AB-INITIO, TEMPLATE-INDEPENDENT SYNTHESIS OF NUCLEIC ACIDS USING THERMOSTABLE ENZYMES

Abstract

The invention relates to the field of nucleic acid synthesis or sequencing, more specifically to methods for ab-initio synthesis of nucleic acids, comprising contacting a nucleotide with a free 3′-hydroxyl group, with at least one nucleoside triphosphate, or a combination of nucleoside triphosphates, in the presence of an archaeal DNA primase or a functionally active fragment and/or variant thereof, thereby covalently binding said nucleoside triphosphate to the free 3′-hydroxyl group of the nucleotide. It also relates to isolated functionally active fragments of archaeal DNA primases which are capable of both ab-initio single-stranded nucleic acid synthesis activity and template-independent terminal nucleotidyl transferase activity.

Claims

1. A method for ab-initio single-stranded nucleic acid synthesis, comprising contacting the free 3′-hydroxyl group of a nucleotide with at least one nucleoside triphosphate, or a combination of nucleoside triphosphates, in the presence of a primase domain of an archaeal DNA primase belonging to the primase-polymerase family or a functionally active fragment and/or variant thereof, wherein said primase domain consists of the N terminal domain of an archaeal DNA primase from an archaeon of the Thermococcus genus, wherein the functionally active fragment and/or variant thereof retains its capabilities of both ab initio single stranded nucleic acid synthesis activity and template-independent terminal nucleotidyl transferase activity, thereby covalently binding said nucleoside triphosphate to the free 3′-hydroxyl group of the nucleotide, and wherein the method is carried out in absence of both complementary nucleic acid template and initiator sequence.

2. (canceled)

3. The method according to claim 1, wherein said archaeal DNA is selected from the group consisting of Thermococcus nautili sp. 30-1 DNA primase, Thermococcus sp. CIR10 DNA primase, Thermococcus peptonophilus DNA primase, and Thermococcus celericrescens DNA primase.

4. The method according to claim 1, wherein said archaeal DNA is: Thermococcus nautili sp. 30-1 DNA primase having the amino acid sequence of SEQ ID NO: 1; Thermococcus sp. CIR10 DNA primase having the amino acid sequence of SEQ ID NO: 14; Thermococcus peptonophilus DNA primase having the amino acid sequence of SEQ ID NO: 17; or Thermococcus celericrescens DNA primase having the amino acid sequence of SEQ ID NO: 19.

5. The method according to claim 1, wherein said primase domain is the primase domain of: the Thermococcus nautili sp. 30-1 DNA primase having the amino acid sequence of any one of SEQ ID NOs: 2 to 13; or the Thermococcus sp. CIR10 DNA primase having the amino acid sequence of any one of SEQ ID NOs: 15 or 16; or the Thermococcus peptonophilus DNA primase having the amino acid sequence of SEQ ID NO: 18; or the Thermococcus celericrescens DNA primase having the amino acid sequence of SEQ ID NO: 20; or a functionally active fragment and/or variant thereof: having at least 70% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 2 to 13, 15, 16, 18 or 20; and being capable of template-independent terminal nucleotidyl transferase activity; and being capable of ab-initio single-stranded nucleic acid synthesis activity.

6. The method according to claim 1, wherein said primase domain of an archaeal DNA primase belonging to the primase-polymerase family is the primase domain of: the Thermococcus nautili sp. 30-1 DNA primase having the amino acid sequence of any one of SEQ ID NOs: 2 to 5; or the Thermococcus sp. CIR10 DNA primase having the amino acid sequence of SEQ ID NO: 15; or the Thermococcus peptonophilus DNA primase having the amino acid sequence of SEQ ID NO: 18; or the Thermococcus celericrescens DNA primase having the amino acid sequence of SEQ ID NO: 20; or a functionally active fragment and/or variant thereof: having at least 70% sequence identity with said amino acid sequence; and being capable of ab-initio single-stranded nucleic acid synthesis activity; and being capable of template-independent terminal nucleotidyl transferase activity.

7. The method according to claim 1, wherein the nucleotide is immobilized onto a support.

8. The method according to claim 1, wherein the ab-initio single-stranded nucleic acid synthesis is carried out at a temperature ranging from about 60° C. to about 95° C.

