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VARIANTS OF THERMOSTABLE DNA PRIMASES AND USES THEREOF

20250043256 ยท 2025-02-06

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to a functionally active mutated primase domain from an archaeal DNA primase belonging to the primase-polymerase family, comprising at least one amino acid substitution, wherein said mutated primase domain retains at least 50% of the template-independent terminal nucleotidyl transferase activity of the corresponding wild-type primase domain.

    Claims

    1. A functionally active mutated primase domain, wherein: said mutated primase domain comprises the N-terminal domain of a Pyrococcus sp. 12-1 archaeal DNA primase belonging to the primase-polymerase family and shares at least 80% of sequence identity with the primase domain of SEQ ID NO: 2 or SEQ ID NO: 3; said mutated primase domain comprises at least one amino acid substitution at position N217, K234, N206, L229, Y233, K236, T230, Y122, F74, F174, F219, 1231, P238, Y235, P228, S68, and/or N232 in the amino acid sequence of SEQ ID NO: 2, or at equivalent positions in SEQ ID NO: 3 as determined by sequence and/or structural alignment; and said mutated primase domain retains at least 50% of the template-independent terminal nucleotidyl transferase activity of the primase domain with SEQ ID NO: 2 or SEQ ID NO: 3.

    2. The functionally active mutated primase domain according to claim 1, wherein the at least one amino acid substitution is at position N217, K234, N206, L229, Y233, K236, T230, Y122, F74, F174, F219, 1231, and/or P238 in the amino acid sequence of SEQ ID NO: 2 or at a positionally equivalent position in SEQ ID NO: 3, wherein said mutated primase domain has at least an equivalent template-independent terminal nucleotidyl transferase activity compared to the corresponding wild-type primase domain.

    3. The functionally active mutated primase domain according to claim 1, wherein the at least one amino acid substitution is at position N217, K234, N206, L229, Y233, K236, T230, Y122, and/or F74 in the amino acid sequence of SEQ ID NO: 2 or at a positionally equivalent position in SEQ ID NO: 3, wherein said mutated primase domain has an improved template-independent terminal nucleotidyl transferase activity compared to the corresponding wild-type primase domain.

    4. The functionally active mutated primase domain according to claim 1, wherein the at least one amino acid substitution comprises: a) a substitution of N217 with an amino acid residue comprising a positively charged side chain such as a lysine (K), arginine (R), or histidine (H) residue; b) a substitution of K234 with an amino acid residue comprising a positively charged side chain such as an arginine (R), or histidine (H) residue; c) a substitution of N206 with an amino acid residue comprising a positively charged side chain such as an arginine (R), histidine (H), or lysine (K) residue; d) a substitution of L229 with an amino acid residue comprising an amine-containing side chain such as an asparagine (N), arginine (R), glutamine (Q), or lysine (K) residue, or a hydrophobic side chain such as an alanine (A), glycine (G), valine (V), isoleucine (I), or methionine (M) residue; e) a substitution of Y233 with an amino acid residue comprising a positively charged side chain such as a lysine (K), histidine (H) or arginine (R) residue, or a hydrophobic side chain such as an alanine (A), glycine (G), valine (V), isoleucine (I), leucine (L), or methionine (M) residue; f) a substitution of K236 with an amino acid residue comprising a positively charged side chain such as an arginine (R), or histidine (H) residue; g) a substitution of T230 with a cysteine (C) residue, or an amino acid residue comprising a hydrophobic side chain such as an alanine (A), glycine (G), valine (V), isoleucine (I), leucine (L), or methionine (M) residue, or a polar uncharged side chain such as a serine (S), asparagine (N), glutamine (Q), or threonine (T) residue; h) a substitution of Y122 with an amino acid residue comprising an aromatic side chain such as a histidine (H), phenylalanine (F), or tryptophan (W) residue, or a hydrophobic side chain such as an alanine (A), glycine (G), valine (V), isoleucine (I), leucine (L), or methionine (M) residue; i) a substitution of F74 with an amino acid residue comprising an aromatic side chain such as a tyrosine (Y), tryptophane (W), or histidine (H) residue, or an amine-containing side chain such as a glutamine (Q), asparagine (N), arginine (R) or lysine (K) residue; j) a substitution of F174 with an amino acid residue comprising an amine-containing side chain such as an arginine (R), glutamine (Q), asparagine (N), or lysine (K) residue, or an amino acid residue comprising an aromatic side chain such as a tyrosine (Y), tryptophane (W), or histidine (H) residue; k) a substitution of F219 with an amino acid residue comprising an aromatic side chain such as a tyrosine (Y), histidine (H), or tryptophan (W) residue; l) a substitution of 1231 with an amino acid residue comprising a hydrophobic side chain such as an alanine (A), glycine (G), valine (V), leucine (L), or methionine (M) residue, or a positively charged side chain such as a lysine (K), arginine (R), or histidine (H) residue; m) a substitution of P238 with an amino acid residue comprising a positively charged side chain such as an arginine (R), histidine (H), or lysine (K) residue; n) a substitution of Y235 with an amino acid residue comprising an aromatic side chain such as a phenylalanine (F), tryptophan (W), or histidine (H) residue; o) a substitution of P228 with an amino acid residue comprising a hydrophobic side chain such as an alanine (A), glycine (G), valine (V), isoleucine (I), leucine (L), or methionine (M) residue, or a polar uncharged side chain such as an asparagine (N), glutamine (Q), threonine (T) or serine (S) residue; p) a substitution of S68 with an amino acid residue comprising a polar uncharged side chain such as an asparagine (N), glutamine (Q), or threonine (T) residue; or q) a substitution of N232 with an amino acid residue comprising an amine-containing side chain such as an arginine (R), asparagine (N), glutamine (Q), lysine (K) or histidine (H) residue; in the amino acid sequence of SEQ ID NO: 2 or at a positionally equivalent position in SEQ ID NO: 3.

