CONTROLLED TEMPLATE-INDEPENDENT SYNTHESIS OF NUCLEIC ACIDS USING THERMOSTABLE ENZYMES

Abstract

The invention relates to methods 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 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 3-end nucleotide. It also relates to isolated functionally active fragments of archaeal DNA primases which are capable of template-independent terminal nucleotidyl transferase activity but are devoid of a template-independent primase activity.

Claims

1. 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 primase domain of the Pyrococcus sp. 12-1 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 3′-end nucleotide, wherein said primase domain has an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:3, and wherein said primase domain has an amino acid sequence of: has at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:3; and is capable of template-independent terminal nucleotidyl transferase activity; and is devoid of an ab-initio single-stranded nucleic acid synthesis activity.

2. The method according to claim 1, wherein said archaeal DNA primase or the functionally active variant thereof is from an archaeon of the Pyrococcus genus.

3. The method according to claim 1 or 2, wherein said archaeal DNA primase or the functionally active variant thereof is Pyrococcus sp. 12-1 DNA primase.

4. The method according to any one of claims 1 to 3, wherein said archaeal DNA primase belonging to the primase-polymerase family or the functionally active variant thereof is Pyrococcus sp. 12-1 DNA primase having the amino acid sequence of SEQ ID NO: 1.

5. The method according to claim 1, wherein said primase domain or functionally active fragment and/or variant thereof has the amino acid sequence of SEQ ID NO: 2, and wherein functionally active fragment and/or variant thereof: has at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2; and is capable of template-independent terminal nucleotidyl transferase activity; and is devoid of an ab-initio single-stranded nucleic acid synthesis activity.

6. The method according to any one of claims 1 to 5, wherein said primase domain of an archaeal DNA primase belonging to the primase-polymerase family is the primase domain of the Pyrococcus sp. 12-1 DNA primase having the amino acid sequence of SEQ ID NO: 2, or a functionally active fragment and/or variant thereof: having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2; and being capable of template-independent terminal nucleotidyl transferase activity; and being devoid of an ab-initio single-stranded nucleic acid synthesis activity.

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

8. The method according to claim 1, wherein the initiator sequence is a single stranded nucleic acid primer.

9. The method according to claim 1, wherein the template-independent synthesis of nucleic acids is carried out at a temperature ranging from about 60° C. to about 95° C.

10. The method according to claim 1, 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.

11. The method according to claim 1, wherein said method is 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.

12. The method according to claim 11, 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 primase domain of the archaeal DNA primase belonging to the primase-polymerase family or the functionally active variant thereof, 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; and thereby obtaining a nucleotide with a free 3′-hydroxyl group.

13. An isolated functionally active fragment of an archaeal DNA primase consisting of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a functionally active fragment and/or variant thereof: having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3; and being capable of template-independent terminal nucleotidyl transferase activity; and being devoid of an ab-initio single-stranded nucleic acid synthesis activity.

14. The isolated functionally active fragment of the archaeal DNA primase or variant thereof according to claim 13, consisting of an amino acid sequence of SEQ ID NO: 2, or a functionally active fragment and/or variant thereof: having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2; and being capable of template-independent terminal nucleotidyl transferase activity; and being devoid of an ab-initio single-stranded nucleic acid synthesis activity.

15. The isolated functionally active fragment of the archaeal DNA primase or variant thereof according to claim 13, consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

16. 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.

17. The nucleic acid according to claim 16, wherein the nucleic acid is operably linked to a promoter.

18. A host cell comprising the expression vector according to claim 17.

19. A method of producing the functionally active fragment of an archaeal DNA primase according to any one of claims 13 to 15, said method comprising: (a) culturing the host cell according to claim 18, under conditions suitable for the expression of said functionally active fragment of the archaeal DNA primase or variant thereof; and (b) isolating said functionally active fragment of the archaeal DNA primase or variant thereof from said host cell.

20. A kit comprising: an initiator sequence comprising a 3′-end nucleotide with a free 3′-hydroxyl group, optionally immobilized onto a support; at least one nucleoside triphosphate, optionally wherein the at least one nucleoside triphosphate is a terminating nucleoside triphosphate comprising a reversible 3′-blocking group; and the isolated functionally active fragment of the archaeal DNA primase according to any one of claims 13 to 15.

21. The method according to claim 12, further comprising the step of: (e) applying a washing solution to remove all reagents, in particular to remove the cleaving agent.

22. The method according to claim 21, further comprising the step of: (f) reiterating steps b) to e) multiple times to synthetize the nucleic acid until desired length and nucleotide sequence.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0240] FIG. 1 is a phylogenetic tree generated using BLOSUM62 with average distance of ten archaeal DNA primases or functionally active fragments thereof, from Pyrococcus sp. 12-1 (fragment PolpP12.sub.Δ297-898), Thermococcus sp. CIR10 (fragment PolpTCIR10.sub.Δ303-928), Thermococcus peptonophilus (fragment PolpTpep.sub.Δ295-914), Thermococcus celericrescens (fragment PolpTcele.sub.Δ295-913), Pyrococcus furiosus (full-length), Thermococcus kodakarensis (full-length), Saccharolobus solfataricus (full-length), Pyrococcus horikoshii (full-length), Archaeoglobus fulgidus (full-length) and Thermococcus nautili sp. 30-1 (fragment PolpTN2A311-923).