9. The method according to claim 1, wherein said method is for ab-initio synthesis of nucleic acids with random nucleotide sequence, and the at least one nucleoside triphosphate does not comprise terminating nucleoside triphosphates.

10. The method according to claim 1, wherein said method is for ab-initio sequence-controlled synthesis of nucleic acids, and the at least one nucleoside triphosphate is a terminating nucleoside triphosphate comprising a reversible 3′-blocking group.

11. The method according to claim 10, comprising the steps of: a) providing the nucleotide with a free 3′-hydroxyl group; b) contacting said nucleotide with a terminating nucleoside triphosphate in the presence of the primase domain of the archaeal DNA primase belonging to the primase-polymerase family or the functionally active fragment and/or variant thereof, thereby covalently binding said terminating nucleoside triphosphate to the free 3′-hydroxyl group of the nucleotide; c) applying a washing solution to remove all reagents, in particular to remove unbound terminating nucleoside triphosphates; d) cleaving the reversible 3′-blocking group of the covalently bound terminating nucleoside triphosphate in the presence of a cleaving agent, and thereby obtaining a nucleotide with a free 3′-hydroxyl group.

12. The method according to claim 1, wherein said method is for cleaning-up contaminating nucleoside triphosphates comprising a free 3′-hydroxyl group in a pool of terminating nucleoside triphosphates.

13. An isolated functionally active fragment of an archaeal DNA primase consisting of an amino acid sequence of any one of SEQ ID NOs: 3 to 13, 15, 16, 18 or 20, or a functionally active fragment and/or variant thereof: having at least 70% sequence identity with said amino acid sequence; being capable of ab-initio single-stranded nucleic acid synthesis activity; and being capable of template-independent terminal nucleotidyl transferase activity.

14. The isolated functionally active fragment of the archaeal DNA primase or variant thereof according to claim 13, consisting of the amino acid sequence of any one of SEQ ID NOs: 3 to 13, 15, 16, 18 or 20.

15. (canceled)

16. (canceled)

17. A nucleic acid encoding the functionally active fragment of an archaeal DNA primase according to claim 13 or an expression vector comprising the nucleic acid operably linked to regulatory elements, or a host cell comprising the expression vector.

18. (canceled)

19. (canceled)

20. (canceled)

21. (canceled)

22. The method according to claim 11, further comprising the step of: (e) applying a washing solution to remove all reagents, in particular to remove the cleaving agent;

23. The method according to claim 22, further comprising the step of: (f) reiterating steps b) to e) multiple times to synthetize the nucleic acid until desired length and nucleotide sequence.

24. The nucleic acid according to claim 17, wherein the nucleic acid is operably linked to a promoter.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0286] FIG. 1 is a photograph of an electrophoresis gel (SDS-PAGE) showing the purification of respectively PolpP12.sub.Δ297-898, PolpTN2.sub.Δ311-923, PolpTCIR10.sub.Δ303-928, PolpTpep.sub.Δ295-914 and PolpTcele.sub.Δ295-913. MW ladder: molecular weight ladder (left lane).

[0287] FIG. 2 is a photograph of a 15% urea-PAGE showing a template-independent nucleic acid synthesis assay using PolpTN2.sub.Δ311-923 [PolpTN2.sub.Δ] or PolpP12.sub.Δ297-898 [PolpP12.sub.Δ], at 60° C., 70° C. or 80° C. [a]: initiator sequence only, no enzyme, no dNTP; [b]: initiator sequence+enzyme, no dNTP; [c]: initiator sequence+enzyme+dNTP mix (unprotected).

[0288] FIGS. 3A-C are a set of three photographs of a 1.5% agarose gel electrophoresis showing a template-independent nucleic acid synthesis assay using PolpP12.sub.Δ297-898 or PolpTN2.sub.Δ311-923 at 70° C., 80° C., 90° C. and 100° C., in comparison with a negative control performed at 70° C. without enzyme [No Enzyme]. dsDNA LF Ladder: SmartLadder 200 to 10000 bp (Eurogentec). [0289] FIG. 3A: red channel, Cy5 fluorescence at 675 nm; [0290] FIG. 3B: green channel, Sybr green II fluorescence at 520 nm; [0291] FIG. 3C: merge of red and green channels.