    5. The functionally active mutated primase domain according to claim1, wherein the at least one amino acid substitution comprises: a) a substitution of N217 with a lysine (K), or arginine (R) residue; preferably with a lysine (K) residue; b) a substitution of K234 with an arginine (R) residue; c) a substitution of N206 with an arginine (R) residue; d) a substitution of L229 with an asparagine (N), alanine (A), glycine (G), or arginine (R) residue; preferably with an asparagine (N) or alanine (A) residue; e) a substitution of Y233 with a lysine (K), histidine (H), arginine (R), or alanine (A) residue; preferably with a lysine (K) or histidine (H) residue; f) a substitution of K236 with an arginine (R) residue; g) a substitution of T230 with a cysteine (C), alanine (A) or serine (S) residue; preferably with a cysteine (C) residue; h) a substitution of Y122 with an alanine (A), or histidine (H) residue; preferably with a histidine (H) residue; i) a substitution of F74 with a tyrosine (Y), or glutamine (Q) residue; preferably with a tyrosine (Y) residue; j) a substitution of F174 with an arginine (R) residue; k) a substitution of F219 with a tyrosine (Y) residue; l) a substitution of 1231 with an alanine (A), lysine (K) or arginine (R) residue; m) a substitution of P238 with an arginine (R) residue; n) a substitution of Y235 with a phenylalanine (F) or tryptophan (W) residue; preferably with a phenylalanine (F) residue; o) a substitution of P228 with an alanine (A) or asparagine (N) residue; p) a substitution of S68 with an asparagine (N) residue; or q) a substitution of N232 with an arginine (R) residue; in the amino acid sequence of SEQ ID NO: 2 or at a positionally equivalent position in SEQ ID NO: 3.

    6. The functionally active mutated primase domain according to claim 1, wherein the at least one amino acid substitution is N217K, N206R, K234R, L229N, Y233H, Y233K, K236R, T230C, Y122H, F74Y, L229A, F174R, Y122A, F219Y, L229G, L229R, T230A, T230S, 1231A, 1231R, 1231K, Y233A, Y233R, P238R, Y235F, Y235W, F74Q, P228N, P228A, N217R, S68N, and/or N232R in the amino acid sequence of SEQ ID NO: 2 or at a positionally equivalent position in SEQ ID NO: 3.

    7. The functionally active mutated primase domain according to claim 1, comprising at least two amino acid substitutions at positions N217 and K234; N217 and K236; N217 and N206; Y122 and Y233; Y122 and N217; Y122 and K234; Y122 and K236; Y122 and N206; Y122 and T230; F74 and N217; F74 and K234; K234 and T230; K236 and T230; N206 and Y233; N206 and T230; or T230 and N217; in the amino acid sequence of SEQ ID NO: 2 or at a positionally equivalent position in SEQ ID NO: 3.