[0241] FIG. 2 is a photograph of an electrophoresis gel (SDS-PAGE) showing the purification of PolpTN2.sub.Δ311-923 (middle lane) and PolpP12.sub.Δ297-898 (right lane). MW ladder: molecular weight ladder (left lane).

[0242] FIG. 3 is a photograph of a 15% urea-PAGE showing a template-independent nucleic acid synthesis assay using PolpP12.sub.Δ297-898 [PolpP12.sub.Δ] or PolpTN2.sub.Δ311-923 [PolpTN2.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).

[0243] 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 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).

[0244] FIG. 4A: red channel, Cy5 fluorescence at 675 nm;

[0245] FIG. 4B: green channel, Sybr green II fluorescence at 520 nm;

[0246] FIG. 4C: merge of red and green channels.

[0247] FIGS. 5A-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 primer (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 PolpTN.sub.Δ231 923, during 15 minutes, followed by the addition of the initiator primer. Left lane shows the MW ladder (SmartLadder 200 to 10000 bp (Eurogentec)).

[0248] FIG. 5A: merge of red and green channels;

[0249] FIG. 5B: green channel, Sybr green II fluorescence at 520 nm;

[0250] FIG. 5C: red channel, Cy5 fluorescence at 675 nm.

[0251] FIGS. 6A-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, in comparison with the wild-type terminal deoxynucleotidyl transferase from calf thymus [TdT]. Reactions were performed at 37° C. or 70° C. using either a dGTP/dCTP mix or a dNTP mix as substrates. SF (dsDNA) Ladder: SmartLadder 100 to 1000 bp (Eurogentec); LF (dsDNA) Ladder: SmartLadder 200 to 10000 bp (Eurogentec).

[0252] FIG. 6A: merge of red and green channels;

[0253] FIG. 6B: red channel, Cy5 fluorescence at 675 nm;

[0254] FIG. 6C: green channel, Sybr green II fluorescence at 520 nm.

[0255] FIG. 7 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.

[0256] FIGS. 8A-C are a set of three figures showing the incorporation by PolpP12.sub.Δ297-898 of labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups at 80° C.

[0257] FIG. 8A: 15% urea-PAGE showing the incorporation of 3′-O-amino dATP or 3′-O-amino dTTP at 80° C.;

[0258] FIG. 8B: analysis report of 3′-O-amino dATP incorporation at 80° C. Rf: relative migration distance;

[0259] FIG. 8C: analysis report of 3′-O-amino dTTP incorporation at 80° C. Rf: relative migration distance.

[0260] FIGS. 9A-C are a set of three figures showing the incorporation by PolpP12.sub.Δ297-898 of nucleoside triphosphates labeled with 3′-O-azidomethylene groups at 80° C.

[0261] FIG. 9A: 15% urea-PAGE showing the incorporation of 3′-O-azidomethyl dATP or 3′-O-azidomethyl dTTP at 80° C.;

[0262] FIG. 9B: analysis report of 3′-O-azidomethyl dATP incorporation at 80° C. R.sub.f: relative migration distance;

[0263] FIG. 9C: analysis report of 3′-O-azidomethyl dTTP incorporation at 80° C. R.sub.f: relative migration distance.

[0264] FIGS. 10A-B are a set of two figures showing the incorporation by PolpP12.sub.Δ297-898 of nucleoside triphosphates labeled with 3′-O-(N-methyl-anthraniloyl) groups at 70° C.

[0265] FIG. 10A: 15% urea-PAGE showing the incorporation of 3′-O-(N-methyl-anthraniloyl)-2′-dATP at 70° C.;

[0266] FIG. 10B: analysis report of 3′-O-(N-methyl-anthraniloyl)-2′-dATP incorporation at 70° C. Rf: relative migration distance.

[0267] FIGS. 11A-B are a set of two figures showing the incorporation by PolpP12.sub.Δ297-898 of nucleoside triphosphates labeled with 3′-O-(2-nitrobenzyl) groups at 70° C.

[0268] FIG. 11A: 15% urea-PAGE showing the incorporation of 3′-O-(2-nitrobenzyl)-2′-dATP at 70° C.;

[0269] FIG. 11B: analysis report of 3′-O-(2-nitrobenzyl)-2′-dATP incorporation at 70° C. Rf: relative migration distance.

[0270] FIG. 12 is a photograph of a 15% urea-PAGE showing the incorporation by PolpP12.sub.Δ297-898 at 80° C. of ribonucleoside triphosphates as well as deoxyuridine triphosphate.

[0271] FIGS. 13A-B are a set of two photographs of a 15% urea-PAGE showing the incorporation by PolpP12.sub.Δ297-898 of ribonucleoside triphosphates labeled with a 3′-O-propargyl group.

[0272] FIG. 13A: incorporation of 3′-O-propargyl GTP by PolpP12.sub.Δ297-898 at 70° C.;

[0273] FIG. 13B: negative control with calf thymus TdT at 37° C.

[0274] FIGS. 14A-D are a set of four figures photographs showing the incorporation by PolpP12.sub.Δ297-898 of deoxyinosine triphosphate and base-modified nucleosides triphosphate at 70° C.