[0292] FIGS. 4A-C are a set of three photographs of a 1.5% agarose gel electrophoresis showing a template-independent nucleic acid synthesis assay using PolpP12.sub.Δ297-898 or PolpTN2.sub.Δ311-923 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 80° C. in the presence or in the absence of each substrate (lanes 1 to 8). Lane 9 shows a two steps reaction in which dNTPs were first incubated with either PolpP12.sub.Δ297-898 or PolpTN2.sub.Δ311-923, during 15 minutes, followed by the addition of the initiator sequence. Left lane shows the MW ladder (SmartLadder 200 to 10000 bp (Eurogentec)). [0293] FIG. 4A: merge of red and green channels; [0294] FIG. 4B: green channel, Sybr green II fluorescence at 520 nm; [0295] FIG. 4C: red channel, Cy5 fluorescence at 675 nm.

[0296] FIGS. 5A-B are a set of six photographs of a 1% agarose gel electrophoresis showing a template-independent nucleic acid synthesis assay using PolpTCIR10.sub.Δ303-928, PolpTpep.sub.Δ295-914 or PolpTcele.sub.Δ295-913 and carried out at 70° C. in the presence or in the absence of a dNTPs mix or a dG/dC mix as well as in the presence or in the absence of the initiator sequence (bearing the Cy5 fluorophore in 5′). MW ladder (SmartLadder 200 to 10000 bp (Eurogentec)). [0297] FIG. 5A: template-independent nucleic acid synthesis assay using PolpTCIR10.sub.Δ303-928 or PolpTpep.sub.Δ295-914;

[0298] FIG. 5B: template-independent nucleic acid synthesis assay using PolpTcele.sub.Δ295-913.

[0299] FIG. 6 is a photograph of an electrophoresis gel (SDS-PAGE) showing the purification of PolpTN2.sub.Δ311-923, PolpTN2.sub.Δ90-96Δ311-923, PolpTN2.sub.Δ205-211Δ311-923 and PolpTN2.sub.Δ248-254Δ311-923. PolpTN2.sub.Δ90-96Δ311-923 and PolpTN2.sub.Δ205-211Δ311-923 are in duplicate. MW ladder: molecular weight ladder.

[0300] FIGS. 7A-C are a set of three photographs of a 1.5% agarose gel electrophoresis showing a template-independent nucleic acid synthesis assay using PolpTN2.sub.Δ311-923, PolpTN2.sub.Δ90-96Δ311-923, PolpTN2.sub.Δ205-211Δ311-923 or PolpTN2.sub.Δ248-254Δ311-923 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. in the presence or in the absence of each substrate. MW ladder is SmartLadder 200 to 10000 bp (Eurogentec). [0301] FIG. 7A: merge of red and green channels; [0302] FIG. 7B: red channel, Cy5 fluorescence at 675 nm; [0303] FIG. 7C: green channel, MidoriGreen direct fluorescence at 520 nm.

[0304] FIG. 8 is a photograph of a 15% urea-PAGE showing the incorporation of protected nucleoside triphosphates (3′-O-amino-dATP and 3′-O-azidomethyl-dATP), using PolpP12.sub.Δ297-898 at 60° C.

[0305] FIGS. 9A-C are a set of three photographs showing the incorporation by PolpP12.sub.Δ297-898 of labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups at 80° C. [0306] FIG. 9A: 15% urea-PAGE showing the incorporation of 3′-O-amino dATP or 3′-O-amino dTTP at 80° C.; [0307] FIG. 9B: Analysis report of 3′-O-amino dATP incorporation at 80° C. R.sub.f: relative migration distance; [0308] FIG. 9C: Analysis report of 3′-O-amino dTTP incorporation at 80° C. R.sub.f: relative migration distance.

[0309] FIGS. 10A-C are a set of three photographs showing the incorporation by PolpP12.sub.Δ297-898 of nucleoside triphosphates labeled with 3′-O-azidomethylene groups at 80° C. [0310] FIG. 10A: 15% urea-PAGE showing the incorporation of 3′-O-azidomethyl dATP or 3′-O-azidomethyl dTTP at 80° C.; [0311] FIG. 10B: Analysis report of 3′-O-azidomethyl dATP incorporation at 80° C. R.sub.f: relative migration distance; [0312] FIG. 10C: Analysis report of 3′-O-azidomethyl dTTP incorporation at 80° C. R.sub.f: relative migration distance.