    8. The functionally active mutated primase domain according to claim 1, comprising at least three amino acid substitutions at positions positionally equivalent to N217, N206 and Y233; N217, N206 and Y122; or N217, N206 and K234; in the amino acid sequence of SEQ ID NO: 2 or at a positionally equivalent position in SEQ ID NO: 3.

    9. The functionally active mutated primase domain according to claim 1, wherein said mutated primase domain is devoid of ab-initio single-stranded nucleic acid synthesis activity.

    10. The functionally active mutated primase domain according to claim 1, wherein the primase domain is fused in N-terminal or C-terminal to a processivity factor through a linker, wherein the processivity factor is a single-stranded DNA-binding protein.

    11. The functionally active mutated primase domain according to claim 10, wherein the single-stranded DNA-binding protein is from Thermotoga neapolitana.

    12. A nucleic acid encoding the functionally active mutated primase domain according to claim 1, an expression vector comprising said nucleic acid, or a host cell comprising said expression vector.

    13. A method for template-independent synthesis of nucleic acids, comprising iteratively contacting an initiator sequence comprising a 3-end nucleotide with a free 3-hydroxyl group, with at least one nucleoside triphosphate, or a combination of nucleoside triphosphates, in the presence of a functionally active mutated primase domain according to claim 1, thereby covalently binding said nucleoside triphosphate to the free 3-hydroxyl group of the 3-end nucleotide.

    14. The method according to claim 13, wherein the template-independent synthesis of nucleic acids is carried out at a temperature ranging from about 60 C. to about 95 C.

    15. The method according to claim 13, wherein the initiator sequence is a single stranded nucleic acid primerimmobilized onto a support.

    16. The method according to claim 15, wherein said method is: for template-independent synthesis of nucleic acids with random nucleotide sequence, and the at least one nucleoside triphosphate, or the combination of nucleoside triphosphates, does not comprise terminating nucleoside triphosphates; or for template-independent sequence-controlled synthesis of nucleic acids, and the at least one nucleoside triphosphate is a terminating nucleoside triphosphate comprising a reversible 3-blocking group.

    17. The method according to claim 13, comprising the steps of: a) providing the initiator sequence comprising a 3-end nucleotide with a free 3-hydroxyl group; b) contacting said 3-end nucleotide with a reversibly terminating nucleoside triphosphate in the presence of the functionally active mutated primase domain according to claim 1, thereby covalently binding said reversibly terminating nucleoside triphosphate to the free 3-hydroxyl group of the 3-end nucleotide; c) applying a washing solution to remove all reagents, in particular to remove unbound reversibly terminating nucleoside triphosphates; d) cleaving the reversible 3-blocking group of the covalently bound terminating nucleoside triphosphate in the presence of a cleaving agent; thereby obtaining a nucleotide with a free 3-hydroxyl group.

    18. The method according to claim 13, further comprising applying a washing solution to remove all reagents, in particular to remove the cleaving agent.

    19. The method according to claim 17, further comprising reiterating steps b) to e) multiple times to synthetize the nucleic acid until desired length and nucleotide sequence is achieved.

    20. A kit comprising: a) an initiator sequence comprising a 3-end nucleotide with a free 3-hydroxyl group, optionally wherein said initiator sequence is immobilized onto a support; b) nucleoside triphosphates; and c) a functionally active mutated primase domain according to claim 1.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0293] FIG. 1 is a bar chart representing the template-independent nucleic acid synthesis relative activity of the purified mutants relative to the activity of PolpP12.sub.297-898 (SHEE-WT), set at 100%. Reactions were performed at 70 C. using a dNTP mix and a 20-nucleotide primer as substrates. Activity represents the length of the elongated DNA.

    [0294] FIG. 2 is a bar chart representing the template-independent nucleic acid synthesis relative activity of double mutants, relative to the activity of PolpP12.sub.297-898 (SHEE-WT), set at 100%. Reactions were performed at 70 C. using a dNTP mix and a 20-nucleotide primer as substrates. Activity represents the length of the elongated DNA.

    [0295] FIG. 3 is a photograph of a 1.5% agarose gel electrophoresis showing a template independent nucleic acid synthesis assay using PolpP12.sub.297-898 (SHEE-WT), in comparison with triple mutants. Reactions were performed at 70 C. using a dNTP mix and a 20-nucleotides primer as substrates. Ladder: SmartLadder 200 to 10000 bp (Eurogentec).