[0275] FIG. 14A: structure of biotin-14-N6-(6-aminohexyl)-dATP;

[0276] FIG. 14B: structure of γ-[N-(biotin-6-amino-hexanoyl)]-(5-aminoallyl)-2′-dUTP;

[0277] FIG. 14C: structure of 5-(3-aminoallyl)-2′-dUTP;

[0278] FIG. 14D: 15% urea-PAGE showing the incorporation by PolpP12.sub.Δ297-898 of deoxyinosine, biotin-14-N.sup.6-(6-aminohexyl)-dATP, γ-[N-(biotin-6-amino-hexanoyl)]-(5-aminoallyl)-2′-dUTP or 5-(3-aminoallyl)-2′-dUTP at 70° C.

[0279] FIG. 15 is a photograph of an electrophoresis gel (SDS-PAGE) showing the purification of PolpTN2.sub.Δ311-923 and PolpTN2.sub.Δ90-96Δ311-923. MW ladder: molecular weight ladder.

[0280] FIG. 16 is a photograph of a 1.5% agarose gel electrophoresis showing a template-independent nucleic acid synthesis assay using PolpTN2.sub.Δ311-923 and PolpTN2.sub.Δ90-96Δ311-923, 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). Red channel, Cy5 fluorescence at 675 nm.

EXAMPLES

[0281] The present invention is further illustrated by the following examples.

Example 1

[0282] Phylogenetic Analysis of Archaeal DNA Primases

[0283] A phylogenetic analysis was performed to highlight the evolutionary relationship between ten selected archaeal DNA primases: [0284] a functionally active fragment of the Pyrococcus sp. 12-1 DNA primase (PolnP12.sub.Δ297-898) having the amino acid sequence of SEQ ID NO: 2; [0285] a functionally active fragment of the Thermococcus sp. CIR10 DNA primase (PolpTCIR10.sub.Δ303-928) having the amino acid sequence of SEQ ID NO: 5; [0286] a functionally active fragment of the Thermococcus peptonophilus DNA primase (PolpTpep.sub.Δ295-914) having the amino acid sequence of SEQ ID NO: 7; [0287] a functionally active fragment of the Thermococcus celericrescens DNA primase (PolpTcele.sub.Δ295-913) having the amino acid sequence of SEQ ID NO: 9; [0288] a functionally active fragment of the Thermococcus nautili sp. 30-1 DNA primase (PolpTN2.sub.Δ311-923) having the amino acid sequence of SEQ ID NO: 11. [0289] Pyrococcus furiosus DNA primase having the amino acid sequence of SEQ ID NO: 12; [0290] Thermococcus kodakarensis DNA primase having the amino acid sequence of SEQ ID NO: 13; [0291] Saccharolobus solfataricus DNA primase having the amino acid sequence of SEQ ID NO: 14; [0292] Pyrococcus horikoshii DNA primase having the amino acid sequence of SEQ ID NO: 15; and [0293] Archaeoglobus fulgidus DNA primase having the amino acid sequence of SEQ ID NO: 16.

[0294] SEQ ID NO: 12 represents the amino acid sequence of the protein “DNA primase catalytic subunit PriS” from Pyrococcus furiosus with NCBI Reference Sequence WP_011011222 version 1 of 2019-06-20.

TABLE-US-00012 SEQ ID NO: 12 MLMREVTKEERSEFYSKEWSAKKIPKFIVDTLESREFGFDHNGEGPSDR KNQYSDIRDLEDYIRATSPYAVYSSVAFYENPREMEGWRGAELVFDIDA KDLPLKRCNHEPGTVCPICLEDAKELAKDTLIILREELGFENIHVVYSG RGYHIRILDEWALQLDSKSRERILAFISASEIENVEEFRRFLLEKRGWF VLKHGYPRVFRLRLGYFILRVNVPHLLSIGIRRNIAKKILDHKEEIYEG FVRKAILASFPEGVGIESMAKLFALSTRFSKAYFDGRVTVDIKRILRLP STLHSKVGLIATYVGTKEREVMKFNPFRHAVPKFRKKEVREAYKLWRES LEYE

[0295] SEQ ID NO: 13 represents the amino acid sequence of the protein “DNA primase catalytic subunit PriS” from Thermococcus kodakarensis with NCBI Reference Sequence WP 011250742 version 1 of 2019-06-15.

TABLE-US-00013 SEQ ID NO: 13 MSKLLREVTPEERRLYYSGEWDAKKLPEFIVESIERREFGFDHTGEGPS DRKNAFSDVRDLEDYIRATAPYAAYSSVAFYRNPQEMEGWLGAELVFDI DAKDLPLRRCQNEHPSGQVCPICLEDAKELARDTLIILKEDFGFENIHV VYSGRGYHIRVIDEWALKLDSKARERILSYVSAAEEVTFDDIQKRYIML SSGYFRVFRLRFGYFIQRINENHLKNIGLKRSTAEKLLDEKTRQDIVEK FVNKGLLAAFPEGVGYRTLLRLFGLSTTFSKAYFDGRVTVDLKRILRLP STLHSKVGLVATYIGSDEKRLEKFDPFKDAVPEFRKEEVQKAYQEWKEL HEG

[0296] SEQ ID NO: 14 represents the amino acid sequence of the protein “DNA primase small subunit PriS” from Saccharolobus solfataricus with NCBI Reference Sequence WP_009989180 version 1 of 2021-03-14.