[0313] FIGS. 11A-C are a set of three scheme showing a clean-up procedure of terminating nucleoside triphosphates, in presence of contaminating nucleoside triphosphates comprising a free 3′-hydroxyl group. [0314] FIG. 11A: first step of ab-initio nucleic acid synthesis carried out in the presence of the DNA primases described herein, and using the contaminating stock of nucleoside triphosphates comprising a free 3′-hydroxyl group; [0315] FIG. 11B: alternative first step of ab-initio nucleic acid synthesis carried out in the presence of the DNA primases described herein, and using the contaminating stock of nucleoside triphosphates comprising a free 3′-hydroxyl group. An excess amount of exogenous dideoxynucleoside triphosphates (ddNTP) is added to avoid incorporating terminating nucleoside triphosphate to the nascent nucleic acid strand. The ddNTP can be functionalized (e.g., with biotin); [0316] FIG. 11C: second step of the process, comprising (1) filing a centrifugal filtration column with the sample obtained after the first step; and (2) spinning the centrifugal filtration column to separate the synthetized single stranded nucleic acid fragments and DNA primase, from the terminating nucleoside triphosphates (3′-blocked nucleoside triphosphates) and buffer.

EXAMPLES

[0317] The present invention is further illustrated by the following examples.

Example 1

PolpTN2.SUB.Δ311-923., PolpTCIR10.SUB.Δ303-928., PolpTpep.SUB.Δ295-914 .and PolpTcele.SUB.Δ295-913 .have an Ab-Initio Single-Stranded Nucleic Acid Synthesis Activity

[0318] The N-terminal domain of the DNA primase from Pyrococcus sp. 12-1 (PolpP12.sub.Δ297-898 having the amino acid sequence of SEQ ID NO: 21), from Thermococcus nautili sp. 30-1 (PolpTN2.sub.Δ311-923 having the amino acid sequence of SEQ ID NO: 2), from Thermococcus sp. CIR10 (PolpTCIR10.sub.Δ303-928 having the amino acid sequence of SEQ ID NO: 15), from Thermococcus peptonophilus (PolpTpep.sub.Δ295-914 having the amino acid sequence of SEQ ID NO: 18) and from Thermococcus celericrescens (PolpTcele.sub.Δ295-913 having the amino acid sequence of SEQ ID NO: 20) were expressed and purified following a protocol adapted from WO2011098588 and Gill et al., 2014 (Nucleic Acids Res. 42(6):3707-3719) (FIG. 1).

TABLE-US-00021 SEQ ID NO: 21 MRPSDIIIDVYKAIQDHPGAGKLAIELRFYPRPTSEWIIVADIEDKAEE LHKVLFKNNVLGKKEAYISMALHDFEEVGKKLEKLRELEEERAQKEGRK PREVTLRNVQGEATGKVHKTVSKYTLTLVVDIDVEEIHKSKVVESEEKA FELAKRAWDELKPKLEGIGVKPRYVFFTGGGVQLWFVAPGLEPIEVIDR ASRVIPPVLNAMLPEGYSVDNIFDRARIVRVPLTINYKYKTPDERPLEI RGRLIEFNDVRTPLGEVLDKLEAYAKEHGISLVTPSQARFIGTVGRYEV DK

[0319] A template-independent nucleic acid synthesis assay was carried out with either PolpTN2.sub.Δ311-923 or PolpP12.sub.Δ297-898, at 60° C., 70° C. and 80° C., using a single stranded nucleic acid primer as initiator sequence (bearing a Cy5 fluorophore in 5′).

[0320] Three different conditions were tested: [0321] a: initiator sequence only; no enzyme, no dNTP; [0322] b: initiator sequence+enzyme; no dNTP; [0323] c: initiator sequence+enzyme+dNTP mix (unprotected).

[0324] As seen on FIG. 2, both PolpTN2.sub.Δ311-923 and PolpP12.sub.Δ297-898 exhibit an untemplated terminal nucleotidyl transferase activity for each tested temperature, when using a mixture of all four dNTPs as substrate. However, it is worth noting that at 70° C. and 80° C., most of the newly synthetized nucleic acids are several hundred bases long, hence cannot be resolved on a 15% urea-PAGE gel and remain in the well.