    [0296] FIG. 4 is a bar chart representing a template independent nucleic acid synthesis activity of triple, relative to the activity of PolpP12.sub.297-898 (SHEE-WT), set at 100%. Activity represents the length of the elongated DNA obtained from the agarose gel depicted in FIG. 4.

    [0297] FIG. 5 is a bar chart representing a template independent nucleic acid synthesis activity of PolpP12.sub.297-898 (SHEE-WT) fused to a DNA-binding domain from Thermotoga neapolitana either at the N-terminus (SHEE-N18) or the C-terminus (SHEE-C18), relative to the activity of PolpP12297-898 (SHEE-WT), set at 100%. Activity represents the length of the elongated DNA.

    [0298] FIG. 6 is a photograph of an electrophoresis gel (SDS-PAGE) showing the purification of respectively PolpTN2311-923, PolpTN2311-923_B, PolpTN2311-923_C and PolpTN2311-923_D. MW ladder: molecular weight ladder (left lane).

    [0299] FIGS. 7A-B are a set of two photographs of a 1.5% agarose gel electrophoresis showing a template-independent nucleic acid synthesis assay or a template-independent ab-inito synthesis assay using PolpTN2.sub.311-923, PolpTN2.sub.311-923_B, PolpTN2.sub.311-923_C or PolpTN2.sub.311-923_D and carried out in the presence or in the absence of dNTPs and/or the initiator sequence (bearing the Cy5 fluorophore in 5). Reactions were performed at 70 C. for 10 min in the presence or in the absence of each substrate. MW ladder is SmartLadder 200 to 10000 bp (Eurogentec). Green channel, MidoriGreen direct fluorescence at 520 nm. Red channel, Cy5 fluorescence at 675 nm. FIG. 7A: in the presence of dNTPs and the initiator sequence (bearing the Cy5 fluorophore in 5); FIG. 7B: in the presence of dNTPs and in the absence of the initiator sequence.

    EXAMPLES

    [0300] The present invention is further illustrated by the following examples.

    [0301] The Inventors have previously identified several archaeal DNA primase domains that are capable of template-independent synthesis, at temperatures between 60 C. and 95 C. (International patent applications WO 2021/250269 and WO 2021/250265).

    [0302] In brief, the data surprisingly showed that: [0303] PolpP12.sub.297-898 has template-independent terminal nucleotidyl transferase activity and is devoid of ab-initio single-stranded nucleic acid synthesis activity (Example 2 of WO 2021/250269); [0304] PolpP12.sub.297-898 has a higher processivity than a member of X-family polymerases, the recombinant terminal deoxynucleotidyl transferase TdT (Example 3 of WO 2021/250269); [0305] PolpP12.sub.297-898 is capable of incorporating protected deoxyribonucleosides triphosphate and protected ribonucleosides triphosphate (Examples 4 and 6 of WO 2021/250269); [0306] PolpP12.sub.297-898 is capable of incorporating ribonucleosides triphosphate and deoxyuridine triphosphate (Example 5 of WO 2021/250269); [0307] PolpP12.sub.297-898 is capable of incorporating deoxyinosine triphosphate and base-modified nucleosides triphosphate (Example 7 of WO 2021/250269); [0308] PolpP12.sub.297-898 or PolpTN2.sub.311-923 variants with internal loop deletions are still functional (Example 8 of WO 2021/250269; Example 2 of WO 2021/250265); and [0309] PolpTN2.sub.311-923, PolpTCIR10.sub.303-928, PolpTpep.sub.295-914 and PolpTcele.sub.295-913 have ab-initio single-stranded nucleic acid synthesis activity (Examples 1 and 3-4 of WO 2021/250265).

    [0310] Here, the Inventors aimed at providing mutants of these archaeal DNA primase domains with at least equivalent if not improved template-independent terminal nucleotidyl transferase activity compared to their wild-type counterparts.

    Example 1

    Single Point Mutations of PolpP12.SUB.297-898 .Modulate the Template-Independent Terminal Nucleotidyl Transferase Activity

    [0311] Around 90 single point mutants of the N-terminal domain of the DNA primase from Pyrococcus sp. 12-1 (PolpP12.sub.297-898 having the amino acid sequence of SEQ ID NO: 2) were generated using the Q5 Site-Directed Mutagenesis kit (New England Biolabs) following manufacturer's protocol. After sequence verification using Sanger's sequencing, PolpP12.sub.297-898 (SHEE-WT) and its corresponding mutants were expressed and purified following a protocol adapted from International patent publication WO2011098588 and Gill et al., 2014 (Nucleic Acids Res. 42(6):3707-3719).