TABLE-US-00014 SEQ ID NO: 14 MGTFTLHQGQTNLIKSFFRNYYLNAELELPKDMELREFALQPFGSDTYV RHLSFSSSEELRDYLVNRNLPLHLFYSSARYQLPSARNMEEKAWMGSDL LFDIDADHLCKLRSIRFCPVCGNAVVSEKCERDNVETLEYVEMTSECIK RGLEQTRNLVEILEDDFGLKPKVYFSGNRGFHVQVDCYGNCALLDSDER KEIAEYVMGIGVPGYPGGSENAPGWVGRKNRGINGVTIDEQVTIDVKRL IRIPNSLHGKSGLIVKRVPNLDDFEFNETLSPFTGYTIFLPYITIETEV LGSIIKLNRGIPIKIKSSIGIYLHLRNLGEVKAYVR

[0297] SEQ ID NO: 15 represents the amino acid sequence of the protein “DNA primase small subunit PriS” from Pyrococcus horikoshii with NCBI Reference Sequence WP_010884304 version 1 of 2019-06-20.

TABLE-US-00015 SEQ ID NO: 15 MLLREVTREERKNFYTNEWKVKDIPDFIVKTLELREFGFDHSGEGPSDR KNQYTDIRDLEDYIRATAPYAVYSSVALYEKPQEMEGWLGTELVFDIDA KDLPLRRCEHEPGTVCPICLNDAKEIVRDTVIILREELGFNDIHIIYSG RGYHIRVLDEWALKLDSKSRERILSFVSASEIEDVEEFRKLLLNKRGWF VLNHGYPRAFRLRFGYFILRIKLPHLINAGIRKSIAKSILKSKEEIYEE FVRKAILAAFPQGVGIESLAKLFALSTRFSKSYFDGRVTVDLKRILRLP STLHSKVGLIAKYVGTNERDVMRFNPFKHAVPKFRKEEVKVEYKKFLES LGT

[0298] SEQ ID NO: 16 represents the amino acid sequence of the protein “DNA primase small subunit PriS” from Archaeoglobus fulgidus with NCBI Reference Sequence WP_048064280 version 1 of 2019-06-15.

TABLE-US-00016 SEQ ID NO: 16 MLTKLFLKKKFEEYYSKNEVELPRKFKNREFAFVPLELLPDFVMHRHIS FRSETDFRAYILSNVPAHIYFSSAYYERPAEDKMENKGWLGADLIFDID ADHLPVKAQSFEKALEMAKREIKKLTAVLRADFGIRDMKIYFSGGRGYH VHVHDEEFLSLGSAERREIVDYLRLNSPKIVVEDRFANSNAAKRVLNYL RKKLEEDERLTSKLKIKPADLKKEKLTKKVIRAVEKFDYSALSIYIDAP VTADVKRLIRLPGSLHGKTGLRVTEVEDIESFNPLKDALAFGDEAVVVK VARKLNLSIGDFSGKIYPGRVKLPEYAAVFLICRGDASYDS

[0299] As seen on the phylogenetic tree (FIG. 1—generated using BLOSUM62 with average distance), we can observe a high degree of evolutionary divergence between the four DNA primases from Pyrococcus sp. 12-1, Thermococcus sp. CIR10, Thermococcus peptonophilus and Thermococcus celericrescens on one hand, and the DNA primases from Pyrococcus furiosus, Thermococcus kodakarensis, Saccharolobus solfataricus, Pyrococcus horikoshii, Archaeoglobus fulgidus and Thermococcus nautili sp. 30-1 on the other hand.

[0300] This evolutionary divergence is strengthened by the identity matrix (Table 1), which shows a very low identity score between the first four DNA primases and the later six DNA primases (maximum 11.1%).

[0301] Although the first four DNA primases show a higher degree of identity with the DNA primase from Thermococcus nautili sp. 30-1, both the phylogenetic tree (FIG. 1) and the

TABLE-US-00017 TABLE 1 P. T. S. P. A. P. sp. T. nautili T. sp. T. T. DNA primase furiosus kodakarensis solfataricus horikoshii fulgidus 12-1 sp. 30-1 CIR10 peptonophilus celericrescens P. ID 0.662 0.12 0.786 0.185 0.074 0.093 0.088 0.111 0.106 furiosus T. 0.662 ID 0.129 0.639 0.187 0.071 0.068 0.077 0.1 0.091 kodakarensis S. 0.12 0.129 ID 0.14 0.221 0.077 0.065 0.08  0.063 0.081 solfataricus P. 0.786 0.639 0.14 ID 0.196 0.08  0.082 0.085 0.1 0.1 horikoshii A. 0.185 0.187 0.221 0.196 ID 0.052 0.049 0.063 0.055 0.046 fulgidus P. 0.074 0.071 0.077 0.08 0.052 ID 0.511 0.739 0.614 0.614 sp. 12-1 T. nautili 0.093 0.068 0.065 0.082 0.049 0.511 ID 0.523 0.503 0.515 sp. 30-1 T. sp. 0.088 0.077 0.08 0.085 0.063 0.739 0.523 ID 0.588 0.611 CIR10 T. 0.111 0.1  0.063 0.1 0.055 0.614 0.503 0.588 ID 0.871 peptonophilus T. 0.106 0.091 0.081 0.1 0.046 0.614 0.515 0.611 0.871 ID celericrescens

[0302] identity matrix (Table 1) still demonstrate their distant relationship. Indeed, from an evolutionary point of view, the DNA primase from Thermococcus nautili sp. 30-1 appears to be out of the group with a maximal identity score of 52.3%.

