent total amount of primer of 35 pmol were washed several times with the buffer L. After these washes, the beads were held on a magnet and the supernatant removed in its entirety.
• the beads are collected by means of a magnet and the supernatant is removed in its entirety.
• the analysis of the activity test is carried out by migration of the different samples in a polyacrylamide gel according to the protocol described in Example 3.

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