    [0312] To evaluate the gain or the loss of activity of PolpP12.sub.297-898 mutants, a template-independent nucleic acid synthesis assay was carried out using PolpP12.sub.297-898 (SHEE-WT) as positive control. All experiments were run at 70 C. using a dNTP mix as substrate and a single stranded nucleic acid primer as initiator sequence. After reaction, samples were resolved on 1.5% agarose gels and nucleic acids were stained using Midori Green Direct. For each mutant, the length of the synthetized DNA was determined using a calibration curve made from the DNA ladder (SmartLadder 200 to 10000 bp, Eurogentec) and relative activities were calculated by setting the activity of PolpP12.sub.297-898 (SHEE-WT) as 100%.

    TABLE-US-00032 TABLE 1 single point mutations of PolpP12.sub.297-898. Amino acid residue positions with reference to SEQ ID NO: 2 numbering. Mutant ID Substitution SHEE-3.12 F219Y SHEE-3.21 Y233A SHEE-3.24 Y233H SHEE-3.32 Y235F SHEE-3.33 Y235W SHEE-4.2 N217R SHEE-4.3 N217K SHEE-9.1 Y122A SHEE-9.2 Y122H SHEE-10.1 F74Y SHEE-11.1 K234R SHEE-12.1 K236R SHEE-13.1 N206R SHEE-15.1 T230C SHEE-15.2 T230A SHEE-17.1 L229A SHEE-18.1 I231A SHEE-20.3 S68N SHEE-21.3 F74Q SHEE-23.2 F174R SHEE-26.2 P228N SHEE-26.3 P228A SHEE-27.1 L229G SHEE-27.2 L229N SHEE-27.3 L229R SHEE-28.1 T230S SHEE-29.2 I231R SHEE-29.3 I231K SHEE-30.1 N232R SHEE-31.1 Y233R SHEE-31.2 Y233K SHEE-33.1 P238R

    [0313] FIG. 1 shows the template-independent nucleic acid synthesis relative activity of the purified mutants of Table 1, relative to the activity of PolpP12.sub.297-898 (SHEE-WT).

    [0314] Strikingly, SHEE-3.24, SHEE-4.3, SHEE-9.2, SHEE-10.1, SHEE-11.1, SHEE-12.1, SHEE-13.1, SHEE-15.1, SHEE-17.1, SHEE-27.2 and SHEE-31.2 showed a clear increase of activity ranging from 120% to 180% compared to wild-type PolpP12.sub.297-898 (SHEE-WT).

    [0315] Surprising, SHEE-4.3, SHEE-10.1 and SHEE-13.1 showed an improved activity, although their mutated amino acid positions (N217K, F74Y and N206R, respectively) are expected to be located outside the catalytic pocket. This phenomenon suggests that these residues are involved in an improved binding of the initiator DNA primer, thus leading to an enhanced template-independent nucleic acid synthesis activity.

    Example 2

    Multiple Point Mutations of PolpP12.sub.297-898 have an Additive Effect on the Improvement of the Template-Independent Terminal Nucleotidyl Transferase Activity

    [0316] Multiple point mutations of the N-terminal domain of the DNA primase from Pyrococcus sp. 12-1 (PolpP12.sub.297-898 having the amino acid sequence of SEQ ID NO: 2) were generated using the Q5 Site-Directed Mutagenesis kit (New England Biolabs) following manufacturer's protocol. Mutants were designed based on the results of single point mutations described in Example 1. After sequence verification using Sanger's sequencing, PolpP12.sub.297-898 (SHEE-WT) and its corresponding mutants were expressed and purified following a protocol adapted from International patent publication WO2011098588 and Gill et al., 2014 (Nucleic Acids Res. 42(6):3707-3719) (Table 2).