Example 2

[0303] PolpP12.sub.Δ297-898 has a Template-Independent Terminal Nucleotidyl Transferase Activity and is Devoid of an Ab-Initio Single-Stranded Nucleic Acid Synthesis Activity

[0304] 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: 2) and from Thermococcus nautili sp. 30-1 (PolpTN2.sub.Δ311-923 having the amino acid sequence of SEQ ID NO: 11) were expressed and purified following a protocol adapted from WO2011098588 and Gill et al., 2014 (Nucleic Acids Res. 42(6):3707-3719) (FIG. 2).

[0305] A template-independent nucleic acid synthesis assay was carried out with either PolpP12.sub.Δ297-898 or PolpTN2.sub.Δ311-923, at 60° C., 70° C. and 80° C., using a single stranded nucleic acid primer as initiator sequence (bearing a Cy5 fluorophore in 5′).

[0306] Three different conditions were tested: [0307] a: initiator sequence only; no enzyme, no dNTP; [0308] b: initiator sequence+enzyme; no dNTP; [0309] c: initiator sequence+enzyme+dNTP mix (unprotected).

[0310] As seen on FIG. 3, both PolpP12.sub.A297-898 and PolpTN2.sub.Δ311-923 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.

[0311] Thus, to analyze the effect of high temperatures on PolpP12.sub.Δ297-898 and PolpTN2.sub.Δ311-923 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. 4). 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).

[0312] As shown on FIGS. 4A and C, both enzymes exhibit a strong template-independent terminal nucleotidyl transferase activity, which is demonstrated by the polymerization of the Cy5-labeled initiator primer (FIG. 4A). 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. 4B), which does not colocalize with the Cy5-labeled initiator primer (Cf. FIGS. 4A 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.

[0313] Interestingly, Béguin et al. have demonstrated that a combination of the full length PolpTN2 primase and the PolB DNA polymerase in presence of deoxyribonucleoside 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.

[0314] To further investigate this phenomenon, both PolpP12.sub.Δ297-898 and PolpTN2.sub.Δ311-923 were subjected to a template-independent nucleic acid synthesis assay (FIG. 5), carried out in the presence or in the absence of dNTPs and/or the initiator primer (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).

[0315] Nine different conditions were tested: [0316] 1: no enzyme, no initiator sequence, no dNTP; [0317] 2: dNTP only; no enzyme, no initiator sequence; [0318] 3: initiator sequence only; no enzyme, no dNTP; [0319] 4: initiator sequence+dNTP mix; no enzyme; [0320] 5: enzyme only, no initiator sequence, no dNTP; [0321] 6: enzyme+dNTP mix; no initiator sequence; [0322] 7: enzyme+initiator sequence; no dNTP; [0323] 8: enzyme+dNTP mix+initiator sequence; [0324] 9: enzyme+dNTP mix+initiator sequence (added after 15 minutes incubation); As shown on FIG. 5, neither PolpP12.sub.Δ297-898 nor PolpTN2.sub.Δ311-923 is able to synthesize nucleic acids in the absence of dNTPs (FIG. 5; lanes 1, 3, 5 and 7) while the association of dNTPs with the initiator primer leads to the synthesis of long nucleic acids fragments (FIG. 5A; lanes 8 and 9). Interestingly, in the absence of the initiator primer (FIG. 5; lanes 1, 2, 5 and 6) PolpTN2.sub.Δ311-923 readily exhibits a strong polymerase activity, upon the addition of dNTPs (FIGS. 5A and 5B; lane 6). Separated channel analysis reveals that this activity is independent of an initiator primer, as demonstrated by the absence of Cy5 fluorescence (FIG. 5C; lane 6), thus confirming the ab-initio single-stranded nucleic acid synthesis activity of PolpTN2.sub.Δ311-923. 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. 5A, 5B and 5C; lane 6), indicating that this enzyme is devoid of an ab-initio single-stranded nucleic acid synthesis activity.

[0325] To further investigate the impact of such ab-initio single-stranded nucleic acid synthesis activity on the ability of PolpP12.sub.Δ297-898 and PolpTN2.sub.Δ311-923 to extend a single stranded nucleic acid fragment, a competition assay was conducted by separating both reactions (FIG. 5, 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 primer for another 15 minutes of incubation, to perform a template-independent primer extension reaction. As expected, PolpP12.sub.Δ297-898 was found to extend the initiator primer (FIG. 5C, lane 9) as previously described, indicating that a pre-incubation with a dNTP mix does not affect its terminal nucleotidyl transferase activity. In contrast, a strong diffuse migration pattern could be observed for PolpTN2.sub.Δ311-923, after Sybr Green II staining (FIG. 5B, lane 9), while the initiator primer was found to migrate up to the dye front (FIG. 5C, lane 9), similarly to the negative control (FIG. 5C, lane 3). Thus, this result indicates that PolpTN2.sub.Δ311-923 is not able to extent the initiator primer in these experimental conditions.