[0325] Thus, to analyze the effect of high temperatures on PolpTN2.sub.Δ311-923 and PolpP12.sub.Δ297-898 activities, a template-independent nucleic acid synthesis assay was performed as previously described, at 70° C., 80° C., 90° C. or 100° C. and resolved by agarose gel electrophoresis (FIG. 3). The terminal transferase activity was specifically evaluated by following the polymerization of the fluorescent primer (bearing a Cy5 fluorophore in 5′), recorded at 675 nm (red channel). Total nucleic acid synthesis and molecular weight markers were stained using Sybr Green II and recorded at 520 nm (green channel).

[0326] As shown on FIGS. 3A and C, both enzymes exhibit a strong template-independent terminal nucleotidyl transferase activity, which is demonstrated by the polymerization of the Cy5-labeled initiator sequence (FIG. 3A). These activities reach a maximum of polymerization at 70° C. and gradually decrease upon increasing temperatures, up to 100° C. However, in contrast to PolpP12.sub.Δ297-898, PolpTN2.sub.Δ311-923 exhibits a diffuse migration pattern at 70° C., 80° C. and 90° C., when stained with Sybr Green II (FIG. 3B), which does not colocalize with the Cy5-labeled initiator sequence (Cf. FIGS. 3A and C). Although this intriguing result may arise from a migration issue, another explanation would be the presence of an unexpected competing activity, such as an ab-initio single-stranded nucleic acid synthesis activity.

[0327] Interestingly, Béguin et al. have demonstrated that a combination of the full length PolpTN2 primase and the PolB DNA polymerase in presence of deoxynucleotide triphosphates leads to the ab-initio synthesis of long double stranded DNA fragments (i.e., without template DNA nor oligonucleotide primer). However, this phenomenon requires the presence of both enzymes and is not observed when only PolpTN2 is reacted with a dNTP mix (Béguin et al., 2015. Extremophiles. 19(1):69-76). In contrast, our results suggest that PolpTN2.sub.Δ311-923 alone might be able to synthesis long fragments of single stranded nucleic acids de novo, i.e., corresponding to an ab-initio activity.

[0328] To further investigate this phenomenon, both PolpTN2.sub.Δ311-923 and PolpP12.sub.Δ297-898 were subjected to a template-independent nucleic acid synthesis assay (FIG. 4), carried out in the presence or in the absence of dNTPs and/or the initiator sequence (bearing the Cy5 fluorophore in 5′). The terminal transferase activity was specifically evaluated by following the polymerization of the fluorescent primer, recorded at 675 nm (red channel). Total nucleic acid synthesis and molecular weight markers were stained using Sybr Green II and recorded at 520 nm (green channel).

[0329] Nine different conditions were tested: [0330] 1: no enzyme, no initiator sequence, no dNTP; [0331] 2: dNTP only; no enzyme, no initiator sequence; [0332] 3: initiator sequence only; no enzyme, no dNTP; [0333] 4: initiator sequence+dNTP mix; no enzyme; [0334] 5: enzyme only, no initiator sequence, no dNTP; [0335] 6: enzyme+dNTP mix; no initiator sequence; [0336] 7: enzyme+initiator sequence; no dNTP; [0337] 8: enzyme+dNTP mix+initiator sequence; [0338] 9: enzyme+dNTP mix+initiator sequence (added after 15 minutes incubation);

[0339] As shown on FIG. 4, neither PolpP12.sub.Δ297-898 nor PolpTN2.sub.Δ311-923 is able to synthesize nucleic acids in the absence of dNTPs (FIG. 4; lanes 1, 3, 5 and 7) while the association of dNTPs with the initiator sequence leads to the synthesis of long nucleic acids fragments (FIG. 4A; lanes 8 and 9).

[0340] Interestingly, in the absence of the initiator sequence (FIG. 4; lanes 1, 2, 5 and 6) PolpTN2.sub.Δ311-923 readily exhibits a strong polymerase activity, upon the addition of dNTPs (FIGS. 4A and 4B; lane 6). Separated channel analysis reveals that this activity is independent of an initiator sequence, as demonstrated by the absence of Cy5 fluorescence (FIG. 4C; lane 6), thus confirming the ab-initio single-stranded nucleic acid synthesis activity of PolpTN2.sub.Δ311-923.

[0341] Conversely, in the same experimental conditions, PolpP12.sub.Δ297-898 does not synthesize nucleic acids, as demonstrated by a total absence of fluorescence in both channels (FIGS. 4A, 4B and 4C; lane 6), indicating that this enzyme is devoid of an ab-initio single-stranded nucleic acid synthesis activity.