    TABLE-US-00033 TABLE 2 multiple point mutations of PolpP12.sub.297-898. Amino acid residue positions with reference to SEQ ID NO: 1 numbering. Mutant ID Substitution SHEE-4.3/11.1 N217K/K234R SHEE-4.3/12.1 N217K/K236R SHEE-4.3/13.1 N217K/N206R SHEE-9.2/3.24 Y122H/Y233H SHEE-9.2/4.3 Y122H/N217K SHEE-9.2/11.1 Y122H/K234R SHEE-9.2/12.1 Y122H/K236R SHEE-9.2/13.1 Y122H/N206R SHEE-9.2/15.1 Y122H/T230C SHEE-10.1/4.3 F74Y/N217K SHEE-10.1/11.1 F74Y/K234R SHEE-11.1/15.1 K234R/T230C SHEE-12.1/15.1 K236R/T230C SHEE-13.1/3.24 N206R/Y233H SHEE-13.1/15.1 N206R/T230C SHEE-15.1/4.3 T230C/N217K SHEE-4.3/13.1/3.24 N217K/N206R/Y233H SHEE-4.3/13.1/9.2 N217K/N206R/Y122H SHEE-4.3/13.1/11.1 N217K/N206R/K234R

    [0317] To evaluate the gain or the loss of activity of the multiple point mutants of PolpP12.sub.297-898, a template-independent nucleic acid synthesis assay was carried out using PolpP12.sub.297-898 (SHEE-WT) as positive control. All experiments were run at 70 C. using a dNTP mix as substrate and a single stranded nucleic acid primer as initiator sequence. After reaction, samples were resolved on 1.5% agarose gels and nucleic acids were stained using Midori Green Direct. For each mutant, the length of the synthetized DNA was determined using a calibration curve made from the DNA ladder (SmartLadder 200 to 10000 bp, Eurogentec) and relative activities were calculated by setting the activity of PolpP12.sub.297-898 (SHEE-WT) as 100%.

    [0318] As shown in FIG. 2, all tested double mutants display at least an equivalent activity to the wild-type PolpP12.sub.297-898 (SHEE-WT).

    [0319] Interestingly, several double mutants even showed an increase of activity when compared to single mutants. Most striking, major improvements are obtained with the 13.1/3.24, 13.1/15.1, 9.2/13.1, 4.3/13.1 and 9.2/4.3, combinations.

    [0320] Likewise, as shown in FIGS. 3 and 4, the three tested triple mutants showed an increased activity ranging from 200% to 300% compared to wild-type PolpP12.sub.297-898 (SHEE-WT).

    [0321] Surprisingly, the activity of these multiple point mutants of PolpP12.sub.297-898 were much higher than the ones obtained for their corresponding single mutants (Example 1), suggesting an additive effect of the mutated residues.

    [0322] Of note, two out of three mutated positions in the triple mutants are expected to be located outside the catalytic pocket (N217K and N206R). This phenomenon thus reinforces the hypothesis of an improved binding of the initiator DNA primer, which leads to an enhanced template-independent nucleic acid synthesis activity.

    Example 3

    Addition of a ssDNA Binding Domain Improves the Template-Independent Terminal Nucleotidyl Transferase Activity

    [0323] In addition to mutagenesis studies, we sought to study the fusion of PolpP12.sub.297-898 (SHEE-WT) with processivity factors to improve template-independent terminal nucleotidyl transferase activity.

    [0324] For that purpose, two constructs were prepared using a DNA-binding domain from Thermotoga neapolitana (with SEQ ID NO: 27), fused either at the N-terminus (SHEE-N18, with SEQ ID NO: 28) or the C-terminus (SHEE-C18, with SEQ ID NO: 29) of the wild-type PolpP12.sub.297 898.