[0326] Therefore, these results demonstrate the negative side effect of the ab-initio single-stranded nucleic acid synthesis activity of PolpTN2.sub.Δ311-923 on its ability to perform a template-independent terminal nucleotidyl transferase reaction, for which a strong competition can be observed. Conversely, PolpP12.sub.Δ297-898 appears devoid of ab-initio single-stranded nucleic acid synthesis activity, and rather acts as a true terminal nucleotidyl transferase, capable of extending an initiator primer to create long nucleic acids fragments, strengthening its application for industrial nucleic acids synthesis.

Example 3

[0327] PolpP12.sub.Δ297-898 has a Higher Processivity than a Member of X-Family Polymerases

[0328] To test whether the use of PolpP12.sub.Δ297-898 presents an industrial advantage over the use of X-family polymerases, a terminal transferase activity assay was carried out at 70° C., and was compared to that of the recombinant terminal deoxynucleotidyl transferase (TdT) from calf thymus at either 37° C. or 70° C. (FIG. 6).

[0329] Experiments were performed using a single-stranded nucleic acid primer as initiator sequence (bearing a Cy5 fluorophore in 5′) and in the presence of a dCTP/dGTP mix or a mixture of all four dNTPs as substrates. 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). TdT was obtained from New England Biolabs (M0315S).

[0330] As seen on FIGS. 6A-C, the transferase activity of PolpP12.sub.Δ297-898 at 70° C. results in the synthesis of long nucleic acid fragments (1.5 kb), either with a dCTP/dGTP mix or with a dNTP mix, when compared to TdT that synthetize only short fragments at 37° C. (400 kb). In addition, no elongation products are observed for TdT at 70° C., neither with a dCTP/dGTP mix nor with a dNTP mix, demonstrating its lack of thermostability.

[0331] Therefore, the industrial use of PolpP12.sub.Δ297-898 appears more promising than the use of TdT for the synthesis of long nucleic acids. This is especially true for the synthesis of GC-rich sequences, such as the one found in microsatellites, which tend to create highly stable secondary structures and that require temperatures higher than 60° C. to break their hydrogen bonding network.

Example 4

[0332] PolpP12.sub.Δ297-898 is Capable of Incorporating Protected Nucleosides Triphosphate

[0333] A terminal transferase activity assay was carried out with PolpP12.sub.Δ297-898 at 60° C. (FIG. 7) 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′).

[0334] Four different conditions were tested: [0335] a: initiator sequence only; no enzyme, no dNTP at 60° C.; [0336] b: initiator sequence+enzyme; no dNTP at 60° C.; [0337] c: initiator sequence+enzyme+3′-O-amino dATP at 60° C.; [0338] d: initiator sequence+enzyme+3′-O-azidomethyl dATP at 60° C.;

[0339] 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. 7). Such a surprising behavior is a very uncommon feature among polymerases, as most of them usually require site directed mutagenesis of their catalytic pocket to relieve the steric hindrance that block the incorporation of 3′-reversible terminating nucleotides.

[0340] 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 at 80° C. using 3′-O-amino dNTPs (FIG. 8) or 3′-O-azidomethyl dNTP (FIG. 9) and a single stranded nucleic acid primer as initiator sequence (bearing a Cy5 fluorophore in 5′).

[0341] Three different conditions were tested in each case: [0342] initiator sequence+enzyme; no dNTP at 80° C.; [0343] initiator sequence+enzyme+3′-O-amino-dATP or 3′-O-azidomethyl dATP at 80° C.; [0344] initiator sequence+enzyme+3′-O-amino dTTP or 3′-O-azidomethyl dTTP at 80° C.

[0345] 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. 8A and 9A).

[0346] 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. 8B and C), while the incorporation of 3′-O-azidomethyl dATP and 3′-O-azidomethyl dTTP led to yields of 66.5% and 82.9%, respectively (FIGS. 9B and C).

[0347] A terminal transferase activity assay was then carried out with PolpP12.sub.Δ297-898 at 70° C. using 3′-reversible terminating nucleotides bearing larger protecting groups, namely 3′-0-(N-methyl-anthraniloyl)-2′-dATP (FIG. 10) or 3′-O-(2-nitrobenzyl)-2′-dATP (FIG. 11), in presence of a single stranded nucleic acid primer as initiator sequence (bearing a Cy5 fluorophore in 5′).

[0348] For each experiment, two different conditions were tested: [0349] initiator sequence+enzyme; no dNTP at 70° C.; [0350] initiator sequence+enzyme+3′-O-(N-methyl-anthraniloyl)-2′-dATP (FIG. 10) or 3′-O-(2-nitrobenzyl)-2′-dATP (FIG. 11) at 70° C.

[0351] PolpP12.sub.Δ297-898 was found to incorporate nucleotides bearing large terminating groups on their 3′ position, as demonstrated by the higher migration pattern of the initiator primer, when compared to the negative control (FIGS. 10 and 11). In addition, these incorporations appear very efficient as judged by the high yields obtained, reaching up to 87.7% and 82% for 3′-O-(N-methyl-anthraniloyl)-2′-dATP and 3′-O-(2-nitrobenzyl)-2′-dATP, respectively.