[0342] To further investigate the impact of such ab-initio single-stranded nucleic acid synthesis activity on the ability of PolpTN2.sub.Δ311-923 and PolpP12.sub.Δ297-898 to extend a single stranded nucleic acid fragment, a competition assay was conducted by separating both reactions (FIG. 4, lane 9). To realize this experiment, both enzymes were first incubated with dNTPs for 15 minutes, to perform a template-independent primase reaction, before adding the initiator sequence for another 15 minutes of incubation, to perform a template-independent primer extension reaction. As expected, a strong diffuse migration pattern could be observed for PolpTN2.sub.Δ311-923, after Sybr Green II staining (FIG. 4B, lane 9), while the initiator sequence was found to migrate up to the dye front (FIG. 4C, lane 9), similarly to the negative control (FIG. 4C, lane 3). In contrast, PolpP12.sub.Δ297-898 was found to extend the initiator sequence (FIG. 4C, lane 9) as previously described, indicating that a pre-incubation with a dNTP mix does not affect its terminal nucleotidyl transferase activity. Thus, this result indicates that PolpTN2.sub.ΔA311-923 is not able to extent the initiator sequence in these experimental conditions.

[0343] We subsequently investigated the ability of PolpTCIR10.sub.Δ303-928, PolpTpep.sub.Δ295-914 and PolpTcele.sub.Δ295-913 to perform a template-independent DNA synthesis reaction in the presence or in the absence of the initiator sequence (bearing the Cy5 fluorophore in 5′). For that purpose, PolpTCIR10.sub.Δ303-928 and PolpTpep.sub.Δ295-914 (FIG. 5A) as well as PolpTcele.sub.Δ295-913 (FIG. 5B) were incubated at 70° C. with or without the initiator sequence and their terminal transferase activity was evaluated by following the polymerization of the fluorescent primer, recorded at 675 nm (red channel) while their ab-initio single-stranded nucleic acid synthesis activity was evaluated through Sybr Green II staining and recorded at 520 nm (green channel).

[0344] As seen on FIGS. 5A and B, both PolpTCIR10.sub.Δ303-928 and PolpTpep.sub.Δ295-914 as well as PolpTcele.sub.Δ295-913 demonstrate an ability to synthesis nucleic acids ab-initio and to extend single-stranded DNA fragments, similarly to PolpTN2.sub.Δ311-923. Furthermore, these activities were strengthened by the ability of PolpTcele.sub.Δ295-913 to catalyze both the terminal nucleotidyl transferase reaction and the ab-initio single-stranded nucleic acid synthesis reaction in the presence of a dC/dG mix (FIG. 5B), indicating that the use of these enzymes is relevant for processes requiring either an ab-initio single-stranded nucleic acid synthesis activity or a terminal nucleotidyl transferase activity.

Example 2

PolpTN2.SUB.Δ311-923 .Variants with Internal Deletions are Still Functional

[0345] Although PolpTN2.sub.Δ311-923, PolpTCIR10.sub.Δ303-928, PolpTpep.sub.Δ295-914 and PolpThele.sub.Δ295-913 present similar activities, it is worth noting that these enzymes are diverging both in term of sequence identity and length. Indeed, protein sequence alignment of these enzymes showed the presence of several loops that we suspected might be dispensable for both terminal nucleotidyl transferase and ab-initio activities in PolpTN2.sub.Δ311-923. These loops are located between amino acid residues 90 to 96, 205 to 211 and 248 to 254 of PolpTN2.sub.Δ311-923 (reference to SEQ ID NO: 2 numbering). One similar loop was also found between amino acid residues 93 to 98 of PolpTCIR10.sub.Δ303-928 (reference to SEQ ID NO: 15 numbering).

[0346] This study was driven by the necessity of providing enzymes that are suitable for industrial applications, and adapted for both upstream and downstream processes. In that respect, the removal of these loops can improve on the one hand protein stability and protein expression yield as it maximizes the presence of structured regions. On the other hand, loop deletion leads to a reduced protein size, which eventually facilitates the removal of the enzyme along with other reagents by ultrafiltration during downstream purification.