    TABLE-US-00034 SEQIDNO:28-SHEE-N18 MSFFNRIILIGRLVRDPEERYTLSGTPVTTFTIAVDRVPRKNAPDDAQTTDFFRV VTFGRLAEFARTYLTKGRLILVEGEMRMRRWETQTGEKRVSPEVVANVVRFM DRKPVEMPSEDIEEKLEIPEEDFTDDTFSEDEPPFRWGSELEMRPSDIIIDVYKAIQ DHPGAGKLAIELRFYPRPTSEWIIVADIEDKAEELHKVLFKNNVLGKKEAYISM ALHDFEEVGKKLEKLRELEEERAQKEGRKPREVTLRNVQGEATGKVHKTVSK YTLTLVVDIDVEEIHKSKVVESEEKAFELAKRAWDELKPKLEGIGVKPRYVFFT GGGVQLWFVAPGLEPIEVIDRASRVIPPVLNAMLPEGYSVDNIFDRARIVRVPLT INYKYKTPDERPLEIRGRLIEFNDVRTPLGEVLDKLEAYAKEHGISLVTPSQARFI GTVGRYEVDKGTGT SEQIDNO:29-SHEE-C18 MRPSDIIIDVYKAIQDHPGAGKLAIELRFYPRPTSEWIIVADIEDKAEELHKVLFK NNVLGKKEAYISMALHDFEEVGKKLEKLRELEEERAQKEGRKPREVTLRNVQG EATGKVHKTVSKYTLTLVVDIDVEEIHKSKVVESEEKAFELAKRAWDELKPKL EGIGVKPRYVFFTGGGVQLWFVAPGLEPIEVIDRASRVIPPVLNAMLPEGYSVD NIFDRARIVRVPLTINYKYKTPDERPLEIRGRLIEFNDVRTPLGEVLDKLEAYAK EHGISLVTPSQARFIGTVGRYEVDKGTMEFEASFFNRIILIGRLVRDPEERYTLSG TPVTTFTIAVDRVPRKNAPDDAQTTDFFRVVTFGRLAEFARTYLTKGRLILVEG EMRMRRWETQTGEKRVSPEVVANVVRFMDRKPVEMPSEDIEEKLEIPEEDFTD DTFSEDEPPF

    [0325] As shown in FIG. 5, both fusion variants of PolpP12.sub.297-898 demonstrate an increased activity, ranging from 250% to more than 350%, for SHEE-C18 and SHEE-N18 respectively, indicating that increasing the binding of DNA drastically influences the template-independent terminal nucleotidyl transferase activity.

    [0326] Thus, these variants might be associated to single point mutations or a combination of mutations, such as the ones described in Examples 1 and 2, to further increase even more their terminal nucleotidyl transferase activity.

    Example 4

    Multiple Point Mutations of PolpP12.sub.297-898 are Transposable to .sub.PolpTN2311-923 for the Improvement of the Template-Independent Terminal Nucleotidyl Transferase Activity

    [0327] To further investigate the potency of multiple point mutations of the N-terminal domain of the DNA primase from Pyrococcus sp. 12-1 described in Example 2 (PolpP12.sub.297-898 having the amino acid sequence of SEQ ID NO: 2) most active mutations were subsequently transposed to the N-terminal domain of the DNA primase from Thermococcus nautili sp. 30-1 (PolpTN2x311-923 having the amino acid sequence of SEQ ID NO: 5) generated from gene synthesis service (Twist Bioscience).

    [0328] N-terminal domain of the DNA primase from Thermococcus nautili sp. 30-1 was considered has a representative member of the Thermococcus genus, including the N-terminal domain of the DNA primase from Thermococcus sp. CIR10, Thermococcus peptonophilus and Thermococcus celericrescens, which were all previously shown to exhibit both template independent nucleotidyl transferase activity and template independent ab-initio activity.

    [0329] PolpTN2.sub.311-923 and its corresponding mutants were expressed and purified following a protocol adapted from International patent publication WO2011098588 and Gill et al., 2014 (Nucleic Acids Res. 42(6):3707-3719) (Table 3).

    TABLE-US-00035 TABLE 3 multiple point mutations of PolpP12.sub.297-898. Amino acid residue positions with reference to SEQ ID NO: 5 numbering. Mutant ID Substitution PolpTN2.sub.311-923 WT PolpTN2.sub.311-923.sub.B F121H/N205R/N222K PolpTN2.sub.311-923.sub.C N205R/N222K/K239R PolpTN2.sub.311-923.sub.D F121H/N205R/N222K/K239R

    [0330] The amino acid sequence of the wild-type primase domain of the Thermococcus nautili sp. 30-1 DNA primase (herein termed PolpTN2.sub.311-923_B), with multiple point mutations F121H, N205R and N222K with reference to SEQ ID NO: 5 numbering, is as set forth in SEQ ID NO: 30.

    [0331] The amino acid sequence of the wild-type primase domain of the Thermococcus nautili sp. 30-1 DNA primase (herein termed PolpTN2.sub.311-923_C), with multiple point mutations N205R, N222K and K239R with reference to SEQ ID NO: 5 numbering, is as set forth in SEQ ID NO: 31.