[0352] Therefore, incorporation of such large functional groups on the 3′ position of nucleotides indicates that steric hinderance is neither a limitation nor a critical parameter for PolpP12.sub.Δ297-898 activity, and further allows a wide range of modifications with a broad spectrum of applications. Indeed, 3′-O-(N-methyl-anthraniloyl)-2′-dATP, also known as MANT-dATP, is a fluorescent nucleotide (κ.sub.exc 355 nm/λ.sub.em 448 nm) which can be used as a quantitative reporter during the nucleic acid synthesis process. Similarly, 3′-O-(2-nitrobenzyl)-2′-dATP exhibits an attractive industrial feature that arise from its photolabile protecting group, indicating that PolpP12.sub.Δ297-898 can be used in a process involving a photo-deprotection step instead of chemical deprotection.

Example 5

[0353] PolpP12.sub.Δ297-898 is Capable of Incorporating Ribonucleosides Triphosphate and Deoxyuridine Triphosphate

[0354] A terminal transferase activity assay was carried out with PolpP12.sub.Δ297-898 at 80° C. (FIG. 12) using rNTP or dUTP and a single stranded nucleic acid primer as initiator sequence (bearing a Cy5 fluorophore in 5′).

[0355] Seven different conditions were tested: [0356] initiator sequence+enzyme, no rNTP; [0357] initiator sequence+enzyme+ATP; [0358] initiator sequence+enzyme+AGP; [0359] initiator sequence+enzyme+UTP; [0360] initiator sequence+enzyme+CTP; [0361] initiator sequence+enzyme, no dNTP; [0362] initiator sequence+enzyme+dUTP;

[0363] PolpP12.sub.Δ297-898 was found to incorporate both ribonucleosides and deoxyuridine at 80° C., as demonstrated by the higher migration pattern of the initiator primer, when compared to the negative controls (FIG. 12).

[0364] This observation is all the more surprising that Gill et al., 2014 (Nucleic Acids Res. 42(6):3707-3719) showed that the DNA primase from Thermococcus nautili sp. 30-1 (having the amino acid sequence of SEQ ID NO: 10) and its truncated version PolpTN2.sub.Δ311-923 (having the amino acid sequence of SEQ ID NO: 11) both failed to incorporate ribonucleosides in a DNA primase activity assay.

Example 6

[0365] PolpP12.sub.Δ297-898 is Capable of Incorporating Protected Ribonucleosides Triphosphate

[0366] To further investigate the ability of PolpP12.sub.Δ297-898 to incorporate 3′-reversible terminating ribonucleotides, a terminal transferase activity assay was carried out at 70° C. using 3′-O-propargyl GTP (FIG. 13A) and a single stranded nucleic acid primer as initiator sequence (bearing a Cy5 fluorophore in 5′). For the sake of comparison, a similar experiment was carried out using the recombinant terminal deoxynucleotidyl transferase (TdT) from calf thymus at 37° C. (FIG. 13B).

[0367] For both experiments, two different conditions were tested: [0368] initiator sequence+no enzyme+3′-O-propargyl GTP at 37° C.; [0369] initiator sequence+calf thymus TdT+3′-O-propargyl GTP at 37° C.; [0370] initiator sequence+no enzyme+3′-O-propargyl GTP at 70° C.; [0371] initiator sequence+PolpP12.sub.Δ297-898+3′-O-propargyl GTP at 70° C.

[0372] Interestingly, in contrast to calf thymus TdT, PolpP12.sub.Δ297-898 was found to incorporate 3′-O terminating ribonucleotides at 70° C., as demonstrated by the higher migration pattern of the initiator primer, when compared to negative controls (FIGS. 13A and B).

[0373] Thus, such an incorporation suggests that PolpP12.sub.Δ297-898 can be used for de novo RNA synthesis and further reinforces its usefulness for the industrial synthesis of nucleic acids.

Example 7

[0374] PolpP12.sub.Δ297-898 is Capable of Incorporating Deoxyinosine Triphosphate and Base-Modified Nucleosides Triphosphate

[0375] Although the incorporation of sugar-modified nucleotides represents a major industrial concern, the synthesis of nucleic acids containing base analogs remains a critical feature for numerous biological applications. Indeed, some nucleotides such as inosine are usually employed for random mutagenesis experiments while biotin-modified bases serve as purification tag.

[0376] A terminal transferase activity assay was carried out with PolpP12.sub.Δ297-898 at 70° C. (FIG. 14) using deoxyinosine, biotin-14-N.sup.6-(6-aminohexyl)-dATP (FIG. 14A), γ-[N-(biotin-6-amino-hexanoyl)]-(5-aminoallyl)-2′-dUTP (FIG. 14B) or 5-(3-aminoallyl)-2′-dUTP (FIG. 14C), and a single stranded nucleic acid primer as initiator sequence (bearing a Cy5 fluorophore in 5′).

[0377] Six different conditions were tested: [0378] initiator sequence+enzyme+dNTP at 70° C.; [0379] initiator sequence only+enzyme, no dNTP at 70° C.; [0380] initiator sequence+enzyme+deoxyinosine at 70° C.; [0381] initiator sequence+enzyme+biotin-14-N.sup.6-(6-aminohexyl)-dATP at 70° C.; [0382] initiator sequence+enzyme+γ-[N-(biotin-6-amino-hexanoyl)]-(5-aminoallyl)-2′-dUTP at 70° C.; [0383] initiator sequence+enzyme+5-(3-aminoallyl)-2′-dUTP at 70° C.