[0347] To investigate the effect of loop deletion and size reduction on terminal nucleotidyl transferase and ab-initio activities, we generated variants of PolpTN2.sub.Δ311-923, which presents the largest size with 310 amino acid residues versus 295 amino acid residues for PolpTpep.sub.Δ295-914 and PolpTcele.sub.Δ295-913. This led to four variants, namely PolpTN2.sub.Δ90-96Δ311-923 (with SEQ ID NO: 3), PolpTN2.sub.Δ205-211Δ311-923 (with SEQ ID NO: 4), PolpTN2.sub.Δ248-254Δ311-923 (with SEQ ID NO: 5), and PolpTN2.sub.Δ243-254Δ311-923 (with SEQ ID NO: 6). The three first ones were expressed and purified as previously described, in simplicate or duplicate (FIG. 6).

[0348] We subsequently investigated the ability of these three variants to perform a template-independent DNA synthesis reaction in the presence or in the absence of the initiator sequence (bearing the Cy5 fluorophore in 5′).

[0349] For that purpose, PolpTN2.sub.Δ90-96Δ311-923, PolpTN2.sub.Δ205-211Δ311-923 and PolpTN2.sub.Δ248-254Δ311-923, along with of PolpTN2.sub.Δ311-923 as control, (FIG. 7) were incubated at 70° C. with or without the initiator sequence and their terminal transferase activity was evaluated by following the polymerization of the fluorescent primer, recorded at 675 nm (red channel) (FIG. 7B) while their ab-initio single-stranded nucleic acid synthesis activity was evaluated through MidoriGreen direct staining and recorded at 520 nm (green channel) (FIG. 7C).

[0350] As seen on FIGS. 7A to C, all tested variants demonstrated an ability to synthesis nucleic acids ab-initio and to extend single-stranded DNA fragments, similarly to PolpTN2.sub.Δ311-923 and as previously demonstrated for PolpTCIR10.sub.Δ303-928, PolpTpep.sub.Δ295-914 and PolpTcele.sub.Δ295-913.

[0351] These results hence demonstrate the possibility of shaping these enzymes to optimally integrate them into industrial processes that require downstream steps, such as ultrafiltration.

[0352] Moreover, since each of the three loop deletions, taken individually, is not detrimental to the activity of the enzyme, it is expectable that: [0353] combinations of two or even three loop deletions in the same PolpTN2.sub.Δ311-923 construct would also lead to a functional enzyme (SEQ ID NOs: 7 to 13); and [0354] deletion of the corresponding loop in PolpTCIR10.sub.Δ303-928 would also lead to a functional enzyme (SEQ ID NO: 16).

Example 3

Commercially Available Protected Nucleoside Triphosphate Pools are not Devoid of Impurities

[0355] A terminal transferase activity assay was carried out with PolpP12.sub.Δ297-898 at 60° C. (FIG. 8) using 3′-O-amino dATP or 3′-O-azidomethyl dATP and a single-stranded nucleic acid primer as initiator sequence (bearing a Cy5 fluorophore in 5′).

[0356] Four different conditions were tested: [0357] a: initiator sequence only; no enzyme, no dNTP at 60° C.; [0358] b: initiator sequence+enzyme; no dNTP at 60° C.; [0359] c: initiator sequence+enzyme+3′-O-amino dATP at 60° C.; [0360] d: initiator sequence+enzyme+3′-O-azidomethyl dATP at 60° C.;

[0361] Thus, PolpP12.sub.Δ297-898 was found to naturally incorporate 3′-reversible terminating nucleotides at 60° C., as demonstrated by the higher migration pattern of the initiator primer, when compared to the negative control (FIG. 8).

[0362] To further investigate the effect of higher temperatures on the ability of PolpP12.sub.Δ297-898 to incorporate 3′-reversible terminating nucleotides, a terminal transferase activity assay was carried out 80° C. using 3′-O-amino dNTPs (FIG. 9) or 3′-O-azidomethyl dNTP (FIG. 10) and a single-stranded nucleic acid primer as initiator sequence (bearing a Cy5 fluorophore in 5′).

[0363] Three different conditions were tested in each case: [0364] initiator sequence+enzyme; no dNTP at 80° C.; [0365] initiator sequence+enzyme+3′-O-amino-dATP or 3′-O-azidomethyl dATP at 80° C.; [0366] initiator sequence+enzyme+3′-O-amino dTTP or 3′-O-azidomethyl dTTP at 80° C.