    [0332] The amino acid sequence of the wild-type primase domain of the Thermococcus nautili sp. 30-1 DNA primase (herein termed PolpTN2.sub.311-923_D), with multiple point mutations F121H, N205R, N222K and K239R with reference to SEQ ID NO: 5 numbering, is as set forth in SEQ ID NO: 32.

    [0333] To evaluate the gain or the loss of activity of the multiple point mutants of PolpTN2.sub.311-923, both a template-independent nucleic acid synthesis assay and an ab-initio synthesis assay were carried out using PolpTN2.sub.311-923 as positive control. All experiments were run at 70 C. using a dNTP mix as substrate and, in the presence, or in the absence of a single stranded nucleic acid primer as initiator sequence. After a short reaction period, samples were resolved on 1.5% agarose gels and nucleic acids were stained using Midori Green Direct.

    [0334] As shown in FIG. 7A, all tested multiple mutants display an increase of template-independent nucleic acid synthesis activity when compared to PolpTN2311-923. This increase of activity is reflected by the higher migration pattern of multiple mutants that can be observed in both green channel (total nucleic acids) and red channel (labeled primer). Such a result thus indicates that the selected mutations of PolpP12.sub.297-898 can be efficiently transposed to PolpTN2.sub.311-923, the representative of the Thermococcus genus.

    [0335] Surprisingly, the ab-initio activity of these multiple point mutants was lower than PolpTN2.sub.311-923, as shown in FIG. 7B, contrasting with the results obtained in the template-independent nucleic acid synthesis experiment (FIG. 7A). Indeed, while the synthesis product of PolpTN2.sub.311-923 remains at the top of the gel, such a decrease of activity is reflected by the lower migration pattern of multiple mutants that can be observed in green channel (total nucleic acids). Most striking, while PolpTN2.sub.311-923_B and PolpTN2.sub.311-923_C exhibit a moderate loss of activity, PolpTN2.sub.311-923_D was found to be inefficient in performing ab-initio synthesis.

    [0336] Although this result appears puzzling at a first sight, it actually reinforces the previous hypothesis that N205R and N222K mutations, which correspond to N206R and N217K in PolpTN2311-923, are involved in an improved binding of the initiator DNA primer, thus leading to an enhanced template-independent nucleic acid synthesis activity (FIG. 7A). In contrast, for both F121H and K239R, which are expected to be located within the active site, their mutation might rather play a role in the lower ab-initio activity observed for PolpTN2.sub.311-923_B and PolpTN2.sub.311-923_C, their association being cumulative as exemplified by PolpTN2.sub.311-923_D, which is almost devoid of ab-initio activity (FIG. 7B).

    CONCLUSION

    [0337] The Inventors have previously discovered that PolpP12.sub.297-898 (SHEE-WT) exhibit a template-independent nucleic acid synthesis activity in presence of an initiator primer and nucleosides triphosphatewhether unprotected or 3-O protected, regardless of the size of the protecting group, and whether deoxyribonucleotides or ribonucleotides, while being devoid of ab-initio nucleic acid synthesis activity.

    [0338] Here, they provide with mutants of PolpP12297-898 that also exhibit a template-independent nucleic acid synthesis activity. Some of the single-point mutants disclosed herein are shown to exhibit improved template-independent terminal nucleotidyl transferase activity compared to PolpP12.sub.297-898 (SHEE-WT); while combinations of two or three single-point mutations, and possibly more, have an additive effect on the improvement of the template-independent terminal nucleotidyl transferase activity.

    [0339] The Inventors had previously discovered that other archaeal DNA primases domains from archaeal DNA primases belonging to the primase-polymerase family also exhibit a template-independent nucleic acid synthesis activity, in particular archaeal DNA primases from Thermococcus nautili sp. 30-1, Thermococcus sp. CIR10, Thermococcus peptonophilus, and Thermococcus celericrescens. Given that the identified positions in PolpP12.sub.297-898 are conserved in these other DNA primases, as assessed by sequence alignments, it is shown that the mutations described in PolpP12.sub.297-898 at positionally equivalent positions in these other DNA primases have the same effect.

    [0340] Finally, the Inventors herein show that fusion of wild-type PolpP12297-898 with oligonucleotide binding domains drastically increase the template-independent terminal nucleotidyl transferase activity. This increase is also expectable with fusions comprising mutants of PolpP12.sub.297-898 or of other DNA primases.