[0384] PolpP12.sub.Δ297-898 was found to naturally incorporate all the tested base analogs nucleotides at 70° C., as demonstrated by the higher migration pattern of the initiator primer, when compared to the negative control without dNTP (FIG. 14D).

[0385] Incorporation of base-modified nucleotides represents another major industrial benefit as it allows a direct synthesis of chemically modified nucleic acids, which in turn saves time and efforts by limiting downstream modifications. For example, direct and controlled incorporation of one or multiple deoxyinosines in DNA coding sequences can be used to generate diversity during random mutagenesis and directed evolution experiments since this nucleotide is able to form wobble base pairs with adenosine, cytosine and uridine. Likewise, biotin is commonly used in molecular biology for both purification and detection. Indeed, biotin-modified nucleic acids can be purified and immobilized using streptavidin-agarose resins or detected and quantified using peroxidase-conjugated streptavidin. Nevertheless, when downstream modifications are still needed, direct incorporation of the aminoallyl group during de novo nucleic acids synthesis is of great importance. Indeed, this chemical modification facilitates specific nucleic acids labeling with biotin and dyes, using amino-reactive compounds, such as N-hydroxysuccinimide ester derivatives.

Example 8

[0386] PolpP12.sub.Δ297-898 Variant with Internal Deletion is Still Functional

[0387] Although PolpP12.sub.Δ297-898, PolpTN2.sub.Δ311-923, PolpTCIR10.sub.Δ303-928, PolpTpep.sub.Δ295-914 and PolpTcele.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 a loop that we suspected might be dispensable for terminal nucleotidyl transferase activity in PolpP12.sub.Δ297-898. This loop is located between amino acid residues 87 to 92 of PolpP12.sub.Δ297-898 (reference to SEQ ID NO: 2 numbering).

[0388] This study was driven by the necessity of providing an enzyme that is suitable for industrial applications, and adapted for both upstream and downstream processes. In that respect, the removal of this loop 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.

[0389] To investigate the effect of loop deletion and size reduction on terminal nucleotidyl transferase activity, we generated variants of PolpTN2.sub.Δ311-923 (which itself also comprises not one but three loops 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: 11 numbering). In particular, the first loop located between amino acid residues 90 to 96 of SEQ ID NO: 11 corresponds to the loop located between amino acid residues 87 to 92 of SEQ ID NO: 2.

[0390] We thus produced among others a PolpTN2.sub.Δ90-96Δ311-923 variant lacking these amino acid residues 90 to 96, which was expressed and purified as previously described (FIG. 15), and we subsequently investigated the ability of this variant 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′).

[0391] For that purpose, PolpTN2.sub.Δ90-96Δ311-923 and PolpTN2.sub.Δ311-923 (as control) 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. 16). Their ab-initio single-stranded nucleic acid synthesis activity was also evaluated through MidoriGreen direct staining and recorded at 520 nm (data not shown).

[0392] As seen on FIG. 16, PolpTN2.sub.Δ90-96Δ311-923 demonstrated an ability to extend single-stranded DNA fragments, similarly to PolpTN2.sub.ΔΔ311-923. It also demonstrated an ab-initio activity (data not shown).

[0393] These results hence demonstrate the possibility of shaping these enzymes to optimally integrate them into industrial processes that require downstream steps, such as ultrafiltration.

[0394] Hence, since this loop deletion is not detrimental to the activity of the enzyme, it is expectable that the deletion of the corresponding loop in PolpP12.sub.Δ297-898 would also lead to a functional enzyme (PolpP12.sub.Δ87-92Δ297-898 with SEQ ID NO: 3).

CONCLUSION

[0395] In conclusion, we were able to show that PolpP12.sub.Δ297-898 exhibit a template-independent nucleic acid synthesis activity in presence of an initiator primer and nucleosides triphosphate, whether unprotected or 3′-O protected, and regardless of the size of the protecting group. Interestingly, this template-independent nucleic acid synthesis activity was not only observed with deoxyribonucleotides but also with ribonucleotides, as well as with base-modified nucleosides triphosphate.

[0396] PolpP12.sub.Δ297-898 is a thermostable enzyme, which makes it especially useful for the synthesis of GC-rich sequences which necessitate temperatures higher than 60° C. to break their stable secondary structures during synthesis. It is also highly processive compared to the classically used TdT, since it was able to synthetize long strands of 1.5 kb at 70° C. when TdT could synthetize only small strands of 400 b at 37° C.

[0397] Moreover, PolpP12.sub.Δ297-898 does not exhibit an ab-initio nucleic acid synthesis activity, contrary to PolpTN2.sub.Δ311-923, which competing activity may be detrimental in several nucleic acid synthesis processes.

[0398] Finally, PolpP12A87-92.sub.Δ297-898 is expected to display the same activity as PolpP12.sub.Δ297-898, while being more stable and producible in higher amounts.

[0399] All these surprising properties and capabilities of PolpP12.sub.Δ297-898 make it thus an excellent resource for nucleic acid synthesis processes.