[0367] As previously shown, PolpP12.sub.Δ297-898 was found to efficiently incorporate 3′-reversible terminating nucleotides at 80° C., as demonstrated by the higher migration pattern of the initiator primer, when compared to negative controls (FIGS. 9A and 10A).

[0368] Furthermore, it was found to incorporate both purine-type and pyrimidine-type nucleobases, with a yield of 76.6% and 80.1% for 3′-O-amino dATP and 3′-O-amino dTTP respectively (FIGS. 9B and C), while the incorporation 3′-O-azidomethyl dATP and 3′-O-azidomethyl dTTP led to yields of 66.5% and 82.9%, respectively (FIGS. 10B and C).

[0369] Despite these performances, FIGS. 8, 9A and 10A evidence that a polyaddition of 2 to 3 nucleoside triphosphates seems to occur at different levels.

[0370] In addition, quality control reports provided by the oxime-blocked nucleoside triphosphates' manufacturers indicate a purity ratio of around 90%. This seems to be consistent with our various observations.

[0371] This small percentage of impurity has extremely detrimental effects in the controlled synthesis of nucleic acids.

Example 4

Clean-Up Procedure of Terminating Nucleotides Prior to their Use in Nucleic Acids Synthesis

[0372] The means and methods described herein can be used for cleaning-up of contaminating nucleoside triphosphates comprising a free 3′-hydroxyl group in a pool of terminating nucleoside triphosphates. Using these means and methods, the costs of the whole clean-up procedure are considerably reduced, since template, primer, or solid support are not needed; moreover, the scope of nucleoside triphosphates that can be purified is wide:deoxyribonucleoside, ribonucleotides, chemical synthesis intermediates, etc.

[0373] Ab-Initio Synthesis Nucleic Acid Synthesis

[0374] Each pool of 3′-blocked nucleoside triphosphates at a concentration ranging from 200 μM to 5 mM is incubated in a buffer comprising 50 mM Tris-HCl (pH 8.0), 5 mM manganese chloride (MnC12), and the functionally active fragment of the DNA primase from Thermococcus nautili sp. 30-1 (with SEQ ID NO: 2), Thermococcus sp. CIR10 (with SEQ ID NO: 15), Thermococcus peptonophilus (with SEQ ID NO: 18), or Thermococcus celericrescens (with SEQ ID NO: 20) at a concentration ranging from 5 μM to 50 μM).

[0375] The targeted concentration of the initial pool of nucleosides triphosphates is calculated to obtain at least the purified 3′-blocked nucleoside triphosphates at a final concentration of 10×, thus ready to be used for different applications such as sequence-controlled, template-independent DNA synthesis.

[0376] The mix is incubated at 70° C. for 1 hour. The enzymatic reaction is then stopped by the addition of 12.5 mM EDTA (FIG. 11A).

[0377] Optionally, exogenous dideoxynucleoside triphosphates can be added in excess, to avoid incorporating terminating nucleoside triphosphate to the nascent nucleic acid strand (FIG. 11B). Such exogenous dideoxynucleoside triphosphates can, e.g., be functionalized to be further affinity purified.

[0378] Isolation of the 3′-Blocked Nucleoside Triphosphates

[0379] In presence of contaminating nucleoside triphosphates comprising a free 3′-hydroxyl group, the enzymatic reaction generates long single stranded nucleic acid fragments ranging from about 15 to hundreds of nucleotides long.

[0380] Purification of the 3′-blocked nucleoside triphosphates can be performed using centrifugal filtration columns, such as, e.g., Amicon® Ultra 0.5 (Merck Millipore) with a molecular weight cut-off ranging from 3 to 30 kD. Such device provides the best balance between recovery and spin time for synthetized nucleic acid and enzyme retention and release of 3′-blocked nucleoside triphosphates (FIG. 11C).

[0381] Hence, at the end of this filtration step, not only the synthetized nucleic acid and enzyme are retained, but above all, the 3′-blocked nucleoside triphosphates are directly recovered in the filtrate at the right concentration (10×), and in the suitable activity buffer for the next step.

[0382] Alternatively, the same result could be obtained using a HPLC system with, e.g., an anion-exchange medium (such as MiniQ™ from Cytiva, formerly GE Healthcare), or an affinity medium (depending on the functional group borne by exogenous dideoxynucleoside triphosphates added in excess).